Disclosed herein are viral vector production systems secreting nuclease
for degradation of residual nucleic acid during viral vector production
and methods of the same. Such a viral vector production system comprises
a viral vector production cell comprising nucleic acid sequences
encoding: 1) viral vector components; and 2) a nuclease, wherein the
nuclease is expressed in the production cell and secreted in cell culture
thereby degrading residual nucleic acid during viral vector production.
Another such viral vector production system comprises 1) a viral vector
production cell comprising nucleic acid sequences encoding viral vector
components; and 2) a nuclease helper cell comprising a nucleic acid
sequence encoding a nuclease, wherein the nuclease is expressed and
secreted in co-culture of the production cell of 1) and the helper cell
of 2), thereby degrading residual nucleic acid during viral vector
production.
| Inventors: |
Farley; Daniel; (Oxford, GB)
; Mitrophanous; Kyriacos; (Oxford, GB)
|
| Applicant: | | Name | City | State | Country | Type |
Oxford BioMedica (UK) Limited |
Oxford | |
GB
| | |
|---|
| Family ID:
|
63921169
|
| Appl. No.:
|
16/980825
|
| Filed:
|
March 15, 2019 |
| PCT Filed:
|
March 15, 2019 |
| PCT NO:
|
PCT/GB2019/050737 |
| 371 Date:
|
September 14, 2020 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 62644373 | Mar 16, 2018 | |
| | 62795222 | Jan 22, 2019 | |
|
|
| Current U.S. Class: |
1/1 |
| Current CPC Class: |
C07K 2319/02 20130101; C12N 2740/15051 20130101; C07K 2319/21 20130101; C12N 5/0686 20130101; C12N 7/00 20130101; C12N 9/22 20130101; C12N 2800/22 20130101; C12N 2710/10051 20130101; C12N 2830/003 20130101; C12N 5/16 20130101; C12N 2510/02 20130101; C07K 2319/04 20130101; C12N 2502/99 20130101; C12N 2740/10051 20130101; C12N 2750/14151 20130101 |
| International Class: |
C12N 7/00 20060101 C12N007/00; C12N 9/22 20060101 C12N009/22; C12N 5/071 20060101 C12N005/071 |
Foreign Application Data
| Date | Code | Application Number |
|---|
| Sep 7, 2018 | GB | 1814590.4 |
Claims
1. A viral vector production system comprising a viral vector production
cell comprising nucleic acid sequences encoding: 1) viral vector
components; and 2) a nuclease, wherein the nuclease is expressed in the
production cell and secreted in cell culture thereby degrading residual
nucleic acid during viral vector production.
2. A viral vector production system comprising: 1) a viral vector
production cell comprising nucleic acid sequences encoding viral vector
components; and 2) a nuclease helper cell comprising a nucleic acid
sequence encoding a nuclease, wherein the nuclease is expressed and
secreted in co-culture of the production cell of 1) and the helper cell
of 2), thereby degrading residual nucleic acid during viral vector
production.
3. A method of producing a viral vector, the method comprising,
transfecting a viral vector production cell with nucleic acid sequences
encoding: 1) viral vector components; and 2) a nuclease, wherein the
viral vector components and the nuclease are expressed in the viral
vector production cell and secreted in cell culture thereby degrading
residual nucleic acid during viral vector production.
4. A method of producing a viral vector, the method comprising contacting
1) a viral vector production cell expressing viral vector components with
2) a nuclease helper cell expressing a nuclease, wherein the nuclease is
expressed in the helper cell and secreted in co-culture of the production
cell with the helper cell thereby degrading residual nucleic acid during
viral vector production.
5. A method of producing a viral vector, the method comprising contacting
1) a viral vector production cell expressing viral vector components with
2) a liquid feed from nuclease helper cell expressing a nuclease, wherein
the nuclease is expressed in the cell and secreted in cell culture
thereby degrading residual nucleic acid during viral vector production.
6. In an improved method of producing a viral vector, the improvement
comprising introducing nucleic acid sequences into a viral vector
production cell, wherein the nucleic acid sequences encode: 1) viral
vector components; and 2) a nuclease, wherein the nuclease is expressed
in the production cell and secreted in cell culture thereby degrading
residual nucleic acid during viral vector production.
7. In an improved method of producing a viral vector, the improvement
comprising contacting in co-culture a viral vector production cell
expressing viral vector components with a nuclease helper cell expressing
a nuclease, wherein the nuclease is expressed in the production cell and
secreted in cell culture thereby degrading residual nucleic acid during
viral vector production.
8. (canceled)
9. (canceled)
10. The viral vector production system of claim 1, wherein the nuclease
is an extracellular nuclease, a sugar-non-specific nuclease, or a
salt-active nuclease.
11-13. (canceled)
14. The viral vector production system of claim 1, wherein the nuclease
is selected from the group consisting of: Vibrio cholerae Endonuclease I
of SEQ ID NO: 1, Vibrio salmonicida Endonuclease I of SEQ ID NO: 2,
Serratia marcescens Nuclease A of SEQ ID NO: 3, BacNucB of SEQ ID NO: 4,
VcEndA-12glc of SEQ ID NO: 5, VcEndA-123glc of SEQ ID NO: 6,
VcEndA-124glc of SEQ ID NO: 7, VcEndA-134glc of SEQ ID NO: 8,
VcEndA-13glc of SEQ ID NO: 9, VcEndA-14glc of SEQ ID NO: 10, and
VcEndA-1glc of SEQ ID NO: 11.
15-31. (canceled)
32. The viral vector production system of claim 1, wherein the viral
vector components comprise a nucleotide of interest (NOI).
33. The viral vector production system of claim 1, wherein the viral
vector components are retroviral vector components.
34. (canceled)
35. The viral vector production system of claim 33, wherein the viral
vector components comprise i) gag-pol; ii) env; iii) optionally the RNA
genome of a retroviral vector; and iv) optional rev, or a functional
substitute thereof.
36-41. (canceled)
42. The viral vector production system of claim 1, wherein expression of
the nuclease is inducible or conditional, and wherein the nucleic acid
encoding the nuclease comprises an inducible or conditional promoter or
regulatory element.
43. A production cell for producing viral vectors comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease,
wherein the nuclease is expressed in the viral vector production cell and
secreted in cell culture thereby degrading residual nucleic acid during
viral vector production, and wherein the production cell is a eukaryotic
production cell.
44. The cell according to claim 43, wherein the nuclease is an
endonuclease, an exonuclease, or an endonuclease fused to an exonuclease.
45-51. (canceled)
52. A production cell for producing viral vectors comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease fusion
protein, wherein the nuclease fusion protein comprises an exonuclease
domain fused to an endonuclease domain, and wherein the nuclease fusion
protein is expressed in the viral vector production cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production, and wherein the production cell is a eukaryotic production
cell.
53. The cell of claim 52, wherein the endonuclease is a VcEndA.
54. (canceled)
55. A cell culture device comprising a viral vector production system
comprising a viral vector production cell comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease,
wherein the nuclease is expressed in the production cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production.
56. (canceled)
57. A variant of a secreted nuclease capable of degrading residual
nucleic acid during viral vector production, said variant comprising the
amino acid sequence of SEQ ID NO: 11.
58. A modified nuclease having increased cell-retention and/or or
cell-association that is expressed through the secretory pathway of a
eukaryotic cell, wherein the modified nuclease comprises a retention
signal at its C-terminus.
59-71. (canceled)
72. The viral vector production system according to claim 1, wherein
activity of secreted nuclease in the cell culture is at least about 1
unit per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
73-76. (canceled)
77. A nuclease helper cell wherein secreted nuclease activity within the
helper cell culture is at least about 10 unit per mL of equivalent
Benzonase.RTM. nuclease activity as determinable by the assay presented
as Assay 1 herein.
78-82. (canceled)
83. The viral vector production system according to claim 1, wherein the
nuclease comprises a cell retention signal.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the production of viral vectors. In
particular, the invention relates to viral vector cell production systems
engineered to express and secrete a nuclease into cell culture media
during the vector manufacturing process.
BACKGROUND TO THE INVENTION
[0002] As indicated above, the present invention relates to production
cells, the preparation thereof and uses thereof. A production cell is
sometimes also referred to as a host cell or host production cell. The
production cells are useful in inter alia gene therapy.
[0003] Gene therapy broadly involves the use of genetic material to treat
disease. It includes the supplementation of cells with defective genes
(e.g. those harbouring mutations) with functional copies of those genes,
the inactivation of improperly functioning genes and the introduction of
new therapeutic genes.
[0004] Therapeutic genetic material may be incorporated into the target
cells of a host using vectors to enable the transfer of nucleic acids.
Such vectors can be generally divided into viral and non-viral
categories.
[0005] Viruses naturally introduce their genetic material into target
cells of a host as part of their replication cycle. Engineered viral
vectors harness this ability to enable the delivery of a nucleotide of
interest (NOI) to a target cell. To date, a number of viruses have been
engineered as vectors for gene therapy. These include retroviruses,
adenoviruses (AdV), adeno-associated viruses (AAV), herpes simplex
viruses (HSV) and vaccinia viruses.
[0006] In addition to modification to carry a nucleotide of interest,
viral vectors are typically further engineered to be replication
defective. As such, the recombinant vectors can directly infect a target
cell, but are incapable of producing further generations of infective
virions. Other types of viral vectors may be conditionally replication
competent within cancer cells only, and may additionally encode a toxic
transgene or pro-enzyme.
[0007] The use of viral vectors for delivery of therapeutic genes is well
known and wide-ranging across indications. In particular, gene therapy
advances and products are now an important part of our global healthcare
markets. Contemporary gene therapy vectors based on RNA viruses such as
.gamma.-Retroviruses and Lentiviruses, and DNA viruses such as Adenovirus
and Adeno-associated virus (AAV) have shown promise in a growing number
of human disease indications. These include ex vivo modification of
patient cells for haematological conditions, and in vivo treatment of
ophthalmic, cardiovascular, neurodegenerative diseases and tumor therapy
or immunotherapy. Other viral vectors such as viruses based on Poxviruses
and Avian viruses are widely used in human and animal vaccinations.
[0008] Retroviral vectors, developed as therapies for various genetic
disorders, continue to show increasing promise in clinical trials and now
a few form the basis of approved therapeutic products. Currently there
are over 459 human clinical trials involving retroviral gene therapy
registered in the Journal of Gene Medicine database; 158 gene therapy
clinical trials are using lentiviral vectors
(http://www.abedia.com/wiley/vectors.php, updated in April, 2017).
Strimvelis received marketing authorisation from the European Commission
on 26 May 2016; Strimvelis is a product for treatment of ADA-SCID based
on patient CD34.sup.+ cells transduced ex vivo with retroviral vectors
expressing the ADA gene. Kymriah (USAN: tisagenlecleucel) received
approval from the FDA on 30 Aug. 2017; Kymriah is a product for the
treatment of patients up to 25 years old with refractory ALL. Papers on
retroviral gene therapy include Wang X, Naranjo A, Brown C E, Bautista C,
Wong C W, Chang W C, Aguilar B, Ostberg J R, Riddell S R, Forman S J,
Jensen M C (2012) J Immunother. 35(9):689-701, Hu Y, Wu Z, Luo Y, Shi J,
Yu J, Pu C, Liang Z, Wei G, Cui Q, Sun J, Jiang J, Xie J, Tan Y, Ni W, Tu
J, Wang J, Jin A, Zhang H, Cai Z, Xiao L, Huang H. (2017) Clin Cancer
Res. 23(13):3297-3306, Galy, A. and A. J. Thrasher (2010) Curr Opin
Allergy Clin Immunol 11(6): 545-550; Porter, D. L., B. L. Levine, M.
Kalos, A. Bagg and C. H. June (2011) N Engl J Med 365(8): 725-733;
Campochiaro, P. A. (2012) Gene Ther 19(2): 121-126; Cartier, N., S.
Hacein-Bey-Abina, C. C. Bartholomae, P. Bougneres, M. Schmidt, C. V.
Kalle, A. Fischer, M. Cavazzana-Calvo and P. Aubourg (2012) Methods
Enzymol 507: 187-198; Sadelain, M., I. Riviere, X. Wang, F. Boulad, S.
Prockop, P. Giardina, A. Maggio, R. Galanello, F. Locatelli and E.
Yannaki (2010) Ann NY Acad Sci 1202: 52-58; DiGiusto, D. L., A. Krishnan,
L. Li, H. Li, S. Li, A. Rao, S. Mi, P. Yam, S. Stinson, M. Kalos, J.
Alvarnas, S. F. Lacey, J. K. Yee, M. Li, L. Couture, D. Hsu, S. J.
Forman, J. J. Rossi and J. A. Zaia (2010) Sci Transl Med 2(36): 36ra43
and Segura M M, M. M., Gaillet B, Gamier A. (2013) Expert opinion in
biological therapy).
[0009] Important examples of such vectors include the gamma-retrovirus
vector system (based on MMLV), the primate lentivirus vector system
(based on HIV-1) and the non-primate lentivirus vector system (based on
EIAV).
[0010] Reverse genetics has allowed these virus-based vectors to be
heavily engineered such that vectors encoding large heterologous
sequences (circa 10 kb) can be produced by transfection of mammalian
cells with appropriate DNA sequences (reviewed in Bannert, K. (2010)
Caister Academic Press: 347-370).
[0011] Engineering and use of retroviral vectors at the research stage
typically involve the production of reporter-gene vectors encoding, for
example, GFP or lacZ. The titres of these clinically irrelevant vectors
are usually in the region of 1.times.10.sup.6 to 1.times.10.sup.7
transducing units per mL (TU/mL) of crude harvest material.
[0012] The manufacture of viral vectors for human gene therapy and
vaccination is well documented over the last several decades in
scientific journals. Well known methods of viral vector manufacture
include the transfection, such as transient transfection, of primary
cells or mammalian/insect cell lines with vector DNA components, followed
by a limited incubation period and then harvest of crude vector from
culture media and/or cells. Transient transfection requires that the
viral genes necessary for the production of viral vectors are introduced
into a production cell (for example, HEK-293) via plasmids by
transfection. Often, each component required for vector production is
encoded by separate plasmids, partly for safety reasons, as it would then
require a number of recombination events to occur for a replication
competent virus particle to be formed through the production process.
[0013] After transfection, incubation & harvest, viral vector virions are
then purified and concentrated from crude material using a generalized
process of some or all of the following steps: 1) clarification, 2)
digest of unwanted or contaminating nucleic acid (e.g., Benzonase.RTM.
treatment), 3) column chromatography (e.g., ion exchange), 4) buffer
exchange/concentration (e.g., ultrafiltration) and further nucleic acid
removal (e.g., second Benzonase.RTM. treatment), 5) polishing (e.g., size
exclusion) and 6) sterile filtration.
[0014] Despite decades of refinement, transient transfection has inherent
drawbacks. The cost of transfection agents/plasmids, and/or process
agents are high and, coupled with the labour-intensive nature of the
transfection technique, this makes transient transfection an expensive
and technically complex process for clinical/commercial vector
production.
[0015] Thus, there is a desire in the art to provide alternative methods
of producing viral vectors which help to address the known issues
associated with the transient transfection process.
[0016] There has been an attempt to generate stable packaging cell lines
in recent years, where viral packaging genes are introduced into
eukaryotic host cells along with selection markers such that these genes
can be stably integrated into the cell. Similarly, stable producer cell
lines also exist where the retroviral genome is also stably integrated.
Both of these allow circumvention of a significant portion of the
transient transfection process.
[0017] Reference is also made to co-pending EP 17210359.0 application
number entitled RETROVIRAL VECTOR, incorporated by reference in its
entirety herein, and which describes viral vector production systems and
methods employing modular constructs comprising at least two of the
nucleic acid components necessary for viral vector production. Such
modular constructs were found to provide levels of vector production
comparable to those with a traditional multi-plasmid transient process,
but allowing for a significant reduction in, for example, the use of
transfection agents.
[0018] Thus, there is a desire in the vector manufacturing sector to
improve both transient vector production processes as well as the
processes for generating stable packaging and producer cell lines.
[0019] The removal of nucleic acids, derived from either production cells
or viral vector components, from the final drug product is an important
aspect of safety and an area of viral vector manufacturing ripe for
improvement. When transformed eukaryotic cell lines, such as HEK293T
cells (which also contain Adenovirus E1 and SV40 T antigen genes) are
used for production, these cells typically harbour genes with
(proto)-oncogenic properties. The inevitable cell death and release of
production cell DNA during viral vector manufacture leads to the presence
of such (partial and contaminating) sequences within crude harvest
material. It is therefore desirable to minimize general DNA contamination
(e.g., longer contaminating dsDNA is degraded to short forms within the
final product) to preclude the potential for unnecessary & potentially
harmful functional gene sequences from being integrated into patient
cells during vector delivery. In addition, typical viral vector
production methods that transiently transfect production cells with large
quantities of plasmid DNA (pDNA) encoding the viral vector components
will result in the majority of the contaminating DNA being of vector
component origin. As such, it is also desirable to remove contaminating,
residual pDNA that could otherwise be taken up and expressed by patient
cells.
[0020] From lab scale to industrial scale, viral vector manufacturers have
turned to the use of recombinant nucleases, such as derived from Serratia
marcescens (e.g., Benzonase.RTM.) or other commercial hydrolytic
nucleases, to treat crude vector material and remove contaminating
nucleic acid during the upstream and downstream purification processes.
For nuclease treatment during upstream processing, a recombinant nuclease
in protein form is typically added to harvest material followed by
incubation at less than or equal to 37.degree. for a limited period of
time. This, however, represents a potentially avoidable additional
processing step, and one in which elevated temperature and increased
incubation time may lead to loss in vector stability. To avoid this as a
stand-alone step after harvest, a recombinant nuclease is often added
into the production cell culture at latter stages of vector production.
However, if the half-life of the nuclease is relatively short or activity
is insufficient to degrade the required amount of residual nucleic acid,
a large amount of nuclease may need to be added at once or continually
during these latter stages of culturing. Unfortunately, the use of
commercially available recombinant nuclease at this stage of the process
often becomes practically burdensome and cost-prohibitive, particularly
as scale is increased to hundreds or thousands of litres.
[0021] Thus, there is a desire to improve viral vector production and
manufacturing processes so as to streamline and make more efficient the
critical step(s) of degrading residual nucleic acid during viral vector
production.
SUMMARY OF THE INVENTION
[0022] The invention disclosed herein describes highly effective and
streamlined viral vector production systems and manufacturing processes
employing the expression and secretion of a hydrolytic nuclease(s) in
viral vector production cells during the production of viral vectors. As
such, secretion of a nuclease degrades unwanted or contaminating
(residual) nucleic acid during viral vector production. In such vector
production systems & processes, the nuclease, encoded by a nucleotide
expression cassette, is either transiently co-transfected with the viral
vector component expression cassette(s) or stably integrated within a
production cell genome or nuclease helper cell genome, or nuclease helper
cells may be generated by transient transfection with the nuclease
expression cassette.
[0023] Due to the complexity of the viral vector production process,
expression and secretion of a nuclease from a gene expression cassette in
conjunction with a viral vector production system and/or during the viral
vector production process is contrary to the state of the art which
demonstrates only the expression/co-expression of nuclease in bacterial
cells and which, in a viral vector context employs the use of adding
commercially available nuclease as a recombinant protein-based enzymatic
treatment of viral vector at burdensome upstream and/or downstream time
points. It was therefore considered, prior to the invention disclosed
herein, that expression and secretion of a nuclease encoded by a nucleic
acid expression cassette in conjunction with viral vector component(s)
expression cassette(s) in a viral vector production system would result
in degradation of necessary vector component DNA thus leading to reduced
expression of the vector components, low titres of produced viral vector,
and/or toxicity to the viral vector production cells.
[0024] However, in an effort to improve viral vector production
manufacturing, the inventors provide the invention disclosed herein:
viral vector production systems and methods of producing viral vector
employing the expression and secretion of a nuclease in viral vector
production cells (producer and/or nuclease helper cells) during the
production of therapeutic viral vectors. The invention offers a more
efficient (and cost-effective) viral vector production system/method
compared to current commercial nuclease techniques. Moreover, the
invention provides the manufacturing of viral vectors in a streamlined
manner, compared to conventional vector manufacturing techniques, by
engineering the secretion of a nuclease for degradation of unwanted or
contaminating residual nucleic acid during viral vector production
without impacting viral titers and/or imparting toxicity to viral vector
production cells. The inventors show that for lentiviral vector
production the application of secreted nuclease in the upstream phase of
production can lead to such efficient degradation of residual DNA within
crude harvest material, that further nuclease treatment during the
downstream process is made unnecessary. Importantly, the levels of
residual DNA within purified/concentrated lentiviral vector material can
be greatly reduced when employing secreted nucleases compared to standard
commercial nucleases, which is of great significance in the gene therapy
field as regulators require ever-increasing improvements in product
quality/safety.
[0025] Accordingly, disclosed herein is a viral vector production system
engineered to secrete a nuclease.
[0026] In one embodiment, the viral vector production system comprises a
viral production cell comprising nucleic acid sequences encoding: 1)
viral vector components; and 2) a nuclease, wherein the nuclease is
expressed in the production cell and secreted in cell culture thereby
degrading residual nucleic acid during viral vector production.
[0027] In another embodiment, the viral vector production system comprises
1) a viral production cell comprising nucleic acid sequences encoding
viral vector components; and 2) a nuclease helper cell comprising a
nucleic acid sequence encoding a nuclease, wherein the nuclease is
expressed and secreted in co-culture of the production cell of 1) and the
helper cell of 2), thereby degrading residual nucleic acid during viral
vector production.
[0028] Also, disclosed herein are methods of producing a viral vector, the
methods comprising expressing and secreting a nuclease during viral
vector production. In particular, such a method of producing a viral
vector comprises the steps of: transfecting (sometimes referred to as
contacting) a viral production cell with nucleic acid sequences encoding:
1) viral vector components; and 2) a nuclease, wherein the nuclease is
expressed in the production cell and secreted in cell culture thereby
degrading residual nucleic acid during viral vector production.
[0029] Additionally, disclosed herein is a method of co-culture comprising
contacting a viral vector production cell expressing vector components
with a nuclease helper cell expressing a nuclease. In a further
embodiment, a liquid feed from the helper cell is contacted with a viral
vector production cell expressing vector components.
[0030] In another embodiment of the invention disclosed herein, is an
improved method of producing a viral vector, the improvement comprising
introducing nucleic acid sequences into a viral vector production cell,
wherein the nucleic acid sequences encode: 1) viral vector components;
and 2) a nuclease, wherein the nuclease is expressed in the production
cell and secreted in cell culture thereby degrading residual nucleic acid
during viral vector production.
[0031] Also disclosed herein is an improved method of producing a viral
vector, the improvement comprising contacting in co-culture a viral
vector production cell comprising viral vector components with a nuclease
helper cell expressing a nuclease, wherein the nuclease is expressed in
the helper cell and secreted in the cell co-culture thereby degrading
residual nucleic acid during viral vector production.
[0032] In another embodiment of the viral vector production system or
methods disclosed herein, cell culture is maintained in a pH range of 6.5
pH to 7.2 pH.
[0033] In another embodiment of the viral vector production system or
methods disclosed herein, the nuclease is a sugar-non-specific nuclease.
[0034] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within the main viral vector production vessel
is at least about 1 Benzonase.RTM. unit equivalents per mL, preferably
wherein said nuclease activity is determinable by the DNAse Alert.TM.
assay provided herein as Assay 1.
[0035] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within the main viral vector production vessel
is at least about 10 Benzonase.RTM. unit equivalents per mL, preferably
wherein said nuclease activity is determinable by the DNAse Alert.TM.
assay provided herein as Assay 1.
[0036] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within the main viral vector production vessel
is at least about 50 Benzonase.RTM. unit equivalents per mL, preferably
wherein said nuclease activity is determinable by the DNAse Alert.TM.
assay provided herein as Assay 1.
[0037] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within the main viral vector production vessel
is at least about 100 Benzonase.RTM. unit equivalents per mL, preferably
wherein said nuclease activity is determinable by the DNAse Alert.TM.
assay provided herein as Assay 1.
[0038] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within nuclease helper cell cultures prior to
inoculating the main viral vector production vessel is at least about 10
Benzonase.RTM. unit equivalents per mL, preferably wherein said nuclease
activity is determinable by the DNAse Alert.TM. assay provided herein as
Assay 1.
[0039] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within nuclease helper cell cultures prior to
inoculating the main viral vector production vessel is at least about 50
Benzonase.RTM. unit equivalents per mL, preferably wherein said nuclease
activity is determinable by the DNAse Alert.TM. assay provided herein as
Assay 1.
[0040] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within nuclease helper cell cultures prior to
inoculating the main viral vector production vessel is at least about 100
Benzonase.RTM. unit equivalents per mL, preferably wherein said nuclease
activity is determinable by the DNAse Alert.TM. assay provided herein as
Assay 1.
[0041] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within nuclease helper cell cultures prior to
inoculating the main viral vector production vessel is at least about
1000 Benzonase.RTM. unit equivalents per mL, preferably wherein said
nuclease activity is determinable by the DNAse Alert.TM. assay provided
herein as Assay 1.
[0042] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within nuclease helper cell cultures prior to
inoculating the main viral vector production vessel is at least about
1500 Benzonase.RTM. unit equivalents per mL, preferably wherein said
nuclease activity is determinable by the DNAse Alert.TM. assay provided
herein as Assay 1.
[0043] In another embodiment of the viral vector production system or
methods or other aspects of the present invention disclosed herein, the
nuclease activity achieved within nuclease helper cell cultures prior to
inoculating the main viral vector production vessel is at least about
2000 Benzonase.RTM. unit equivalents per mL, preferably wherein said
nuclease activity is determinable by the DNAse Alert.TM. assay provided
herein as Assay 1.
[0044] In another embodiment of the viral vector production system or
methods, the nuclease is selected from the group consisting of SmNucA,
VsEndA, VcEndA, and BacNucB.
[0045] In a further embodiment, the nuclease is Vibrio cholerae
Endonuclease I of SEQ ID NO: 1 or variant thereof having at least 90%
amino acid identity to SEQ ID NO: 1.
[0046] In yet a further embodiment, the nuclease variant is VcEndA variant
of any of SEQ ID NOS: 5-11.
[0047] In a further embodiment, the nuclease comprises a salt-active
nuclease.
[0048] In a further embodiment, the salt-active nuclease is Vibrio
salmonicida Endonuclease I of SEQ ID NO: 2 or a variant thereof having at
least 90% amino acid identity to SEQ ID NO: 2.
[0049] In a further embodiment, the nuclease comprises Serratia marcescens
Nuclease A of SEQ ID NO: 3 or a variant thereof having at least 90%
sequence identity to SEQ ID NO: 3.
[0050] In a further embodiment, the nuclease comprises Bacillus BacNucB of
SEQ IS NO: 4 or a variant thereof having at least 90% amino acid identity
to SEQ ID NO: 4.
[0051] In another embodiment, the nuclease comprises an N-terminal
secretory signal.
[0052] In another embodiment of the invention disclosed herein, the
nucleic acid sequences encoding a nuclease are sequence-optimised to
remove potential splice sites and/or unstable elements.
[0053] In another embodiment, the nucleic acid sequences encoding a
nuclease are codon-optimised for expression in the production cell.
[0054] In a further embodiment, the production systems and/or methods
comprise one or more additional nucleases.
[0055] In a further embodiment, the production systems and/or methods
comprise a fusion protein of two or more nucleases or nuclease domains.
[0056] In an even further embodiment, the fusion protein comprises an
endonuclease and an exonuclease.
[0057] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 5 litres (L) of medium.
[0058] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 10 litres (L) of medium.
[0059] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 15 litres (L) of medium.
[0060] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 20 litres (L) of medium.
[0061] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 25 litres (L) of medium.
[0062] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 30 litres (L) of medium.
[0063] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 35 litres (L) of medium.
[0064] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 40 litres (L) of medium.
[0065] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 45 litres (L) of medium.
[0066] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 50 litres (L) of medium.
[0067] In another embodiment of the systems or methods, the cell culture
comprises a volume of at least about 60 litres (L) of medium.
[0068] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 70 litres (L) of medium.
[0069] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 80 litres (L) of medium.
[0070] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 90 litres (L) of medium.
[0071] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 100 litres (L) of medium.
[0072] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 200 litres (L) of medium.
[0073] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of at least
about 500 litres (L) of medium.
[0074] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises a volume of up to, or
at least, about 1000 litres (L) of medium.
[0075] In another embodiment of the systems or methods or other aspects of
the present invention, the cells are adherent.
[0076] In another embodiment of the systems or methods or other aspects of
the present invention, the cells are in suspension.
[0077] In another embodiment of the systems or methods or other aspects of
the present invention, the cells are adherent in suspension.
[0078] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture comprises serum.
[0079] In another embodiment of the systems or methods or other aspects of
the present invention, the cell culture is serum-free.
[0080] In another embodiment of the systems or methods or other aspects of
the present invention, the host cell is a mammalian host cell.
[0081] In another embodiment of the systems or methods or other aspects of
the present invention, the host cell is a human host cell.
[0082] In another embodiment of the systems or methods or other aspects of
the present invention, the human host cell is a HEK293 cell, or a
derivative thereof. Examples of HEK293 derivatives include HEK293S,
HEK293SG, HEK293SGGD, HEK293FTM and HEK293T. These derivatives may also
be referred to as variants of HEK293.
[0083] In one embodiment of the systems or methods or other aspects of the
present invention, the HEK293 cell is a HEK293T cell.
[0084] In another embodiment of the systems or methods or other aspects of
the present invention, the viral vector components comprise a nucleotide
of interest (NOI).
[0085] In another embodiment of the systems or methods or other aspects of
the present invention, the viral vector components are retroviral vector
components.
[0086] In another embodiment of the systems or methods or other aspects of
the present invention, the retroviral vector components are lentiviral
vector components.
[0087] In another embodiment of the systems or methods or other aspects of
the present invention, the viral vector components comprise i) gag-pol;
ii) env; iii) optionally the RNA genome of a retroviral vector; and iv)
optionally rev, or a functional substitute thereof.
[0088] In another embodiment of the systems or methods or other aspects of
the present invention, wherein at least two of the nucleic acid sequences
encoding the viral vector components are located at the same genetic
locus.
[0089] In another embodiment of the systems or methods or other aspects of
the present invention, at least two of the nucleic acid sequences
encoding the viral vector components are in reverse and/or alternating
orientations.
[0090] In another embodiment of the systems or methods or other aspects of
the present invention, at least two of the nucleic acid sequences
encoding gag-pol and/or env are associated with at least one regulator
element.
[0091] In another embodiment of the systems or methods or other aspects of
the present invention, the env is a VSV-G env.
[0092] In another embodiment of the systems or methods or other aspects of
the present invention, the viral vector is an adenoviral vector
[0093] In another embodiment of the systems or methods or other aspects of
the present invention, the viral vector is an adeno-associated viral
vector.
[0094] In another embodiment of the systems or methods or other aspects of
the present invention, the nuclease is expressed and secreted from the
cell after transient transfection.
[0095] In another embodiment of the systems or methods or other aspects of
the present invention, the nuclease is expressed and secreted from the
cell after stable integration of the cell.
[0096] In another embodiment of the systems or methods or other aspects of
the present invention, the expression of the nuclease is inducible or
conditional, and wherein the nucleic acid encoding the nuclease comprises
an inducible or conditional promoter or regulatory element.
[0097] In another embodiment of the systems or methods or other aspects of
the present invention, the nuclease is an extracellular nuclease.
[0098] In an additional embodiment, the invention disclosed herein
provides a transient or stable production cell for producing viral
vectors comprising nucleic acid sequences encoding: 1) viral vector
components; and 2) a nuclease, wherein the nuclease is expressed in the
production cell and secreted in cell culture thereby degrading residual
DNA during viral vector production.
[0099] In another embodiment, the invention provides a transient or stable
production cell for producing viral vectors comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease fusion
protein, wherein the nuclease fusion protein comprises an exonuclease
domain fused to an endonuclease domain, and wherein the nuclease fusion
protein is expressed in the viral vector production cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production, and wherein the production cell is a eukaryotic production
cell.
[0100] In another embodiment, the invention disclosed herein provides a
cell culture device, such as a bioreactor, comprising the viral vector
production cells expressing and secreting a nuclease.
[0101] In addition, disclosed herein is a variant of a secreted nuclease
capable of degrading residual nucleic acid during viral vector
production, the variant comprising the amino acid sequence of any of SEQ
ID NOS: 5-11. In particular, the variant comprises SEQ ID NO: 7, SEQ ID
NO: 10, or SEQ ID NO: 11.
[0102] Also disclosed herein is a modified nuclease having increased
cell-retention and/or or cell-association that is expressed through the
secretory pathway of a eukaryotic cell, the modified nuclease comprising
a retention signal at its C-terminus.
[0103] In another embodiment, the modified nuclease localizes to the
endoplasmic reticulum (ER) and/or the golgi compartments thereby
resulting in increased cell retention compared to cell retention of a
corresponding unmodified nuclease. In an additional embodiment, the
modified nuclease is bound to ER receptors of the ER retention-defective
complementation group.
[0104] In another embodiment, the retention signal is at its C-terminus of
consensus [KRHQSA]-[DENQ]-E-L or KKXX and in a further embodiment, the
retention signal is at its C-terminus of consensus KDEL.
[0105] In other embodiments, a eukaryotic cell expresses the modified
nuclease and a viral vector production system comprising the cell
expressing the modified nuclease.
DESCRIPTION OF THE FIGURES
[0106] FIGS. 1A-1B illustrate two concepts of the invention disclosed
herein showing secretion of nuclease from eukaryotic cells during
production of a viral vector production system. In these concepts, the
cells expressing secreted nuclease can be either transiently transfected
with the nuclease expression cassette or may be stably integrated into
the genomes of the cells and may be optionally inducible. In either
concept, the mode of viral vector production may be adherent cell culture
or suspension cell culture. FIG. 1A shows a culture of viral vector
production cells spiked with nuclease helper cells that are capable of
expressing secreted nuclease. FIG. 1B shows a viral vector production
system wherein the production cells are capable of expressing secreted
nuclease themselves without the use of nuclease helper cells.
[0107] FIG. 2 illustrates a work flow step-wise approach for modification
of a nuclease for genetic engineering in a production cell for use in the
invention disclosed herein.
[0108] FIGS. 3A-3D illustrate schematic nuclease expression cassettes for
use in the invention disclosed herein. FIG. 3A shows, without limitation,
an exemplary expression cassette for SmNucA. FIG. 3B shows, without
limitation, an exemplary expression cassette for VsEndA and VcEndA. FIG.
3C shows, without limitation, an exemplary expression cassette for
BacNucB. FIG. 3D shows, without limitation, an exemplary expression
cassette for a regulated secreted nuclease cassette for stable cell
development.
[0109] FIGS. 4A-4C show that post-transfection expression and secretion of
a nuclease from HEK293T cells correlated with reduced DNA in culture
media. FIG. 4A shows transfected cell lysates immunoblotted for
anti-histidine tagged smNucAH6. FIG. 4B shows an immuno-dot blot of
transfected cell culture media probing using anti-histidine tagged
SmNucAH6. FIG. 4C shows residual DNA analysis within the media.
[0110] FIG. 5 shows transient lentiviral vector production in adherent or
suspension HEK293T cell culture comparing secreted tagged and untagged
nucleases (SmNucA) from expression cassettes versus Benzonase.RTM. &
untreated vector harvests.
[0111] FIG. 6 shows transient lentiviral vector production in adherent
HEK293T using SmNucA expression cassettes with the native signal peptide
and without, wherein exemplary eukaryotic ER signal peptides replace the
native signal.
[0112] FIG. 7 shows, without limitation, examples of secreted nuclease
(e.g., VsEndA & SmNucA) via expression cassette (1% of total input pDNA)
during the generation of lentiviral vectors in adherent HEK293T cell
cultures during transient transfection with vector components.
[0113] FIG. 8 shows, without limitation, examples of secreted nuclease
(e.g., VsEndA & SmNucA) via expression cassette (1% of total input pDNA)
during the generation of lentiviral vectors in adherent HEK293T producer
cell line cultures by dox induction of vector components, during
transient transfection with nuclease plasmid.
[0114] FIGS. 9A-9B show secreted nuclease during vector production in
suspension cultures. FIG. 9A shows, without limitation, examples of
secreted nuclease (e.g., VsEndA & SmNucA) via expression cassette (5% of
total input pDNA) during the generation of lentiviral vectors in HEK293T
cell culture in suspension during transient transfection of vector
components. FIG. 9B shows concentrated vector supernatant by SDS-PAGE and
immunoblotting using anti-histidine tag antibody to detect secreted
nuclease proteins.
[0115] FIGS. 10A-D show in silico analysis and, without limitation,
exemplary modifications to BacNucB for use in the invention disclosed
herein. FIG. 10A shows the primary sequence of BacNucB and transmembrane
prediction analysis. FIG. 10B shows analysis of the secretory peptide
signal of BacNucB. FIG. 10C shows analysis of BacNucB regions of
alpha-helices, beta-strands and coils, and positions of novel N.times.S/T
sequons engineered into four different variants. FIG. 10D shows the
analysis of the primary sequence of BacNucB with the four N.times.S/T
sequon variant positions, and their predicted use as N-glycan sites.
[0116] FIGS. 11A-11B show enhancement of clearance of residual plasmid DNA
in lentiviral vector production cell cultures by engineered BacNucB. FIG.
11A shows the use of BacNucB variants expression plasmids (5% total pDNA)
in suspension, serum-free HEK293T cells during lentiviral vector
production via transient transfection to reduce pDNA whilst maintaining
high vector titres. FIG. 11B shows concentrated vector supernatant by
SDS-PAGE and immunoblotting using anti-histidine tag antibody to detect
secreted nuclease proteins.
[0117] FIGS. 12A-12C show enhancement of clearance of residual plasmid DNA
in lentiviral vector production cell cultures by use of the engineered
variant VcEndA-1glc', containing mutated N.times.S/T sequons. FIG. 12A
shows VcEndA variants expression plasmids (5% total pDNA) in suspension,
serum-free HEK293T cells during lentiviral vector production via
transient transfection, whilst maintaining high vector titres. FIG. 12B
shows the degree of residual DNA from vector supernatants via
agarose-electrophoresis. FIG. 12C shows concentrated vector supernatant
or post-production cell lysates by SDS-PAGE and immunoblotting using
anti-histidine tag antibody to detect secreted nuclease proteins.
[0118] FIG. 13 shows degradation of residual (plasmid) DNA from lentiviral
vector production bioreactors at 0.5 L scale using secreted VsEndA from
expression cassette.
[0119] FIGS. 14A-14B: FIG. 14A shows degradation of residual DNA from
lentiviral vector production bioreactors at 5 L scale using VsEndA from
expression cassette, and total KanR/TUs through subsequent downstream
processing. FIG. 14B shows analysis of concentrated samples from some of
the process steps (in A) as analysed by gel electrophoresis (ethidium
bromide stain for DNA).
[0120] FIGS. 15A-15B show degradation of residual DNA in lentiviral vector
production cell cultures using a tetR-regulated VsEndA expression
cassette during co-transfection. FIG. 15A shows that vector production
titers remain high. FIG. 15B shows the degree of residual DNA from vector
supernatants via agarose-electrophoresis.
[0121] FIGS. 16A-16B show degradation of residual DNA in lentiviral vector
production cell cultures co-cultured with nuclease helper cells
expressing tetR-regulated VsEndA or SmNucA. FIG. 16A shows that vector
production titers remain high. FIG. 16B shows the degree of residual DNA
from vector supernatants via agarose-electrophoresis.
[0122] FIGS. 17A-17B summarise the N.times.S/T N-glycan sequon mutants of
VcEndA. FIG. 17A displays a protein alignment of VsEndA and VcEndA,
highlighting the N.times.S/T sequons within VcEndA that are potential
sites of N-glycosylation when expressed in eukaryotic cells. Only the
first N-glycan sequon (position 102NCT) of VcEndA is shared with VsEndA,
and so the amino acid sequence at the equivalent positions within VsEndA
relating to sequons 2, 3 and 4 of VcEndA provide a useful guide for
mutation of these sequons. Accordingly, variants of VcEndA were generated
(displayed in FIG. 17B; [constructs 9, 19-25]) containing a combination
of modifications comprising 119NLT>NLV, and/or 130NRS>DRS and/or
133NFS>NFR, resulting is SEQ ID NOs 1, 5-11.
[0123] FIGS. 18A-18B show the expression of VcEndAH6 variants relative to
SmNucAH6 and VsEndAH6 in post-production cell lysates and culture media
in serum free, suspension mode LV production cultures. FIG. 18A displays
an immunoblot of LV post-production cell lysates probed against tubulin
(loading control) and anti-His6 (nucleases). FIG. 18B displays an
immunoblot of concentrated crude vector harvest probed against VSVG
(secretion control) and anti-His6 (nucleases). The molecular weights of
the nucleases is modulated by N-glycan status (typically a single glycan
adds .about.2.5 kDa); the unmodified sizes of the nucleases are:
VcEndAH6--27.6 kDa; VsEndAH6--27.9 kDa; SmNucAH6--29.7 kDa. Secreted
nuclease plasmids were spiked-in at 0.5% or 5% total plasmid DNA at
transfection. The VcEndAH6 variants are described in FIG. 17, and SEQ ID
NOs 1, 5-11.
[0124] FIGS. 19A-19B display the LV-CMV-GFP vector titres produced in the
presence of secreted nucleases described in FIGS. 17 and 18, as well as
the resulting impact on residual DNA in crude vector harvest. At LV
harvest, production cells were removed by centrifugation, and
supernatants filtered (0.22 .mu.m) before titration by transduction of
HEK293T cells followed by flow cytometry; LV titres were calculated
accordingly (FIG. 19A). Further, supernatants were concentrated by
centrifugation using 3K cut-off Amicon-15 devices, and 50% of this
material loaded onto a 2% agarose gel for residual DNA electrophoresis
(FIG. 19B; ethidium bromide stained gel). The data show that LV titres
are minimally impacted by secreted nucleases, and that engineering of
VcEndA N-glycan sequons results in improved secretion of the nuclease,
and enhanced residual DNA clearance.
[0125] FIGS. 20A-20B display confirmed pH set points (FIG. 20A) and cell
viability (FIG. 20B) during evaluation of VcEndAH6-1glc, VsEndAH6 and
SmNucAH6 in production of LVs in serum-free, suspension cultures under
acidic or alkaline culturing conditions.
[0126] FIGS. 21A-21B show the expression of VcEndAH6-1glc, SmNucAH6 and
VsEndAH6 in post-production cell lysates and culture media in serum free,
suspension mode LV production cultures under acid or alkaline culturing
conditions. FIG. 21A displays an immunoblot of LV post-production cell
lysates probed against tubulin (loading control) and anti-His6
(nucleases). FIG. 21B displays an immunoblot of concentrated crude vector
harvest probed against VSVG (secretion control) and anti-His6
(nucleases). The molecular weights of the nucleases is modulated by
N-glycan status (typically a single glycan adds .about.2.5 kDa); the
unmodified sizes of the nucleases are: VcEndAH6--27.6 kDa; VsEndAH6--27.9
kDa; SmNucAH6--29.7 kDa. Secreted nuclease plasmids were spiked-in at 5%
total plasmid DNA at transfection.
[0127] FIGS. 22A-22B display the LV-CMV-GFP vector titres produced in the
presence of secreted nucleases VcEndAH6-1glc, SmNucAH6 and VsEndAH6, as
well as the resulting impact on residual DNA in crude vector harvest. At
LV harvest, production cells were removed by centrifugation, and
supernatants filtered (0.22 .mu.m) before titration by transduction of
HEK293T cells followed by flow cytometry; LV titres were calculated
accordingly (FIG. 22A). Further, supernatants were concentrated by
centrifugation using 3K cut-off Amicon-15 devices, and 50% of this
material loaded onto a 2% agarose gel for residual DNA electrophoresis
(FIG. 22B; ethidium bromide stained gel). The data show that the
VcEndAH6-1glc variant displays improved clearance of residual DNA under
acidic compared to VsEndAH6, whilst maintaining high titre LV production.
[0128] FIGS. 23A-23B display confirmed pH set points (FIG. 23A) and cell
viability (FIG. 23B) during evaluation of VcEndAH6-124glc, VcEndAH6-1glc,
and VsEndAH6 in production of LVs in serum-free, suspension cultures
under acidic or alkaline culturing conditions. Interestingly, these data
indicate a potential benefit of VcEndAH6-based nuclease expression on
cell viability at the latter stages of LV production (also observed in
FIG. 20).
[0129] FIGS. 24A-24B display the LV-CMV-GFP vector titres produced in the
presence of secreted nucleases VcEndAH6-124glc, VcEndAH6-1glc, and
VsEndAH6, as well as the resulting impact on residual DNA in crude vector
harvest. At LV harvest, production cells were removed by centrifugation,
and supernatants filtered (0.22 .mu.m) before titration by transduction
of HEK293T cells followed by flow cytometry; LV titres were calculated
accordingly (FIG. 24A). Further, supernatants were concentrated by
centrifugation using 3K cut-off Amicon-15 devices, and 50% of this
material loaded onto a 2% agarose gel for residual DNA electrophoresis
(FIG. 24B; ethidium bromide stained gel). The data show that the
VcEndAH6-124glc and VcEndAH6-1glc variants displays improved clearance of
residual DNA under acidic compared to VsEndAH6, whilst maintaining high
titre LV production.
[0130] FIG. 25 displays a schematic showing how nucleases can be modified
in order to retain more of the nuclease within cells for use in
production of cell-associated viral vectors such as AAVs and AdVs. For
some embodiments of the present invention some, preferably all, of the
nucleases should be targeted to the secretory pathway by use of a
functional ER signal peptide (SP) encoded on the N-terminus of the
protein, which is cleaved by Signal Peptidase (SPase) allowing release
into the ER lumen. However, by optionally appending the C-terminus of the
nuclease with an ER-retention signal such as the sequence `KDEL` (reading
N-to-C), the modified nuclease will be bound by one of the `ER
retention-defective [ERD] complementation group` protein receptors that
will allow retention of more nuclease within the vector production cell.
For production of cell-associated viral vectors such as AAVs and AdVs,
this allows production cells to be isolated away (i.e. filtered,
precipitated or centrifuged) from culture media prior to cell lysis
whilst enabling the nuclease to remain with cells and to be present at
the point of cell lysis in order to degrade the production cell DNA.
[0131] FIGS. 26A-26B display data from the use of secreted and ER-retained
nucleases during the production of scAAV2-GFP vector in suspension,
serum-free HEK293T cells. Replicate scAAV2 vector production cultures
were set up by transfecting cells with Genome, Repcap2 and helper
plasmids, and either 5% (total input pDNA) of the indicated nuclease
plasmid or no nuclease (pBlueScript; AAV-NEG). The nucleases vcEndAH6
[wt] and vcEndAH6-1glc were optionally engineered with a C-terminal
ER-retention signal (KDEL; see FIG. 25), and compared to smNucAH6. Two
days post-transfection, cells were harvested by centrifugation and
subject to lysis. To the lysate magnesium chloride was added and
incubated for 1 hour; for the positive control, SAN was added to AAV-NEG
lysate and incubated in parallel. Debris was pelleted by centrifugation,
supernatant filtered and analysed directly for vector titre on HEK293T
cells [FIG. 26A] or loaded onto an agarose gel to assess residual DNA
degradation [FIG. 26B]. Without wishing to be bound by theory, the level
of residual DNA within the `AAV-NEG` sample lane may be lower than
actually present within untreated cell lysate because much of the
released DNA was lost during sample filtration, which was difficult to
perform due to viscosity. Therefore, whilst the vcEndAH6 [wt] variant
appears to have a lower impact on residual DNA compared to other nuclease
variants, it in fact is likely to have substantial activity.
[0132] FIG. 27 displays densitometry analysis of the residual DNA agarose
gel image presented in FIG. 26B. Using ImageLab, the gel image was
divided into multiple channels such that each sample lane was overlaid by
2-3 channels. A representative channel for the stated sample lanes is
presented, indicating relative band intensity (arbitrary unit) compared
to an empty lane (Background). The resulting band `profiles` show the
residual DNA within the gel well and within the sample lane. As can be
seen from the image of the gel in FIG. 26B, the short, DNAse resistant
forms can be seen, and a correlative peak can be observed within the lane
profiles. The vcEndAH6-1glc-KDEL variant is able to achieve greater
clearance of residual DNA over the other variants and SAN.
[0133] FIGS. 28A-28B display the results of a transfection optimization
experiment to identify conditions that achieve close-to-maximal levels of
nuclease expression within nuclease helper cell cultures. Transfections
of serum-free, suspension HEK293T cells were mediated by mixing
lipofectamine 2000CD (at the same ratio per mass of plasmid DNA at each
condition) and nuclease encoding plasmid, at 100, 300, 500, 700 or 900 ng
per mL of final transfected cell culture. VcEndAH6-1glc or SmNucAH6
expression plasmids driven by a strong CMV or weaker SV40 promoter were
used. As a comparison, replicate cultures were also co-transfected with
each of the nuclease plasmids at 5% total plasmid input with 95% input of
pBluescript, to mimic the type of transfection mix when co-transfecting
cells for viral vector production (i.e. nuclease and vector made in the
same cell); this was effectively 62ng of nuclease plasmid per mL.
Replicate transfections were induced by sodium butyrate (10 mM final
concentration) .about.20 hour post-transfection to mimic conditions
within a viral vector production culture. Two days post-transfection,
helper cell culture supernatants were filtered (0.2 .mu.m) and
concentrated by 3K cut-off spin columns, and cell lysates generated prior
to immunoblotting for the nucleases (anti-HisTag), and GAPDH (cell
lysates only). [A] Normalised immunoblot band intensities plotted for all
conditions. [B] Immunoblots of secreted nuclease protein within helper
cells cultures.
[0134] FIGS. 29A-29C display the results of lentiviral vector (LV-CMV-GFP)
production in serum-free, suspension HEK293T cultures at 40 mL shake
flask scale using nuclease helper cells generated by transient
transfection in parallel to the main vector production culture. Nuclease
helper cells were generated by transfection of HEK293T cells with 750ng
(per mL of final culture) of pSV40-VcEndAH6-1glc or pSV40-SmNucAH6 and in
parallel to transfection of HEK293T cells with vector components under
conditions used in previous Examples. Approximately 20 hours
post-transfection, cultures were counted and helper cells added to the
main vector production vessel to achieve the desired target percentage
proportion of total cells in the main vector production vessel. In this
Example, helper cell proportion ranged from 1% to 10% total cells. Sodium
butyrate was added at the same time to a final concentration of 10 mM.
Two days post-transfection, vector supernatants were harvested, clarified
(0.2 .mu.m) and subjected to further analysis. [A] Vector supernatants
were titrated by transduction of adherent HEK293T cells followed by FACS
two days post-transduction. [B] helper cell and vector supernatants were
concentrated by 3K cut-off spin columns and analysed for nuclease
expression (anti-HisTag), or for residual DNA analysis on an agarose gel
(Ethidium bromide; vector supernatants only) [C].
[0135] FIGS. 30A-30B display the analysis of production of lentiviral
vectors (LV-CMV-GFP) in serum-free, suspension HEK293T cells when using
nuclease helper cells to clear residual DNA. In parallel to the main
vector production cultures, serum-free suspension HEK293T cells were
transfected with pCMV-VcEndAH6-1glc at 750ng per mL culture media. At the
point of sodium butyrate induction, nuclease helper cells were inoculated
into the main vector production vessel at the indicated proportion
(percentage total cells). Standard vector production continued until two
days post-transfection, when vector supernatants were taken for titration
on adherent HEK293T cells followed by FACS [A], and also concentrated by
3K cut-off spin columns for agarose gel analysis (ethidium bromide) [B].
[0136] FIGS. 31A-31B display the correlation between achievable nuclease
secretion levels and the specific nuclease activity within lentiviral
vector production culture media, when spiking-in nuclease helper cells;
the vector preps produce are the same as those in FIG. 30. Culture
supernatants were analysed for nuclease activity by DNAse Alert assay,
using Benzonase.RTM. as a standard curve [A]. Concentrated culture
supernatants from the experiment were analysed by immunoblotting to
nuclease (anti-HisTag) [B].
[0137] FIGS. 32A-32B present the results of lentiviral vector (LV-CMV-GFP)
production in serum-free, suspension HEK293T cultures at 5 L bioreactor
scale using VcEndAH6-1glc in co-transfection. pSV40-VcEndAH6-1glc was
mixed with LV component plasmids at 5% total input, whereas the
`standard` control bioreactor was transfected with pBlueScript at 5%
total input. Production of vectors proceeded as explained in Example 21,
maintaining the pH at 6.7 post-transfection [A]. Approximately 2.5 L of
clarified harvest material was taken through the downstream process, with
sampling at each stated stage: In-bio (prior to 1 hour incubation with
MgCl.sub.2+/-Benzonase.RTM.), CLH (clarified harvest; post-treatment with
MgCl.sub.2+/-Benzonase.RTM.), IEX Eluate (Eluted material from IEX column
[diluted to 0.6M NaCl], HFF Pre (buffer exchanged IEX Eluate by
dia-/ultra-filtration using hollow fibre cartridge prior to
Benzonase.RTM. addition), HFF Post (post-Benzonase.RTM.-treated, buffer
exchanged). Samples were concentrated by 3K cut-off spin columns before
loading onto a 2% agar gel containing ethidium bromide [B].
[0138] FIG. 33 displays a stained SDS-PAGE gel loaded with cell lysates of
serum-free, suspension HEK293T cells transfected with
pSV40-VcEndAH6-1glc, pSV40-VcEndAH6-1glc-KDEL or untransfected cells.
VcEndAH6-1glc-KDEL was enriched within cell extracts compared to
VcEndAH6-1glc.
DETAILED DESCRIPTION OF THE INVENTION
[0139] The practice of the invention disclosed herein will employ, unless
otherwise indicated, conventional techniques of chemistry, molecular
biology, microbiology and immunology, which are within the capabilities
of a person of ordinary skill in the art. Such techniques are explained
in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T.
Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition,
Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.
(1995 and periodic supplements) Current Protocols in Molecular Biology,
Ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.; B. Roe, J.
Crabtree, and A. Kahn (1996) DNA Isolation and Sequencing: Essential
Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee (1990) In
Situ Hybridization: Principles and Practice; Oxford University Press; M.
J. Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL
Press; and, D. M. J. Lilley and J. E. Dahlberg (1992) Methods of
Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA
Methods in Enzymology, Academic Press. Each of these general texts is
herein incorporated by reference.
[0140] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of skill in
the art to which this invention belongs. Unless otherwise specified,
"rev" and "gag-pol" refer to the proteins and/or genes of lentiviral
vectors.
Nuclease-Secreting Viral Vector Production System
[0141] The invention disclosed herein is a nuclease-secreting viral vector
production system comprising a set of nucleic acid sequences encoding the
components required for production of the viral vector and expression and
secretion of at least one nuclease, thereby degrading
unwanted/contaminating i.e., residual, nucleic acid during viral vector
production.
[0142] Accordingly, the invention disclosed herein provides an improved
vector production system which expresses and secretes a nuclease by viral
vector production cells during the production of viral vectors or
virus-based vaccines. As a result, unwanted (contaminating), residual
nucleic acid (e.g., pDNA) otherwise associated with the crude harvest
material is degraded during the production process in a streamlined
manner rather than what is typically required with burdensome upstream
and/or downstream commercial nuclease enzymatic treatment steps which can
sometimes impair to some extent produced viral vector quality and
quantity. See well known upstream and downstream processing steps in
Merten, O-W., Schweizer, M., Chahal, P., & Kamen, A. A. Manufacturing of
viral vectors for gene therapy: part I. Upstream processing.
Pharmaceutical Bioprocessing 2, 183-203 (2014); Merten, O-W., Schweizer,
M., Chahal, P., & Kamen, A. A. Manufacturing of viral vectors: part II.
Downstream processing and safety aspects Pharmaceutical Bioprocessing 2,
237-251 (2014); Gousseinov, E., Kools, W., and Pattnaik, P. Nucleic Acid
Impurity Reduction in Viral Vaccine Manufacturing. BioProcess
International 12, 59-68 (2014). For the same reasons, the invention
disclosed herein is also useful in providing improved exosome and gesicle
production systems.
[0143] The nucleic acid sequence(s) encoding the nuclease of the invention
disclosed herein may be in the form of a nucleotide expression
cassette(s) or plasmid. Such an expression cassette(s) is either
co-transfected with the viral vector component expression cassette(s)
and/or is stably integrated within the viral vector production cell DNA
(or cell genome).
[0144] In one embodiment, the nucleic acid sequence(s) of the invention
disclosed herein is an expression construct encoding one or more
nucleases. In another embodiment, the nucleases may be expressed as a
fusion protein in such a manner so that two nucleases (or nuclease
domains) are secreted during viral vector production. In a further
embodiment, the nuclease fusion protein comprises an endonuclease domain
and an exonuclease domain, whereby, during viral vector production, the
secreted endonuclease degrades circular, plasmid DNA whilst the secreted
exonuclease targets and degrades linear DNA from 5' and/or 3' termini in
order to more efficiently degrade DNA wrapped around the histone
proteins, that may be otherwise inaccessible to endonucleases.
[0145] The nuclease expression cassette comprises a promoter sequence
and/or a regulatory element(s) which drive expression of the nuclease.
Such promoters may drive constitutive, inducible, and/or conditional
expression of the nuclease. In addition, the nuclease comprises a native
and/or a non-native N-terminal secretory signal for secretion of the
nuclease into the cell culture.
[0146] In an alternative embodiment of the invention disclosed herein, the
nuclease expression cassette is used to transiently transfect or stably
integrate within a nuclease helper cell used in conjunction (or
co-culture) with a viral vector production cell. Such a nuclease helper
cell is then spiked-in with viral vector production cells during viral
vector manufacture and co-cultured until the end of viral vector
production.
[0147] The approach of using cultured cells of the invention disclosed
herein to secrete a nuclease during the upstream viral vector production
process has the advantages of 1) immediate degradation of unwanted or
contaminating (residual) nucleic acid within the culture media as soon as
viral vector virions are being produced, 2) perpetual secretion of the
nuclease throughout viral production without the need for further
burdensome manipulations, 3) removing the step of treatment of viral
vector crude harvest material with commercial nuclease (e.g.,
Benzonase.RTM.), 4) substantially reduced residual DNA within purified
vector product, and 5) lower the cost associated with adding recombinant
nuclease during downstream viral vector purification/processing.
[0148] The invention disclosed herein provides viral vector production
systems (in both transient and stable vector production aspects)
expressing secreted nucleases in adherent and/or in suspension,
serum-free cell culture. Surprisingly, considering the complex biology
involved in the assembly of viral vector virions during viral vector
production, the inventors show herein that expression and secretion of a
nuclease in cell culture during viral vector production overcomes
numerous issues in upstream and downstream viral vector manufacturing
whilst still maintaining high production titres. The inventors also
demonstrate herein scalability from lab scale or small scale viral vector
production to viral vector production in bioreactors.
[0149] The invention disclosed herein provides evidence that a secreted
nuclease degrades residual DNA within crude vector harvests with improved
performance over commercially available nucleases such as Benzonase.RTM.
and SAN (salt-active nuclease). The invention disclosed herein shows that
widely divergent nucleases (e.g., derived from gram-negative and positive
bacteria) degrades residual DNA during viral vector production. Because
nuclease-appended C-terminal histidine tags are shown herein to not
impact functionality of nuclease secretion, anti-histidine-tag based
ELISAs were used to measure residual nuclease in final viral vector
formulations.
[0150] Importantly, the invention disclosed herein provides evidence that
the timing of nuclease secretion is temporally linked to gene expression
from co-transfected viral vector component expression cassettes. In this
manner, nuclease expression initiates at the earliest point at which
residual plasmid DNA degradation is desired. Moreover, in another
embodiment, inducible expression of nuclease allows for the temporal
control of nuclease expression in the viral vector production system
disclosed herein.
[0151] In one aspect of the invention, a viral vector production cell line
is transfected with viral vector expression cassette(s) and an inducible
nuclease expression cassette, such as is based on a tetracycline-based
approach, which allows for stable secretion of the nuclease into the cell
culture media upon addition of the inducer during viral vector
production.
[0152] In another aspect of the invention, the nuclease cassette is
introduced into a viral vector production cell stably expressing viral
vector components. In this manner, stable secretion of the nuclease can
be constitutive secretion or inducible secretion into the cell culture
media depending on the regulatory elements of choice. In the aspect where
some of the vector components are also regulated, the nuclease cassette
may be under control of the same or separate regulatory elements of the
viral vector components, the latter separating temporal control of the
nuclease and the vector components.
[0153] In another aspect of the invention, the nuclease cassette expresses
and secretes nuclease in a retroviral or lentiviral vector production
system for degradation of residual nucleic acid during vector production.
[0154] In an alternative aspect relating to regulation of nuclease
expression in a delayed setting, HIV-1 rev or an analogous retrovirus
RNA-export protein is used for modulation of nuclease secretion instead
or in addition to the use of a transcription regulatory control
element(s) to regulate nuclease expression. In this manner,
nuclease-encoding mRNA comprises an HIV-1 rev-responsive element (RRE,
which is bound by HIV-1 rev) or analogous retrovirus RNA-export
protein-responsive element. The nuclease ORF and RRE are placed within
the same intron within the expression cassette and therefore in the
absence of HIV-1 rev (or analogous retrovirus RNA-export protein) the
amount of nuclease-encoding mRNA within the cytoplasm is reduced compared
to amounts of nuclease in the presence of rev. HIV-1 rev or the analogous
retrovirus RNA-export protein is co-expressed at a desired time point
during production of the vector in order to increase expression of the
nuclease.
[0155] Disclosed herein is evidence that a nuclease helper cell can be
introduced during the viral vector production upstream process to
co-culture with the viral vector production cells. In this regard,
nuclease is supplied in parallel.
[0156] In another aspect of the invention, the nuclease-helper cell or
helper cell can be cultured in parallel to the viral vector production
culture, and then media comprising secreted nuclease is fed to the viral
vector production cell culture.
[0157] In another aspect, the nuclease helper cell or helper cell can be
cultured in parallel to the exosome production culture, and then media
comprising secreted nuclease is fed to the exosome production cell
culture.
[0158] In another aspect, the nuclease helper cell or helper cell can be
cultured in parallel to the gesicle production culture, and then media
comprising secreted nuclease is fed to the exosome gesicle cell culture.
[0159] In another aspect of the invention disclosed herein, the nuclease
cassette expresses and secretes nuclease in an AAV or adenoviral vector
production system for degradation of residual nucleic acid during vector
production.
[0160] Typically, AAV-based vectors are produced in mammalian cell lines
(e.g. HEK293-based) or through use of the baculovirus/Sf9 insect cell
system. AAV vectors can be produced by transient transfection of vector
component encoding DNAs, typically together with helper functions from
Adenovirus or Herpes Simplex virus (HSV), or by use of cell lines stably
expressing AAV vector components. Adenoviral vectors are typically
produced in mammalian cell lines that stably express Adenovirus E1
functions (e.g. HEK293-based). Adenoviral vectors are also typically
`amplified` via helper-function-dependent replication through serial
rounds of `infection` using the production cell line. For the invention
disclosed herein, the common feature of AAV vector and Adenovirus vector
production is the step of cell lysis in order to more efficiently release
vector virions that are cell-associated. Some methods, such as
freeze-thaw, allow for cell lysis within the harvest media, whilst at
larger scales it may be desirable to concentrate production cells (e.g.
by centrifugation or filtration) before cell lysis by freeze-thaw or
mechanical or chemical treatment. At the cell lysis step, the crude
vector typically becomes substantially contaminated with production cell
DNA, and usually a commercial source of recombinant nuclease protein
(such as Benzonase.RTM. or SAN-HQ) is used either immediately prior to
and/or subsequently to cell lysis in order to minimise the amount of DNA;
this reduces viscosity and facilitates subsequent downstream processing.
[0161] Therefore, the invention disclosed herein includes, without
limitation, the use of secreted nuclease in place of commercial nuclease
in AAV and Adenovirus vector production systems and manufacturing
processes. In the context of such systems and processes, the secreted
nuclease is present within either the media (if cell lysis is occurring
within the culture media) and/or within the vector production cells as a
`steady-state` pool of protein, since the nuclease is continually
expressed and secreted into the ER-Golgi network as a mature nuclease.
Nuclease present within the production cells is present if cells are
concentrated-away from culture media but is released upon cell lysis, and
therefore has access to contaminating (or residual) DNA. Alternatively,
clarified culture supernatant comprising the secreted nuclease as well as
`free` vector virions may be `added-back` to the bulk cell lysate to
cause DNA degradation prior to further downstream processing. In one
aspect of the invention, nuclease secretion in such AAV- or
Adenovirus-based vector production (or indeed any viral vector platform
wherein vector virions remain chiefly cell associated) where cell lysis
is required, may utilise a eukaryotic cell line containing an inducible,
stable nuclease expression cassette. In another aspect, nuclease
secretion may be achieved via transient transfection of the nuclease
plasmid (with other vector components) for AAV vector production or
production of Adenovirus vector master seed stock when performing
recombination within transfected cells (e.g. RapAd.RTM. system).
Nucleases
[0162] Nuclease structure and function is well known in the field (Yang,
Q. Rev. Biophys. 2011 February; 44(1)L1-93). Nucleases are a ubiquitous
class of enzymes that hydrolyse nucleic acids--DNA and RNA. These enzymes
mediate the hydrolysis of the phosphodiester bond within the backbone of
polynucleotides resulting in cleavage. Many nucleases require metal ions
for their maximal activity. Nucleases can be categorised into two broad
groups governed by their mode off attack: [1] Endonucleases and [2]
Exonucleases.
[0163] Endonucleases attack the phosphodiester bond within the
polynucleotide chain (interior/`endo`); some endonucleases are
non-specific meaning that they cleave between any nucleotide, whilst
others may have site preference at specific di-nucleotides.
[0164] Exonucleases digest from the termini of the nucleic acids present
at either 5' or 3' ends of the polynucleotide chains. Some nucleases, for
example Nuclease Bal-31, may have both endo- and exon-nuclease
properties, being able to both digest DNA from termini and within the
polynucleotide, depending on the nature of the target (i.e. whether the
target is dsDNA or ssDNA).
[0165] Given the lack of requirement for the target polynucleotide to
possess free 5' or 3' termini, endonucleases are able to digest circular
forms of DNA (such as plasmid DNA). As such, a nuclease of the invention
disclosed herein is a nuclease from the `sugar-non-specific` nucleases,
since these enzymes can non-specifically degrade ssDNA, dsDNA, ssRNA
and/or dsRNA, in a sequence-independent manner. This group includes the
`Serratia` family of nucleases, which includes bacteria nucleases (NucA)
and eukaryotic mitochondrial endonuclease G (Endo G), which is also known
as Nuc1p in yeast. The Serriatia nucleases are structurally very similar;
the catalytic .beta..beta..alpha. motif forms a structural subdomain and
is packed against one face of a six-stranded antiparallel .beta.-sheet.
The `sugar-non-specific` nucleases also include the bacterial periplasmic
endonuclease I family of nucleases e.g. VsEndA.
[0166] Nucleases secreted from eukaryotic or prokaryotic cells share
analogous secretion pathways which are governed by similar features of
all secreted proteins of classical secretion pathways. A relatively short
peptide (typically less than 30 residues) located at the N-terminus of
the pre-mature nuclease protein emerging from the ribosome, is recognised
by cellular machinery and physically relocated from the cytoplasm/cytosol
to the membrane of the endoplasmic reticulum (ER) or plasma membrane of
eukaryotes or prokaryotes, respectively, where translation continues
through the membrane into a separate cellular compartment. After protein
translation, the secretory peptide is cleaved where protein folding is
completed and the mature nuclease is contained within the ER (eukaryotes)
or periplasm (prokaryotes). The nuclease will then typically be released
into the extracellular space.
[0167] The invention disclosed herein includes, without limitation, the
use of wild-type and modified or mutated (i.e., variant) nucleases
capable of being secreted in viral vector production. Such nucleases may
optionally include eukaryotic secretory signals to replace bacterial or
native secretory peptides. Exemplary secretory signal peptides include,
without limitation, those listed in the following table:
TABLE-US-00001
Secrecon MWWRLWWLLLL Synthetic
LLLLWPMVWA (Barash et al.
(SEQ ID NO: 12) Biochem
Biophys
Res Commun,
2002
Jun. 21;
294(4):
835-842.)
Mouse METDTLLLWV Mouse Ig
IgKVIII LLLWVPGSTG kappa
(SEQ ID NO: 13)
Human MDMRVPAQLLG Human Ig
1gKVIII LLLLWLRGARC kappa
(SEQ ID NO: 14)
CD33 MPLLLLLP Human CD3
LLWAGALA
(SEQ ID NO: 15)
tPA MDAMKRGLCCVL Human tPA
LLCGAVFVSPS
(SEQ ID NO: 16)
Albumin MKWVTFIS Human
LLFSSAYS serum
(SEQ ID NO: 17) albumin
VSVG MKCLLYLA VSV
FLFIGVNC Glycoprotein
(SEQ ID NO: 18)
[0168] Nucleases for use in the invention disclosed herein may include
modifications and/or mutations relating to function-disrupting N-glycan
sequons or creation of novel N-glycan sequon sites. Secretion of a
protein with nuclease activity that can be efficiently secreted from a
eukaryotic cell through the classical secretory pathway necessitates the
presence of a functional secretory signal-peptide encoded at the
beginning of the open-reading frame (ORF), and often the secreted protein
will comprise at least one asparagine-linked (N-linked) glycan so that
secretion is made more efficient. Glycosylation occurs at the asparagine
residue of N.times.S or N.times.T sequons (where x can be any amino acid
except proline) during export through the secretory pathway found in
eukaryotic cells, bacterial secreted nucleases are not glycosylated in
this manner. However, since the N.times.S/T motif also occurs often in
bacterial nucleases, these sites in principle could be utilized if
bacterial nucleases are expressed from eukaryotic cells, as per the
invention disclosed herein. N-glycan site predictor programs (such as
NetNGlyc) can be helpful in identifying which N.times.S/T site might be
most likely to be utilized. Additionally, determination can be made as to
the proximity of N-glycan to the DNAse active site and any impact on DNA
activity. Indeed, the inventors show that nucleases can be mutated to
modulate or modify N.times.S/T sequons so as to improve secretion in the
context of the viral vector production system and/or per the production
cells as disclosed herein.
[0169] A nuclease of the invention disclosed herein may include
modification of a C-terminal appendage of a nuclease with a retention
signal, such as an ER retention signal, which improves cell-retention or
cell-association by localising the nuclease to the ER and/or golgi
compartments. In this manner, the nuclease binds to the ER receptors of
the ER retention-defective complementation group. Such a modification
improves residual DNA clearance from cell lysate during vector
production, particularly during AAV vector production. Retention signals
include, without limitation, a retention signal of consensus
[KRHQSA]-[DENQ]-E-L or KKXX, and/or a retention signal of consensus KDEL.
C-terminal retention signals having KDEL motifs are known in the art
(e.g., Raykhel et al., J. Cell. Biol., 2007 Dec. 7, 179(6): 1193-1204).
[0170] Alternative modifications to the nucleases of the invention for
achieving cell-retention/association include, without limitation, use of
a GPI anchor or transmembrane domain at the C-terminus of the nuclease.
As such, in alternative embodiments, a modified nuclease comprises a GPI
anchor and/or a transmembrane domain at its C-terminus.
[0171] A nuclease of the invention disclosed herein, without limitation,
is a nuclease with an optimal pH or pH range for functionality in a viral
vector production system. Secreted nucleases of the invention function at
an optimal pH or optimal pH range for the degradation of residual nucleic
acid during viral vector production. Optimal pH of a secreted nuclease of
the invention disclosed herein is, without limitation, about pH6, about
pH6.5, about pH6.6, about pH6.8, about pH7, about pH7.2, about pH7.5. An
optimal pH range of a secreted nuclease of the invention is, without
limitation, about pH6-about pH7, about pH6-about pH6.5, about pH6-about
pH7.2, about pH6.5-about pH7.2, about pH6.5-about pH7.5, about
pH6.5-about pH7, about pH6.5-about pH6.8, about pH6.6-about pH7.2, about
pH6.6-about pH7.5, about pH6.6-about pH7.2, about pH6.6-about pH7, about
pH6.6-about pH6.8, about pH6.8-about pH7, about pH6.8-about pH7.2, about
pH7-about pH7.2, about pH7-about pH7.5, about pH7.2-about pH7.5.
[0172] A nuclease of the invention disclosed herein is, without
limitation, a hydrolytic nuclease capable of being secreted, such as,
without limitation, an extracellular nuclease or a nuclease modified to
function as an extracellular nuclease, in a viral vector production
system.
[0173] Widespread use of commercial nuclease (protein-form) occurs in a
manufacturing context when there is a need to remove as much as possible
of the contaminating (or residual) nucleic acids (both DNA and RNA) from
a production system. It is well known that the requirement to remove
nucleic acids is particularly important if production occurs
intracellularly or if cells are lysed during production. As a result of
this, large amounts of contaminating nucleic acids are released which
make further purification processes, such as filtration and/or
chromatography, more challenging due to, e.g., increased viscosity of the
sample.
[0174] As such, a nuclease of the invention disclosed herein is, without
limitation, a nuclease that cleaves (i.e., degrades) contaminating (or
unwanted) residual nucleic acid (preferably DNA but may also have RNAse
activity) whilst exhibiting nuclease functionality and stability in the
context of a viral vector production system.
[0175] A nuclease of the invention disclosed herein is, without
limitation, a prokaryotic nuclease, a eukaryotic nuclease, and/or a
functional variant or derivative thereof that is capable of degrading
residual nucleic acid in a viral vector production system.
[0176] A nuclease of the invention disclosed herein is, without
limitation, a sugar-non-specific nuclease, and/or a functional variant,
domain, or derivative thereof that is capable of degrading dsDNA, ssDNA,
dsRNA and/or ssRNA in a viral vector production system.
[0177] A nuclease of the invention herein is, without limitation, a
nuclease derived from an organism selected from the group consisting of
Vibrio cholerae, Vibrio salmonicida, Serratia marcescens, and Bacillus
licheniformis, variants thereof, and combinations thereof.
[0178] A nuclease of the invention herein is, without limitation, any
known nuclease or nuclease derived from a known organism. (See Wang,
supra.) For example, without limitation, a nuclease of the invention is a
nuclease derived from Vibrio cholerae, Vibrio salmonicida, Serratia
marcescens, and Bacillus licheniformis. Furthermore, without limitation,
a nuclease of the invention is any known nuclease in the following
categories: [0179] Predatory bacterial nucleases e.g. Bdellovibrio
bacteriovorus Bd1244, Bd1934 [0180] Plant nucleases e.g. Hordeum vulgare
L microspore nuclease (Barley) [0181] Snase family nucleases e.g.
Staphylococcus aureus NucA [0182] Intestinal nucleases e.g. Pancreatic
DNAse I [0183] Low pH active e.g. Mycobacterium smegmatis Rv0888 (low pH
nuclease)
[0184] A nuclease of the invention disclosed herein is, without
limitation, selected from the group consisting of smNucA (NCBI Reference
Sequence: WP_047571650.1), VsEndA (GenBank: CAQ78235.1), VcEndA (NCBI
Reference Sequence: WP_000972597.1), BacNucB (NCBI Reference Sequence:
WP_003182220.1), variants thereof, and combinations thereof.
[0185] A nuclease of the invention disclosed herein is, without
limitation, a modified variant of Endonuclease I from Vibrio cholerae
(herein referred to as VcEndA). The reference sequence for wild-type
VcEndA [Predicted eukaryotic signal peptide in bold, N.times.S/T sequons
underlined] is provided herein as SEQ ID NO: 1:
TABLE-US-00002
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVK
IYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVR
KNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKN
CTRTSPEFNQMEADLHNLTPAIGEVNGNRSNFSF
SQWNGIDGVTYGQCEMQVNFKERTAMPPERARGA
IARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVS
EWECVRDQKIEKVQGNSNRFVREQCPN
[0186] Without limitation, variants of VcEndA with referenced mutations
are as follows:
TABLE-US-00003
SEQ ID NO: 5 'VcEndA-12glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRS
NFRFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
SEQ ID NO: 6 'VcEndA-123glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGNRS
NFRFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
SEQ ID NO: 7 VcEndA-124glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRS
NFSFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
SEQ ID NO: 8 'VcEndA-134glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGEVNGNRS
NFSFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
SEQ ID NO: 9 'VcEndA-13glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGEVNGNRS
NFRFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
SEQ ID NO: 10 'VcEndA-14glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGEVNGDRS
NFSFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
SEQ ID NO: 11 'VcEndA-1glc':
MMIFRFVTTLAASLPLLTFAAPISFSHAKNEAVKIYRDHPVSFY
CGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQF
GHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGEVNGDRS
NFRFSQWNGIDGVTYGQCEMQVNFKERTAMPPERARGAIARTYL
YMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGN
SNRFVREQCPN
[0187] VcEndA is a species of curved rod-shaped gram-negative bacteria in
the family Vibrionaceae. This bacterial nuclease comprises a bacteria
secretory signal that functions as a eukaryotic secretory signal peptide.
The optimal salt concentration of this nuclease is 150-200 mM, and it is
active over a wide range of pH, making it an ideal candidate
extracellular nuclease for secretion into viral vector production cell
media. Whilst the unmodified VcEndA has detectable secretion and DNAse
activity when expressed from vector production cells, ablation of the
N.times.S/T motifs starting at N.sup.119, N.sup.130, N.sup.133 (residue
numbers relative to pre-mature native VcEndA) enables improved secretion
of VcEndA and clearance of DNA in the invention disclosed herein.
[0188] A nuclease of the invention disclosed herein is, without
limitation, a `salt-active` nuclease, which is typically derived from a
halophilic microbial organism. Unlike other nucleases, salt-active
nucleases maintain activity in hyper-physiological salt concentration
(e.g. >0.15M [NaCl]). As such, post-harvest clarified vector material
and the salt-active nuclease can be incubated with hyper-physiological
salt concentration to potentially aid the dissociation of nucleic acid
bound to protein within the harvest material, making it more accessible
to nuclease cleavage.
[0189] A salt-active nuclease of the invention disclosed herein is,
without limitation, `Endonuclease from Vibrio salmonicida (herein
referred to as VsEndA`). See Altermark B1, Niiranen L, Willassen N P,
Smalas A O, Moe E. Comparative studies of endonuclease I from
cold-adapted Vibrio salmonicida and mesophilic Vibrio cholerae. FEBS J.
274(1):252-63 (2007). The reference sequence for wild-type VsEndA
(predicted eukaryotic signal peptide above in bold, N.times.S/T sequons
underlined) is provided herein as SEQ ID NO: 2:
TABLE-US-00004
Endonuclease I [V.Salmonicida] ('VsEndA')
MKLIRLVISLIAVSFTVNVMAAPPSSFSKAKKEAVKIYLDYPTS
FYCGCDITWKNKKKGIPELESCGYQVRKQEKRASRIEWEHVVPA
WQFGHQRQCWQKGGRKNCTRNDKQFKSMEADLHNLVPAIGEVNG
DRSNFRFSQWNGSKGAFYGQCAFKVDFKGRVAEPPAQSRGAIAR
TYLYMNNEYKFNLSKAQRQLMEAWNKQYPVSTWECTRDERIAKI
QGNHNQFVYKACTK
[0190] VsEndA is a species of curved rod-shaped gram-negative bacteria in
the family Vibrionaceae. This bacterial nuclease contains a bacteria
secretory signal that functions as a eukaryotic secretory signal peptide.
[0191] A nuclease of the invention disclosed herein is, without
limitation, `Nuclease A` from Serratia marcescens (hereinafter `SmNucA`),
which is a species of rod-shaped gram-negative bacteria in the family
Enterobacteriaceae. The reference sequence for wild-type SmNucA
(predicted eukaryotic signal peptide above in bold, N.times.S/T sequons
underlined) is provided herein as SEQ ID NO: 3:
TABLE-US-00005
Nuclease A [S.marcescens] ('SmNucA')
MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSI
VRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNP
ADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNI
TPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMG
KLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGAD
FCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGC
KN
[0192] A nuclease of the invention disclosed herein is, without
limitation, `Nuclease B` from Bacillus licheniformis, hereinafter
`BacNucB`, which is a species of rod-shaped gram-positive bacteria in the
family Enterobacteriaceae. The reference sequence for wild-type BacNucB
(predicted eukaryotic signal peptide above in bold) is provided herein as
SEQ ID NO: 4:
TABLE-US-00006
Endonuclease B [Bacillus sp. E.g.
B.licheniformis] ('BacNuca')
MIKKWAVHLLFSALVLLGLSGGAAYSPQHAEGAARYDDILYFPA
SRYPETGAHISDAIKAGHSDVCTIERSGADKRRQESLKGIPTKP
GFDRDEWPMAMCEEGGKGASVRYVSSSDNRGAGSWVGNRLSGFA
DGTRILFIVQ
Vectors
[0193] The invention disclosed herein relates to viral vectors and the
manufacturing and/or production of the same.
[0194] A vector is a tool that allows or facilitates the transfer of an
entity from one environment to another. In accordance with the invention,
and by way of example, some vectors used in recombinant nucleic acid
techniques allow entities, such as a segment of nucleic acid (e.g. a
heterologous DNA segment, such as a heterologous cDNA segment), to be
transferred into and expressed by a target cell. The vector may
facilitate the integration of the nucleic acid/nucleotide of interest
(NOI) to maintain the NOI and its expression within the target cell.
Alternatively, the vector may facilitate the replication of the vector
through expression of the NOI in a transient system.
[0195] The vectors of the invention are viral vectors, in particular
retroviral vectors, with a promoter for the expression of the said NOI
and optionally a regulator of the NOI. The vectors may contain one or
more selectable marker genes (e.g. a neomycin resistance gene) and/or
traceable marker gene(s) (e.g. a gene encoding green fluorescent protein
(GFP)). Vectors may be used, for example, to infect and/or transduce a
target cell.
[0196] The viral vector may be used to express the NOI in a compatible
target cell in vitro. Thus, the invention provides a method of making
proteins in vitro by introducing a vector of the invention into a
compatible target cell in vitro and growing the target cell under
conditions which result in expression of the NOI. Protein and NOI may be
recovered from the target cell by methods well known in the art. Suitable
target cells include mammalian cell lines and other eukaryotic cell
lines.
[0197] The vector may be an expression vector. Expression vectors as
described herein comprise regions of nucleic acid containing sequences
capable of being transcribed. Thus, sequences encoding mRNA, tRNA and
rRNA are included within this definition.
[0198] In some aspects the vectors may have "insulators"--genetic
sequences that block the interaction between promoters and enhancers, and
act as a barrier reducing read-through from an adjacent gene.
[0199] In one embodiment the insulator is present between one or more of
the retroviral nucleic acid sequences to prevent promoter interference
and read-thorough from adjacent genes. If the insulators are present in
the modular construct between one or more of the retroviral nucleic acid
sequences, then each of these insulated genes may be arranged as
individual expression units.
[0200] In one aspect, the invention provides a cell for producing
retroviral vectors comprising nucleic acid sequences encoding: [0201]
i) gag-pol; [0202] ii) env; [0203] iii) optionally the RNA genome of the
retroviral vector; and [0204] iv) optionally rev, or a functional
substitute thereof,
[0205] In another aspect, vectors of the invention comprise at least two
nucleic acid sequences that are located at the same genetic locus;
wherein the at least two nucleic acid sequences are in reverse and/or
alternating orientations; and wherein the nucleic acid sequences encoding
gag-pol and/or env are associated with at least one regulatory element.
[0206] In another embodiment, the retrovirus is derived from a foamy
virus.
[0207] In another embodiment, the retroviral vector is derived from a
lentivirus.
[0208] In another embodiment, the lentiviral vector is derived from HIV-1,
HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna lentivirus.
Retroviral and Lentiviral Vectors
[0209] The retroviral vector of the invention disclosed herein may be
derived from or may be derivable from any suitable retrovirus. A large
number of different retroviruses have been identified. Examples include:
murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse
mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma
virus (FuSV), Moloney murine leukemia virus (Mo MLV), FBR murine
osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV),
Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29
(MC29) and Avian erythroblastosis virus (AEV). A detailed list of
retroviruses may be found in Coffin et al. (1997) "Retroviruses", Cold
Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus
pp 758-763.
[0210] Retroviruses may be broadly divided into two categories, namely
"simple" and "complex". Retroviruses may even be further divided into
seven groups. Five of these groups represent retroviruses with oncogenic
potential. The remaining two groups are the lentiviruses and the
spumaviruses. A review of these retroviruses is presented in Coffin et al
(1997) ibid.
[0211] The basic structure of retroviral and lentiviral genomes share many
common features such as a 5' LTR and a 3' LTR, between or within which
are located a packaging signal to enable the genome to be packaged, a
primer binding site, integration sites to enable integration into a
target cell genome and gag/poi and env genes encoding the packaging
components--these are polypeptides required for the assembly of viral
particles. Lentiviruses have additional features, such as the rev gene
and RRE sequences in HIV, which enable the efficient export of RNA
transcripts of the integrated provirus from the nucleus to the cytoplasm
of an infected target cell.
[0212] In the provirus, these genes are flanked at both ends by regions
called long terminal repeats (LTRs). The LTRs are responsible for
proviral integration, and transcription. LTRs also serve as
enhancer-promoter sequences and can control the expression of the viral
genes.
[0213] The LTRs themselves are identical sequences that can be divided
into three elements, which are called U3, R and U5. U3 is derived from
the sequence unique to the 3' end of the RNA. R is derived from a
sequence repeated at both ends of the RNA and U5 is derived from the
sequence unique to the 5' end of the RNA. The sizes of the three elements
can vary considerably among different retroviruses.
[0214] In a typical retroviral vector of the invention, at least part of
one or more protein coding regions essential for replication may be
removed from the virus; for example, gag/pol and env may be absent or not
functional. This makes the viral vector replication-defective.
[0215] Lentiviruses are part of a larger group of retroviruses. A detailed
list of lentiviruses may be found in Coffin et al (1997) "Retroviruses"
Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E
Varmus pp 758-763). In brief, lentiviruses can be divided into primate
and non-primate groups. Examples of primate lentiviruses include but are
not limited to: the human immunodeficiency virus (HIV), the causative
agent of human auto-immunodeficiency syndrome (AIDS), and the simian
immunodeficiency virus (SIV). The non-primate lentiviral group includes
the prototype "slow virus" visna/maedi virus (VMV), as well as the
related caprine arthritis-encephalitis virus (CAEV), equine infectious
anaemia virus (EIAV), feline immunodeficiency virus (FIV), Maedi visna
virus (MVV) and bovine immunodeficiency virus (BIV).
[0216] The lentivirus family differs from retroviruses in that
lentiviruses have the capability to infect both dividing and non-dividing
cells (Lewis et al (1992) EMBO J 11(8):3053-3058 and Lewis and Emerman
(1994) J Virol 68 (1):510-516). In contrast, other retroviruses, such as
MLV, are unable to infect non-dividing or slowly dividing cells such as
those that make up, for example, muscle, brain, lung and liver tissue.
[0217] A lentiviral vector, as used herein, is a vector which comprises at
least one component part derivable from a lentivirus. Preferably, that
component part is involved in the biological mechanisms by which the
vector infects or transduces target cells and expresses NOI.
[0218] The lentiviral vector may be derived from either a primate
lentivirus (e.g. HIV-1) or a non-primate lentivirus (e.g. EIAV).
[0219] In general terms, a typical retroviral vector production system
involves the separation of the viral genome from the essential viral
packaging functions. These vector components are typically provided to
the production cells on separate DNA expression cassettes (alternatively
known as plasmids, expression plasmids, DNA constructs or expression
constructs).
[0220] The vector genome comprises the NOI. Vector genomes typically
require a packaging signal (ip), the internal expression cassette
harbouring the NOI, (optionally) a post-transcriptional element (PRE),
the 3'-ppu and a self-inactivating (SIN) LTR. The R-U5 regions are
required for correct polyadenylation of both the vector genome RNA and
NOI mRNA, as well as the process of reverse transcription. The vector
genome may optionally include an open reading frame, as described in WO
2003/064665, which allows for vector production in the absence of rev.
[0221] The packaging functions include the gag/poi and env genes. These
are required for the production of vector particles by the production
cell. Providing these functions in trans to the genome facilitates the
production of replication-defective viral vectors.
[0222] Production systems for gamma-retroviral vectors are typically
3-component systems requiring genome, gag/pol and env expression
constructs. Production systems for HIV-1-based lentiviral vectors may
additionally require the accessory gene rev to be provided and for the
vector genome to include the rev-responsive element (RRE). EIAV-based
lentiviral vectors do not require rev to be provided in trans if an
open-reading frame (ORF) is present within the genome (see WO
2003/064665).
[0223] Usually both the "external" promoter (which drives the vector
genome cassette) and "internal" promoter (which drives the NOI cassette)
encoded within the vector genome cassette are strong eukaryotic or virus
promoters, as are those driving the other vector system components.
Examples of such promoters include CMV, EF1a, PGK, CAG, TK, SV40 and
Ubiquitin promoters. Strong `synthetic` promoters, such as those
generated by DNA libraries (e.g. JeT promoter) may also be used to drive
transcription. Alternatively, tissue-specific promoters such as rhodopsin
(Rho), rhodopsin kinase (RhoK), cone-rod homeobox containing gene (CRX),
neural retina-specific leucine zipper protein (NRL), Vitelliform Macular
Dystrophy 2 (VMD2), Tyrosine hydroxylase, neuronal-specific
neuronal-specific enolase (NSE) promoter, astrocyte-specific glial
fibrillary acidic protein (GFAP) promoter, human al-antitrypsin (hAAT)
promoter, phosphoenolpyruvate carboxykinase (PEPCK), liver fatty acid
binding protein promoter, Flt-1 promoter, INF-.beta. promoter, Mb
promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40/hAlb
promoter, SV40/CD43, SV40/CD45, NSE/RU5' promoter, ICAM-2 promoter, GPIIb
promoter, GFAP promoter, Fibronectin promoter, Endoglin promoter,
Elastase-1 promoter, Desmin promoter, CD68 promoter, CD14 promoter and
B29 promoter may be used to drive transcription.
[0224] Production of retroviral vectors involves either the transient
co-transfection of the production cells with these DNA components or use
of stable production cell lines wherein all the components are stably
integrated within the production cell genome (e.g. Stewart H J, Fong-Wong
L, Strickland I, Chipchase D, Kelleher M, Stevenson L, Thoree V, McCarthy
J, Ralph G S, Mitrophanous K A and Radcliffe P A. (2011). Hum Gene Ther.
March; 22 (3):357-69). An alternative approach is to use a stable
packaging cell (into which the packaging components are stably
integrated) and then transiently transfect in the vector genome plasmid
as required (e.g. Stewart, H. J., M. A. Leroux-Carlucci, C. J. Sion, K.
A. Mitrophanous and P. A. Radcliffe (2009). Gene Ther. June; 16
(6):805-14). It is also feasible that alternative, not complete,
packaging cell lines could be generated (just one or two packaging
components are stably integrated into the cell lines) and to generate
vector the missing components are transiently transfected. The production
cell may also express regulatory proteins such as a member of the tet
repressor (TetR) protein group of transcription regulators (e.g. T-Rex,
Tet-On, and Tet-Off), a member of the cumate inducible switch system
group of transcription regulators (e.g. cumate repressor (CymR) protein),
or an RNA-binding protein supplied to repress transgene expression during
production (e.g. TRAP--tryptophan-activated RNA-binding protein).
[0225] In one embodiment of the invention disclosed herein, the viral
vector is derived from EIAV. EIAV has the simplest genomic structure of
the lentiviruses. In addition to the gag/pol and env genes, EIAV encodes
three other genes: tat, rev, and S2. Tat acts as a transcriptional
activator of the viral LTR (Derse and Newbold (1993) Virology
194(2):530-536 and Maury et al (1994) Virology 200(2):632-642) and rev
regulates and coordinates the expression of viral genes through
rev-response elements (RRE) (Martarano et al. (1994) J Virol
68(5):3102-3111). The mechanisms of action of these two proteins are
thought to be broadly similar to the analogous mechanisms in the primate
viruses (Martarano et al. (1994) J Virol 68(5):3102-3111). The function
of S2 is to antagonize SERINC3/5 (Chande et al., PNAS, 2016 Nov. 15;
113(46):13197-13202. In addition, an EIAV protein, Ttm, has been
identified that is encoded by the first exon of tat spliced to the env
coding sequence at the start of the transmembrane protein. In an
alternative embodiment of the invention disclosed herein the viral vector
is derived from HIV: HIV differs from EIAV in that it does not encode S2
but unlike EIAV it encodes vif, vpr, vpu and nef.
[0226] The term "recombinant retroviral or lentiviral vector" (RRV) refers
to a vector with sufficient retroviral genetic information to allow
packaging of an RNA genome, in the presence of packaging components, into
a viral particle capable of transducing a target cell. Transduction of
the target cell may include reverse transcription and integration into
the target cell genome. The RRV carries non-viral coding sequences which
are to be delivered by the vector to the target cell. A RRV is incapable
of independent replication to produce infectious retroviral particles
within the target cell. Usually the RRV lacks a functional gag/pol and/or
env gene, and/or other genes essential for replication.
[0227] Preferably the RRV vector of the invention disclosed herein has a
minimal viral genome.
[0228] As used herein, the term "minimal viral genome" means that the
viral vector has been manipulated so as to remove the non-essential
elements whilst retaining the elements essential to provide the required
functionality to infect, transduce and deliver a NOI to a target cell.
Further details of this strategy can be found in WO 1998/17815 and WO
99/32646. A minimal EIAV vector lacks tat, rev and S2 genes and neither
are these genes provided in trans in the production system. A minimal HIV
vector lacks vif, vpr, vpu, tat and nef.
[0229] The expression plasmid used to produce the vector genome within a
production cell may include transcriptional regulatory control sequences
operably linked to the retroviral genome to direct transcription of the
genome in a production cell/packaging cell. These regulatory sequences
may be the natural sequences associated with the transcribed retroviral
sequence, i.e. the 5' U3 region, or they may be a heterologous promoter
such as another viral promoter, for example the CMV promoter, as
discussed below. Some lentiviral vector genomes require additional
sequences for efficient virus production. For example, particularly in
the case of HIV, RRE sequences may be included. However, the requirement
for RRE (and dependence on rev which is provided in trans) may be reduced
or eliminated by codon optimisation. Further details of this strategy can
be found in WO 2001/79518.
[0230] Alternative sequences which perform the same function as the
rev/RRE system are also known. For example, a functional analogue of the
rev/RRE system is found in the Mason Pfizer monkey virus. This is known
as the constitutive transport element (CTE) and comprises an RRE-type
sequence in the genome which is believed to interact with a factor in the
infected cell. The cellular factor can be thought of as a rev analogue.
Thus, CTE may be used as an alternative to the rev/RRE system. Any other
functional equivalents of the Rev protein which are known or become
available may be relevant to the invention. For example, it is also known
that the Rex protein of HTLV-I can functionally replace the Rev protein
of HIV-1. Rev and RRE may be absent or non-functional in the vector for
use in the methods of the invention disclosed herein; in the alternative
rev and RRE, or functionally equivalent systems, may be incorporated in
vector systems herein.
[0231] As used herein, the term "functional substitute" means a protein or
sequence having an alternative sequence which performs the same function
as another protein or sequence. The term "functional substitute" is used
interchangeably with "functional equivalent" and "functional analogue"
herein with the same meaning.
SIN Vectors
[0232] The retroviral vectors of the invention may be used in a
self-inactivating (SIN) configuration in which the viral enhancer and
promoter sequences have been deleted. SIN vectors can be generated and
transduce non-dividing target cells in vivo, ex vivo or in vitro with an
efficacy similar to that of non-SIN vectors. The transcriptional
inactivation of the long terminal repeat (LTR) in the SIN provirus should
prevent mobilisation by replication-competent virus. This should also
enable the regulated expression of genes from internal promoters by
eliminating any cis-acting effects of the LTR.
[0233] By way of example, self-inactivating retroviral vector systems have
been constructed by deleting the transcriptional enhancers or the
enhancers and promoter in the U3 region of the 3' LTR. After a round of
vector reverse transcription and integration, these changes are copied
into both the 5' and the 3' LTRs producing a transcriptionally inactive
provirus. However, any promoter(s) internal to the LTRs in such vectors
will still be transcriptionally active. This strategy has been employed
to eliminate effects of the enhancers and promoters in the viral LTRs on
transcription from internally placed genes. Such effects include
increased transcription or suppression of transcription. This strategy
can also be used to eliminate downstream transcription from the 3' LTR
into genomic DNA. This is of particular concern in human gene therapy
where it is highly desirable, in some instances important, to prevent the
adventitious activation of any endogenous oncogene. Yu et al., (1986)
PNAS 83: 3194-98; Marty et al., (1990) Biochimie 72: 885-7; Naviaux et
al., (1996) J. Virol. 70: 5701-5; Iwakuma et al., (1999) Virol. 261:
120-32; Deglon et al., (2000) Human Gene Therapy 11: 179-90. SIN
lentiviral vectors are described in U.S. Pat. Nos. 6,924,123 and
7,056,699.
Replication-Defective Lentiviral Vectors
[0234] In the genome of a replication-defective lentiviral vector the
sequences of gag/pol and/or env may be mutated and/or not functional.
[0235] In a typical lentiviral vector of the invention disclosed herein,
at least part of one or more coding regions for proteins essential for
virus replication may be removed from the vector. This makes the viral
vector replication-defective. Portions of the viral genome may also be
replaced by a NOI in order to generate a vector comprising an NOI which
is capable of transducing a non-dividing target cell and/or integrating
its genome into the target cell genome.
[0236] In one embodiment the lentiviral vectors are non-integrating
vectors as described in WO 2006/010834 and WO 2007/071994.
[0237] In a further embodiment the vectors have the ability to deliver a
sequence which is devoid of or lacking viral RNA. In a further embodiment
a heterologous binding domain (heterologous to gag) located on the RNA to
be delivered and a cognate binding domain on Gag or GagPol can be used to
ensure packaging of the RNA to be delivered. Both of these vectors are
described in WO 2007/072056.
Adenoviral and Adeno-Associated Viral Vectors
[0238] The use of recombinant adeno-associated viral (AAV) and Adenovirus
based viral vectors for gene therapy is widespread, and manufacture of
the same has been well documented. Typically, AAV-based vectors are
produced in mammalian cell lines (e.g. HEK293-based) or through use of
the baculovirus/Sf9 insect cell system. AAV vectors can be produced by
transient transfection of vector component encoding DNAs, typically
together with helper functions from Adenovirus or Herpes Simplex virus
(HSV), or by use of cell lines stably expressing AAV vector components.
[0239] An AAV vector it is commonly understood to be a vector derived from
an adeno-associated virus serotype, including without limitation, AAV-1,
AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8. AAV vectors can have
one or more of the AAV wild-type genes deleted in whole or part,
preferably the rep and/or cap genes, but retain functional flanking ITR
sequences. Functional ITR sequences are necessary for the rescue,
replication and packaging of the AAV virion. Thus, an AAV vector is
defined herein to include at least those sequences required in cis for
replication and packaging (e.g., functional ITRs) of the virus. The ITRs
need not be the wild-type nucleotide sequences, and may be altered, e.g.,
by the insertion, deletion or substitution of nucleotides, so long as the
sequences provide for functional rescue, replication and packaging. An
`AAV vector` also refers to its protein shell or capsid, which provides
an efficient vehicle for delivery of vector nucleic acid to the nucleus
of target cells. AAV production systems require helper functions which
typically refers to AAV-derived coding sequences which can be expressed
to provide AAV gene products that, in turn, function in trans for
productive AAV replication. As such, AAV helper functions include both of
the major AAV open reading frames (ORFs), rep and cap. The Rep expression
products have been shown to possess many functions, including, among
others: recognition, binding and nicking of the AAV origin of DNA
replication; DNA helicase activity; and modulation of transcription from
AAV (or other heterologous) promoters. The Cap expression products supply
necessary packaging functions. AAV helper functions are used herein to
complement AAV functions in trans that are missing from AAV vectors. It
is understood that a AAV helper construct refers generally to a nucleic
acid molecule that includes nucleotide sequences providing AAV functions
deleted from an AAV vector which is to be used to produce a transducing
vector for delivery of a nucleotide sequence of interest. AAV helper
constructs are commonly used to provide transient expression of AAV rep
and/or cap genes to complement missing AAV functions that are necessary
for AAV replication; however, helper constructs lack AAV ITRs and can
neither replicate nor package themselves. AAV helper constructs can be in
the form of a plasmid, phage, transposon, cosmid, virus, or virion. A
number of AAV helper constructs have been described, such as the commonly
used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap
expression products. See, e.g., Samulski et al. (1989) J. Virol.
63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945,
incorporated herein by reference. A number of other vectors have been
described which encode Rep and/or Cap expression products. See, e.g.,
U.S. Pat. Nos. 5,139,941 and 6,376,237, incorporated herein by reference.
In addition, it is common knowledge that the term "accessory functions"
refers to non-AAV derived viral and/or cellular functions upon which AAV
is dependent for its replication. Thus, the term captures proteins and
RNAs that are required in AAV replication, including those moieties
involved in activation of AAV gene transcription, stage specific AAV mRNA
splicing, AAV DNA replication, synthesis of Cap expression products and
AAV capsid assembly. Viral-based accessory functions can be derived from
any of the known helper viruses such as adenovirus, herpesvirus (other
than herpes simplex virus type-1) and vaccinia virus.
[0240] The following publications, incorporated herein by reference,
describe various aspects of adeno-associated virus biology and/or
techniques relating to the production of adeno-associated viral vectors.
Aponte-Ubillus, et al. Molecular design for recombinant adeno-associated
virus (rAAV) vector production. Appl Microbiol Biotechnol. 2018; 102(3):
1045-1054; Wang Q, et al. A Robust System for Production of Superabundant
VP1 Recombinant AAV Vectors. Mol Ther Methods Clin Dev. 2017 Dec. 15; 7:
146-156; Adamson-Small L, et al. A scalable method for the production of
high-titer and high-quality adeno-associated type 9 vectors using the HSV
platform. Mol Ther Methods Clin Dev. 2016; 3: 16031; Clement N and
Grieger J C. Manufacturing of recombinant adeno-associated viral vectors
for clinical trials. Mol Ther Methods Clin Dev. 2016; 3: 16002; Guo P, et
al. Rapid and simplified purification of recombinant adeno-associated
virus. J Virol Methods. 2012 August; 183(2): 139-146; Shin J-H, et al.
Recombinant Adeno-Associated Viral Vector Production and Purification.
Methods Mol Biol. 2012; 798: 267-284; Cecchini S, et al. Reproducible
High Yields of Recombinant Adeno-Associated Virus Produced Using
Invertebrate Cells in 0.02-to 200-Liter Cultures. Hum. Gene Ther. 22(8)
2011: 1021-1030; Wright J F. Adeno-Associated Viral Vector Manufacturing:
Keeping Pace with Accelerating Clinical Development. Hum. Gene Ther.
22(8) 2011: 913-915; Kotin R L. Large-scale recombinant adeno-associated
virus production. Hum Mol Genet. 2011 Apr. 15; 20(R1): R2-R6; Thomas D L,
et al. Scalable Recombinant Adeno-Associated Virus Production Using
Recombinant Herpes Simplex Virus Type 1 Coinfection of Suspension-Adapted
Mammalian Cells. Hum. Gene Ther. 20(8) 2009: 861-870; Thorne B A, et al.
Manufacturing Recombinant Adeno-Associated Viral Vectors from Producer
Cell Clones. Hum. Gene Ther. 20(7) 2009: 707-714; Wright F. Transient
Transfection Methods for Clinical Adeno-Associated Viral Vector
Production. Hum. Gene Ther. 20(7) 2009: 698-706; Urabe M, et al. Insect
Cells As a Factory to Produce AdenoAssociated Virus Type 2 Vectors. Hum.
Gene Ther. 13(16) 2002: 1935-1943.
[0241] Adenoviral vectors are typically produced in mammalian cell lines
that stably express Adenovirus E1 functions (e.g. HEK293-based).
Adenoviral vectors are also typically `amplified` via
helper-function-dependent replication through serial rounds of
`infection` using the production cell line. An adenoviral vector and
production system thereof comprises a polynucleotide comprising all or a
portion of an adenovirus genome. It is well known that an adenovirus is,
without limitation, an adenovirus derived from Ad2, Ad5, Ad12, and Ad40.
An adenoviral vector is typically in the form of DNA encapsulated in an
adenovirus coat or adenoviral DNA packaged in another viral or viral-like
form (such as herpes simplex, and AAV).
[0242] The following publications, incorporated herein by reference,
describe various aspects of adenovirus biology and/or techniques relating
to the production of adenoviral vectors. Graham and Van de Eb (1973)
Virology 52:456-467; Takiff et al. (1981) Lancet ii:832-834; Berkner and
Sharp (1983) Nucleic Acid Research 6003-6020; Graham (1984) EMBO J
3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; and Bett et al.
(1994) Proc. Natl. Acad Sci. USA 91:8802-8806. Adenoviruses have been
genetically modified to produce replication-defective gene transfer
vectors. In such vectors, the early adenovirus gene products E1A and E1B
are deleted and provided in trans by the packaging cell line 293
developed by Frank Graham (Graham et al. (1987) J. Gen. Birol. 36:59-72
and Graham (1977) J. Genetic Virology 68:937-940). The gene to be
transduced is commonly inserted into the adenovirus in the deleted E1A
and E1B region of the virus genome. Bett et al. (1994), supra. Adenoviral
vectors and the production thereof have been described by
Stratford-Perricaudet (1990) Human Gene Therapy 1:2-256; Rosenfeld (1991)
Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309:61-66;
Jaffe et al. (1992) Nat Gent. 1:372-378; Quantin et al. (1992) Proc Natl.
Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992) Cell 68:143-155;
Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal
La Salle et al. (1993) Science 259:988-990; Mastrangeli et al. (1993) J.
Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650; Hayaski
et al. (1994) J. Biol. Chem. 269:23872-23875; Lee C S, et al.
Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and
Cell-Based Therapies in the New Era of Personalized Medicine. Genes Dis.
2017 June; 4(2):43-63; Kallel H, Kamen A A. Large-Scale Adenovirus and
Poxvirus-Vectored Vaccine Manufacturing to Enable Clinical Trials.
Biotechnol. J. 10(5) 2015: 741-747; Miravet S, et al. Construction,
production, and purification of recombinant adenovirus vectors. Methods
Mol Biol. 2014; 1089:159-73; Silva A C, et al. Scalable production of
adenovirus vectors. Methods Mol Biol. 2014; 1089:175-96; Kreppel F.
Production of high-capacity adenovirus vectors. Methods Mol Biol. 2014;
1089:211-29; Xie L, et al. Large-Scale Propagation of a
Replication-Defective Adenovirus Vector in Stirred-Tank Bioreactor
PER.C6.TM. Cell Culture Under Sparging Conditions. Biotechnol. Bioeng.
83(1) 2003: 45-52; Gamier A, et al. Scale-Up of the Adenovirus Expression
System for the Production of Recombinant Protein in Human 293S Cells.
Cell Culture Engineering IV. Buckland B C, Ed. Springer Science and
Business Media: Rotterdam, The Netherlands, 1994; 145-155.
NOI and Polynucleotides
[0243] Polynucleotides of the invention may comprise DNA or RNA. They may
be single-stranded or double-stranded. It will be understood by a skilled
person that numerous different polynucleotides can encode the same
polypeptide as a result of the degeneracy of the genetic code. In
addition, it is to be understood that skilled persons may, using routine
techniques, make nucleotide substitutions that do not affect the
polypeptide sequence encoded by the polynucleotides of the invention to
reflect the codon usage of any particular host organism in which the
polypeptides of the invention are to be expressed.
[0244] The polynucleotides may be modified by any method available in the
art. Such modifications may be carried out in order to enhance the in
vivo activity or lifespan of the polynucleotides of the invention.
[0245] Polynucleotides such as DNA polynucleotides may be produced
recombinantly, synthetically or by any means available to those of skill
in the art. They may also be cloned by standard techniques.
[0246] Longer polynucleotides will generally be produced using recombinant
means, for example using polymerase chain reaction (PCR) cloning
techniques. This will involve making a pair of primers (e.g. of about 15
to 30 nucleotides) flanking the target sequence which it is desired to
clone, bringing the primers into contact with mRNA or cDNA obtained from
an animal or human cell, performing PCR under conditions which bring
about amplification of the desired region, isolating the amplified
fragment (e.g. by purifying the reaction mixture with an agarose gel) and
recovering the amplified DNA. The primers may be designed to contain
suitable restriction enzyme recognition sites so that the amplified DNA
can be cloned into a suitable vector.
Proteins
[0247] As used herein, the term "protein" includes single-chain
polypeptide molecules as well as multiple-polypeptide complexes where
individual constituent polypeptides are linked by covalent or
non-covalent means. As used herein, the terms "polypeptide" and "peptide"
refer to a polymer in which the monomers are amino acids and are joined
together through peptide or disulfide bonds.
Variants, Derivatives, Analogues, Homologues and Fragments
[0248] In addition to the specific proteins and nucleotides mentioned
herein, the invention disclosed herein also encompasses the use of
variants, derivatives, analogues, homologues and fragments thereof.
[0249] In the context of the invention herein, a variant of any given
sequence is a sequence in which the specific sequence of residues
(whether amino acid or nucleic acid residues) has been modified in such a
manner that the polypeptide or polynucleotide in question retains at
least one of its endogenous functions. A variant sequence can be obtained
by addition, deletion, substitution, modification, replacement and/or
variation of at least one residue present in the naturally-occurring
protein.
[0250] The term "derivative" as used herein, in relation to proteins or
polypeptides of the invention includes any substitution of, variation of,
modification of, replacement of, deletion of and/or addition of one (or
more) amino acid residues from or to the sequence providing that the
resultant protein or polypeptide retains at least one of its endogenous
functions.
[0251] The term "analogue" as used herein, in relation to polypeptides or
polynucleotides includes any mimetic, that is, a chemical compound that
possesses at least one of the endogenous functions of the polypeptides or
polynucleotides which it mimics.
[0252] Typically, amino acid substitutions may be made, for example from
1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence
retains the required activity or ability. Amino acid substitutions may
include the use of non-naturally occurring analogues.
[0253] Proteins used in the invention disclosed herein may also have
deletions, insertions or substitutions of amino acid residues which
produce a silent change and result in a functionally equivalent protein.
Deliberate amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues as long as
the endogenous function is retained. For example, negatively charged
amino acids include aspartic acid and glutamic acid; positively charged
amino acids include lysine and arginine; and amino acids with uncharged
polar head groups having similar hydrophilicity values include
asparagine, glutamine, serine, threonine and tyrosine.
[0254] Conservative substitutions may be made, for example according to
the table below. Amino acids in the same block in the second column and
preferably in the same line in the third column may be substituted for
each other:
TABLE-US-00007
ALIPHATIC Non-polar G A P
I L V
Polar-uncharged C S T M
N Q
Polar-charged D E
K R H
AROMATIC F W Y
[0255] The term "homologue" means an entity having a certain homology with
the wild type amino acid sequence and the wild type nucleotide sequence.
The term "homology" can be equated with "identity".
[0256] In the present context, a homologous sequence is taken to include
an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or
90% identical, preferably at least 95%, 97 or 99% identical to the
subject sequence. Typically, the homologues will comprise the same active
sites etc. as the subject amino acid sequence. Although homology can also
be considered in terms of similarity (i.e. amino acid residues having
similar chemical properties/functions), in the context of the invention
disclosed herein it is preferred to express homology in terms of sequence
identity.
[0257] In the present context, a homologous sequence is taken to include a
nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90%
identical, preferably at least 95%, 97% or 99% identical to the subject
sequence. Although homology can also be considered in terms of
similarity, in the context of the invention disclosed herein it is
preferred to express homology in terms of sequence identity.
[0258] Homology comparisons can be conducted by eye, or more usually, with
the aid of readily available sequence comparison programs. These
commercially available computer programs can calculate percentage
homology or identity between two or more sequences.
[0259] Percentage homology may be calculated over contiguous sequences,
i.e. one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino acid in
the other sequence, one residue at a time. This is called an "ungapped"
alignment. Typically, such ungapped alignments are performed only over a
relatively short number of residues.
[0260] Although this is a very simple and consistent method, it fails to
take into consideration that, for example, in an otherwise identical pair
of sequences, one insertion or deletion in the nucleotide sequence may
cause the following codons to be put out of alignment, thus potentially
resulting in a large reduction in percent homology when a global
alignment is performed. Consequently, most sequence comparison methods
are designed to produce optimal alignments that take into consideration
possible insertions and deletions without penalising unduly the overall
homology score. This is achieved by inserting "gaps" in the sequence
alignment to try to maximise local homology.
[0261] However, these more complex methods assign "gap penalties" to each
gap that occurs in the alignment so that, for the same number of
identical amino acids, a sequence alignment with as few gaps as possible,
reflecting higher relatedness between the two compared sequences, will
achieve a higher score than one with many gaps. "Affine gap costs" are
typically used that charge a relatively high cost for the existence of a
gap and a smaller penalty for each subsequent residue in the gap. This is
the most commonly used gap scoring system. High gap penalties will of
course produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is preferred
to use the default values when using such software for sequence
comparisons. For example when using the GCG Wisconsin Bestfit package the
default gap penalty for amino acid sequences is -12 for a gap and -4 for
each extension.
[0262] Calculation of maximum percentage homology therefore firstly
requires the production of an optimal alignment, taking into
consideration gap penalties. A suitable computer program for carrying out
such an alignment is the GCG Wisconsin Bestfit package (University of
Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Research 12:387).
Examples of other software that can perform sequence comparisons include,
but are not limited to, the BLAST package (see Ausubel et al. (1999)
ibid--Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and
the GENEWORKS suite of comparison tools. Both BLAST and FASTA are
available for offline and online searching (see Ausubel et al. (1999)
ibid, pages 7-58 to 7-60). However, for some applications, it is
preferred to use the GCG Bestfit program. Another tool, called BLAST 2
Sequences is also available for comparing protein and nucleotide
sequences (see FEMS Microbiol Lett (1999) 174(2):247-50; FEMS Microbiol
Lett (1999) 177(1):187-8).
[0263] Although the final percentage homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score matrix
is generally used that assigns scores to each pairwise comparison based
on chemical similarity or evolutionary distance. An example of such a
matrix commonly used is the BLOSUM62 matrix--the default matrix for the
BLAST suite of programs. GCG Wisconsin programs generally use either the
public default values or a custom symbol comparison table if supplied
(see user manual for further details). For some applications, it is
preferred to use the public default values for the GCG package, or in the
case of other software, the default matrix, such as BLOSUM62.
[0264] Once the software has produced an optimal alignment, it is possible
to calculate percentage homology, preferably percentage sequence
identity. The software usually does this as part of the sequence
comparison and generates a numerical result.
[0265] "Fragments" are also variants and the term typically refers to a
selected region of the polypeptide or polynucleotide that is of interest
either functionally or, for example, in an assay. "Fragment" thus refers
to an amino acid or nucleic acid sequence that is a portion of a
full-length polypeptide or polynucleotide.
[0266] Such variants may be prepared using standard recombinant DNA
techniques such as site-directed mutagenesis. Where insertions are to be
made, synthetic DNA encoding the insertion together with 5' and 3'
flanking regions corresponding to the naturally-occurring sequence either
side of the insertion site may be made. The flanking regions will contain
convenient restriction sites corresponding to sites in the
naturally-occurring sequence so that the sequence may be cut with the
appropriate enzyme(s) and the synthetic DNA ligated into the break. The
DNA is then expressed in accordance with the invention to make the
encoded protein. These methods are only illustrative of the numerous
standard techniques known in the art for manipulation of DNA sequences
and other known techniques may also be used.
[0267] All variants, fragments or homologues of the regulatory protein
suitable for use in the cells and/or modular constructs of the invention
will retain the ability to bind the cognate binding site of the NOI such
that translation of the NOI is repressed or prevented in a viral vector
production cell.
[0268] All variants fragments or homologues of the binding site will
retain the ability to bind the cognate RNA-binding protein, such that
translation of the NOI is repressed or prevented in a viral vector
production cell.
Codon Optimisation
[0269] The polynucleotides used in the invention disclosed herein
(including the NOI and/or components of the vector production system) may
be codon-optimised. Codon optimisation has previously been described in
WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of
particular codons. This codon bias corresponds to a bias in the relative
abundance of particular tRNAs in the cell type. By altering the codons in
the sequence so that they are tailored to match with the relative
abundance of corresponding tRNAs, it is possible to increase expression.
By the same token, it is possible to decrease expression by deliberately
choosing codons for which the corresponding tRNAs are known to be rare in
the particular cell type. Thus, an additional degree of translational
control is available.
[0270] Many viruses, including retroviruses, use a large number of rare
codons and changing these to correspond to commonly used mammalian
codons, increases expression of a gene of interest, e.g. a NOI or
packaging components in mammalian production cells, can be achieved.
Codon usage tables are known in the art for mammalian cells, as well as
for a variety of other organisms.
[0271] Codon optimisation of viral vector components has a number of other
advantages. By virtue of alterations in their sequences, the nucleotide
sequences encoding the packaging components of the viral particles
required for assembly of viral particles in the producer cells/packaging
cells have RNA instability sequences (INS) eliminated from them. At the
same time, the amino acid sequence coding sequence for the packaging
components is retained so that the viral components encoded by the
sequences remain the same, or at least sufficiently similar that the
function of the packaging components is not compromised. In lentiviral
vectors codon optimisation also overcomes the Rev/RRE requirement for
export, rendering optimised sequences Rev-independent. Codon optimisation
also reduces homologous recombination between different constructs within
the vector system (for example between the regions of overlap in the
gag-pol and env open reading frames). The overall effect of codon
optimisation is therefore a notable increase in viral titre and improved
safety.
[0272] In one embodiment only codons relating to INS are codon optimised.
However, in a much more preferred and practical embodiment, the sequences
are codon optimised in their entirety, with some exceptions, for example
the sequence encompassing the frameshift site of gag-pol (see below).
[0273] The gag-pol gene of lentiviral vectors comprises two overlapping
reading frames encoding the gag-pol proteins. The expression of both
proteins depends on a frameshift during translation. This frameshift
occurs as a result of ribosome "slippage" during translation. This
slippage is thought to be caused at least in part by ribosome-stalling
RNA secondary structures. Such secondary structures exist downstream of
the frameshift site in the gag-pol gene. For HIV, the region of overlap
extends from nucleotide 1222 downstream of the beginning of gag (wherein
nucleotide 1 is the A of the gag ATG) to the end of gag (nt 1503).
Consequently, a 281 bp fragment spanning the frameshift site and the
overlapping region of the two reading frames is preferably not codon
optimised. Retaining this fragment will enable more efficient expression
of the Gag-Pol proteins. For EIAV the beginning of the overlap has been
taken to be nt 1262 (where nucleotide 1 is the A of the gag ATG) and the
end of the overlap to be nt 1461. In order to ensure that the frameshift
site and the gag-pol overlap are preserved, the wild type sequence has
been retained from nt 1156 to 1465.
[0274] Derivations from optimal codon usage may be made, for example, in
order to accommodate convenient restriction sites, and conservative amino
acid changes may be introduced into the Gag-Pol proteins.
[0275] In one embodiment, codon optimisation is based on lightly expressed
mammalian genes. The third and sometimes the second and third base may be
changed.
[0276] Due to the degenerate nature of the genetic code, it will be
appreciated that numerous gag-pol sequences can be achieved by a skilled
worker. Also there are many retroviral variants described which can be
used as a starting point for generating a codon-optimised gag-pol
sequence. Lentiviral genomes can be quite variable. For example there are
many quasi-species of HIV-1 which are still functional. This is also the
case for EIAV. These variants may be used to enhance particular parts of
the transduction process. Examples of HIV-1 variants may be found at the
HIV Databases operated by Los Alamos National Security, LLC at
http://hiv-web.lanl.gov. Details of EIAV clones may be found at the
National Center for Biotechnology Information (NCBI) database located at
http://www.ncbi.nlm.nih.gov.
[0277] The strategy for codon-optimised gag-pol sequences can be used in
relation to any retrovirus. This would apply to all lentiviruses,
including EIAV, FIV, BIV, CAEV, VMV SIV, HIV-1 and HIV-2. In addition
this method could be used to increase expression of genes from HTLV-1,
HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV and other
retroviruses.
[0278] Codon optimisation can render gag-pol expression Rev-independent.
In order to enable the use of anti-rev or RRE factors in the lentiviral
vector, however, it would be necessary to render the viral vector
generation system totally Rev/RRE-independent. Thus, the genome also
needs to be modified. This is achieved by optimising vector genome
components. Advantageously, these modifications also lead to the
production of a safer system absent of all additional proteins both in
the producer and in the transduced cell.
Common Vector Elements
Promoters and Enhancers
[0279] Expression of a NOI and polynucleotide may be controlled using
control sequences for example transcription regulation elements or
translation repression elements, which include promoters, enhancers and
other expression regulation signals (e.g. tet repressor (TetR) system) or
the Transgene Repression In vector Production cell system (TRIP) or other
regulators of NOIs described herein.
[0280] Prokaryotic promoters and promoters functional in eukaryotic cells
may be used. Tissue-specific or stimuli-specific promoters may be used.
Chimeric promoters may also be used comprising sequence elements from two
or more different promoters.
[0281] Suitable promoting sequences are strong promoters including those
derived from the genomes of viruses, such as polyoma virus, adenovirus,
fowlpox virus, bovine papilloma virus, avian sarcoma virus,
cytomegalovirus (CMV), retrovirus and Simian Virus 40 (SV40), or from
heterologous mammalian promoters, such as the actin promoter, EF1a, CAG,
TK, SV40, ubiquitin, PGK or ribosomal protein promoter. Alternatively,
tissue-specific promoters such as rhodopsin (Rho), rhodopsin kinase
(RhoK), cone-rod homeobox containing gene (CRX), neural retina-specific
leucine zipper protein (NRL), Vitelliform Macular Dystrophy 2 (VMD2),
Tyrosine hydroxylase, neuronal-specific neuronal-specific enolase (NSE)
promoter, astrocyte-specific glial fibrillary acidic protein (GFAP)
promoter, human al-antitrypsin (hAAT) promoter, phosphoenolpyruvate
carboxykinase (PEPCK), liver fatty acid binding protein promoter, Flt-1
promoter, INF-6 promoter, Mb promoter, SP-B promoter, SYN1 promoter, WASP
promoter, SV40/hAlb promoter, SV40/CD43, SV40/CD45, NSE/RU5' promoter,
ICAM-2 promoter, GPIIb promoter, GFAP promoter, Fibronectin promoter,
Endoglin promoter, Elastase-1 promoter, Desmin promoter, CD68 promoter,
CD14 promoter and B29 promoter may be used to drive transcription.
[0282] Transcription of a NOI may be increased further by inserting an
enhancer sequence into the vector. Enhancers are relatively orientation-
and position-independent; however, one may employ an enhancer from a
eukaryotic cell virus, such as the SV40 enhancer and the CMV early
promoter enhancer. The enhancer may be spliced into the vector at a
position 5' or 3' to the promoter, but is preferably located at a site 5'
from the promoter.
[0283] The promoter can additionally include features to ensure or to
increase expression in a suitable target cell. For example, the features
can be conserved regions e.g. a Pribnow Box or a TATA box. The promoter
may contain other sequences to affect (such as to maintain, enhance or
decrease) the levels of expression of a nucleotide sequence. Suitable
other sequences include the Sh1-intron or an ADH intron. Other sequences
include inducible elements, such as temperature, chemical, light or
stress inducible elements. Also, suitable elements to enhance
transcription or translation may be incorporated herein.
Regulators of NOIs
[0284] A complicating factor in the generation of retroviral
packaging/producer cell lines and retroviral vector production is that
constitutive expression of certain retroviral vector components and NOIs
are cytotoxic leading to death of cells expressing these components and
therefore inability to produce vector. Therefore, for some embodiments of
the present invention, the expression of these components (e.g. gag-pol
and envelope proteins such as VSV-G) has to be regulated. The expression
of other non-cytotoxic vector components can also be regulated to
minimise the metabolic burden on the cell. The modular constructs and/or
cells of the invention may comprise cytotoxic and/or non-cytotoxic vector
components associated with at least one regulatory element. As used
herein, the term "regulatory element" refers to any element capable of
affecting, either increasing or decreasing, the expression of an
associated gene or protein. A regulatory element includes a gene switch
system, transcription regulation element and translation repression
element
[0285] A number of prokaryotic regulator systems have been adapted to
generate gene switches in mammalian cells. Many retroviral packaging and
producer cell lines have been controlled using gene switch systems (e.g.
tetracycline and cumate inducible switch systems) thus enabling
expression of one or more of the retroviral vector components to be
switched on at the time of vector production. Gene switch systems include
those of the (TetR) protein group of transcription regulators (e.g.
T-Rex, Tet-On, and Tet-Off) and those of the cumate inducible switch
system group of transcription regulators (e.g. CymR protein).
[0286] One such tetracycline-inducible system is the tetracycline
repressor (TetR) system based on the T-REx.TM. system. By way of example,
in such a system tetracycline operators (TetO.sub.2) are placed in a
position such that the first nucleotide is 10 bp from the 3' end of the
last nucleotide of the TATATAA element of the human cytomegalovirus major
immediate early promoter (hCMVp) then TetR alone is capable of acting as
a repressor (Yao F, Svensjo T, Winkler T, Lu M, Eriksson C, Eriksson E.
Tetracycline repressor, tetR, rather than the tetR-mammalian cell
transcription factor fusion derivatives, regulates inducible gene
expression in mammalian cells. 1998. Hum Gene Ther 9: 1939-1950). In such
a system the expression of the NOI can be controlled by a CMV promoter
into which two copies of the TetO.sub.2 sequence have been inserted in
tandem. TetR homodimers, in the absence of an inducing agent
(tetracycline or its analogue doxycycline [dox]), bind to the TetO.sub.2
sequences and physically block transcription from the upstream CMV
promoter. When present, the inducing agent binds to the TetR homodimers,
causing allosteric changes such that it can no longer bind to the
TetO.sub.2 sequences, resulting in gene expression. The TetR gene used in
the Examples disclosed herein has been codon optimised as this was found
to improve translation efficiency resulting in tighter control of
TetO.sub.2 controlled gene expression.
[0287] The TRiP system is described in WO 2015/092440 and provides another
way of repressing expression of the NOI in the production cells during
vector production. The TRAP-binding sequence (e.g. TRAP-tbs) interaction
forms the basis for a transgene protein repression system for the
production of retroviral vectors, when a constitutive and/or strong
promoter, including a tissue-specific promoter, driving the transgene is
desirable and particularly when expression of the transgene protein in
production cells leads to reduction in vector titres and/or elicits an
immune response in vivo due to viral vector delivery of transgene-derived
protein (Maunder et al, Nat Commun. (2017) March 27; 8).
[0288] Briefly, the TRAP-tbs interaction forms a translational block,
repressing translation of the transgene protein (Maunder et al, Nat
Commun. (2017) March 27; 8). The translational block is only effective in
production cells and as such does not impede the DNA- or RNA-based vector
systems. The TRiP system is able to repress translation when the
transgene protein is expressed from a constitutive and/or strong
promoter, including a tissue-specific promoter from single- or multi
cistronic mRNA. It has been demonstrated that unregulated expression of
transgene protein can reduce vector titres and affect vector product
quality. Repression of transgene protein for both transient and stable
PaCL/PCL vector production systems is beneficial for production cells to
prevent a reduction in vector titres: where toxicity or molecular burden
issues may lead to cellular stress; where transgene protein elicits an
immune response in vivo due to viral vector delivery of transgene-derived
protein; where the use of gene-editing transgenes may result in on/off
target affects; where the transgene protein may affect vector and/or
envelope glycoprotein exclusion.
Envelope and Pseudotyping
[0289] In one preferred aspect, the retroviral vector of the invention
disclosed herein has been pseudotyped. In this regard, pseudotyping can
confer one or more advantages. For example, the env gene product of the
HIV based vectors would restrict these vectors to infecting only cells
that express a protein called CD4. But if the env gene in these vectors
has been substituted with env sequences from other enveloped viruses,
then they may have a broader infectious spectrum (Verma and Somia (1997)
Nature 389(6648):239-242). By way of example, workers have pseudotyped an
HIV based vector with the glycoprotein from VSV (Verma and Somia (1997)
Nature 389(6648):239-242).
[0290] In another alternative, the Env protein may be a modified Env
protein such as a mutant or engineered Env protein. Modifications may be
made or selected to introduce targeting ability or to reduce toxicity or
for another purpose (Valsesia-Wittman et al 1996 J Virol 70: 2056-64;
Nilson et al (1996) Gene Ther 3(4):280-286; and Fielding et al (1998)
Blood 91(5):1802-1809 and references cited therein).
[0291] The vector may be pseudotyped with any molecule of choice.
[0292] As used herein, "env" shall mean an endogenous lentiviral envelope
or a heterologous envelope, as described herein.
VSV-G
[0293] The envelope glycoprotein (G) of Vesicular stomatitis virus (VSV),
a rhabdovirus, is an envelope protein that has been shown to be capable
of pseudotyping certain enveloped viruses and viral vector virions.
[0294] Its ability to pseudotype MoMLV-based retroviral vectors in the
absence of any retroviral envelope proteins was first shown by Emi et al.
(1991) Journal of Virology 65:1202-1207). WO 1994/294440 teaches that
retroviral vectors may be successfully pseudotyped with VSV-G. These
pseudotyped VSV-G vectors may be used to transduce a wide range of
mammalian cells. More recently, Abe et al. (1998) J Virol 72(8) 6356-6361
teach that non-infectious retroviral particles can be made infectious by
the addition of VSV-G.
[0295] Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-7)
successfully pseudotyped the retrovirus MLV with VSV-G and this resulted
in a vector having an altered host range compared to MLV in its native
form. VSV-G pseudotyped vectors have been shown to infect not only
mammalian cells, but also cell lines derived from fish, reptiles and
insects (Burns et al. (1993) ibid). They have also been shown to be more
efficient than traditional amphotropic envelopes fora variety of cell
lines (Yee et al., (1994) Proc. Natl. Acad. Sci. USA 91:9564-9568, Emi et
al. (1991) Journal of Virology 65:1202-1207). VSV-G protein can be used
to pseudotype certain retroviruses because its cytoplasmic tail is
capable of interacting with the retroviral cores.
[0296] The provision of a non-retroviral pseudotyping envelope such as
VSV-G protein gives the advantage that vector particles can be
concentrated to a high titre without loss of infectivity (Akkina et al.
(1996) J. Virol. 70:2581-5). Retrovirus envelope proteins are apparently
unable to withstand the shearing forces during ultracentrifugation,
probably because they consist of two non-covalently linked subunits. The
interaction between the subunits may be disrupted by the centrifugation.
In comparison the VSV glycoprotein is composed of a single unit. VSV-G
protein pseudotyping can therefore offer potential advantages for both
efficient target cell infection/transduction and during manufacturing
processes.
[0297] WO 2000/52188 describes the generation of pseudotyped retroviral
vectors, from stable producer cell lines, having vesicular stomatitis
virus-G protein (VSV-G) as the membrane-associated viral envelope
protein, and provides a gene sequence for the VSV-G protein.
Ross River Virus
[0298] The Ross River viral envelope has been used to pseudotype a
non-primate lentiviral vector (FIV) and following systemic administration
predominantly transduced the liver (Kang et al (2002) J Virol
76(18):9378-9388). Efficiency was reported to be 20-fold greater than
obtained with VSV-G pseudotyped vector, and caused less cytotoxicity as
measured by serum levels of liver enzymes suggestive of hepatotoxicity.
Baculovirus GP64
[0299] The baculovirus GP64 protein has been shown to be an alternative to
VSV-G for viral vectors used in the large-scale production of high-titre
virus required for clinical and commercial applications (Kumar M, Bradow
B P, Zimmerberg J (2003) Hum Gene Ther. 14(1):67-77). Compared with
VSV-G-pseudotyped vectors, GP64-pseudotyped vectors have a similar broad
tropism and similar native titres. Because, GP64 expression does not kill
cells, HEK293T-based cell lines constitutively expressing GP64 can be
generated.
Alternative Envelopes
[0300] Other envelopes which give reasonable titre when used to pseudotype
EIAV include Mokola, Rabies, Ebola and LCMV (lymphocytic choriomeningitis
virus). Intravenous infusion into mice of lentivirus pseudotyped with
4070A led to maximal gene expression in the liver.
Packaging Sequence
[0301] As utilized within the context of the invention the term "packaging
signal", which is referred to interchangeably as "packaging sequence" or
"psi", is used in reference to the non-coding, cis-acting sequence
required for encapsidation of retroviral RNA strands during viral
particle formation. In HIV-1, this sequence has been mapped to loci
extending from upstream of the major splice donor site (SD) to at least
the gag start codon. In EIAV the packaging signal comprises the R region
into the 5' coding region of Gag.
[0302] As used herein, the term "extended packaging signal" or "extended
packaging sequence" refers to the use of sequences around the psi
sequence with further extension into the gag gene. The inclusion of these
additional packaging sequences may increase the efficiency of insertion
of vector RNA into viral particles.
[0303] Feline immunodeficiency virus (FIV) RNA encapsidation determinants
have been shown to be discrete and non-continuous, comprising one region
at the 5' end of the genomic mRNA (R-U5) and another region that mapped
within the proximal 311 nt of gag (Kaye et al., J Virol. October;
69(10):6588-92 (1995).
Internal Ribosome Entry Site (IRES)
[0304] Insertion of IRES elements allows expression of multiple coding
regions from a single promoter (Adam et al (as above); Koo et al (1992)
Virology 186:669-675; Chen et al 1993 J. Virol 67:2142-2148). IRES
elements were first found in the non-translated 5' ends of picornaviruses
where they promote cap-independent translation of viral proteins (Jang et
al (1990) Enzyme 44: 292-309). When located between open reading frames
in an RNA, IRES elements allow efficient translation of the downstream
open reading frame by promoting entry of the ribosome at the IRES element
followed by downstream initiation of translation.
[0305] A review on IRES is presented by Mountford and Smith (TIG May 1995
vol 11, No 5:179-184). A number of different IRES sequences are known
including those from encephalomyocarditis virus (EMCV) (Ghattas, I. R.,
et al., Mol. Cell. Biol., 11:5848-5859 (1991); BiP protein [Macejak and
Sarnow, Nature 353:91 (1991)]; the Antennapedia gene of Drosophila (exons
d and e) [Oh, et al., Genes & Development, 6:1643-1653 (1992)] as well as
those in polio virus (PV) [Pelletier and Sonenberg, Nature 334: 320-325
(1988); see also Mountford and Smith, TIG 11, 179-184 (1985)].
[0306] IRES elements from PV, EMCV and swine vesicular disease virus have
previously been used in retroviral vectors (Coffin et al, as above).
[0307] The term "IRES" includes any sequence or combination of sequences
which work as or improve the function of an IRES. The IRES(s) may be of
viral origin (such as EMCV IRES, PV IRES, or FMDV 2A-like sequences) or
cellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES).
[0308] In order for the IRES to be capable of initiating translation of
each polynucleotide it should be located between or prior to the
polynucleotides in the modular construct.
[0309] Vector constructs disclosed utilised for development of stable cell
lines require the addition of selectable markers for selection of cells
where stable integration has occurred. These selectable markers can be
expressed as a single transcription unit within the modular construct or
it may be preferable to use IRES elements to initiate translation of the
selectable marker in a polycistronic message (Adam et al 1991 J. Virol.
65, 4985). As such, a number of the modular constructs were designed such
that the selectable maker was placed downstream of one of the retroviral
vector components utilising an IRES element.
Genetic Orientation and Insulators
[0310] It is well known that nucleic acids are directional and this
ultimately affects mechanisms such as transcription and replication in
the cell. Thus genes can have relative orientations with respect to one
another when part of the same nucleic acid construct.
[0311] As such, at least two nucleic acid sequences present at the same
locus in the cell or construct can be in a reverse and/or alternating
orientations. In other words, at this particular locus, the pair of
sequential genes will not have the same orientation. Alternating
orientations benefits retroviral vector production when the nucleic acids
required for vector production are based at the same genetic locus within
the production cell. This can help prevent both transcriptional and
translational read-through when the region is expressed within the same
physical location of the production cell. This in turn can also improve
the safety of the resulting constructs in preventing the generation of
replication-competent retroviral vectors. When nucleic acid sequences are
in reverse and/or alternating orientations the use of insulators can
prevent inappropriate expression or silencing of a NOI from its genetic
surroundings.
[0312] The term "insulator" refers to a class of DNA sequence elements
that when bound to insulator-binding proteins possess an ability to
protect genes from surrounding regulator signals. There are two types of
insulators: an enhancer blocking function and a chromatin barrier
function. When an insulator is situated between a promoter and an
enhancer, the enhancer-blocking function of the insulator shields the
promoter from the transcription-enhancing influence of the enhancer
(Geyer and Corces 1992; Kellum and Schedl 1992). The chromatin barrier
insulators function by preventing the advance of nearby condensed
chromatin which would lead to a transcriptionally active chromatin region
turning into a transcriptionally inactive chromatin region and resulting
in silencing of gene expression. Insulators which inhibit the spread of
heterochromatin, and thus gene silencing, recruit enzymes involved in
histone modifications to prevent this process (Yang J, Corces V G.
Chromatin Insulators: A Role in Nuclear Organization and Gene Expression.
Advances in cancer research. 2011; 110:43-76.
doi:10.1016/6978-0-12-386469-7.00003-7; Huang, Li et al. 2007; Dhillon,
Raab et al. 2009). An insulator can have one or both of these functions
and the chicken .beta.-globin insulator (cHS4) is one such example. This
insulator is the most extensively studied vertebrate insulator, is highly
rich in G+C and has both enhancer-blocking and heterochromatic barrier
functions (Chung J H, Whitely M, Felsenfeld G. Cell. 1993; 74:505-514).
Other such insulators with enhancer blocking functions are not limited to
but include the following: human .beta.-globin insulator 5 (HS5), human
.beta.-globin insulator 1 (HS1), and chicken .beta.-globin insulator
(cHS3) (Farrell CM1, West A G, Felsenfeld G., Mol Cell Biol. 2002 June;
22(11):3820-31; J Ellis et al. EMBO J. 1996 Feb. 1; 15(3): 562-568). In
addition to reducing unwanted distal interactions the insulators also
help to prevent promoter interference (i.e. where the promoter from one
transcription unit impairs expression of an adjacent transcription unit)
between adjacent retroviral nucleic acid sequences. If the insulators are
used between each of the retroviral vector nucleic acid sequences, then
the reduction of direct read-through will help prevent the formation of
replication-competent retroviral vector particles. An insulator can be
present between each of the retroviral nucleic acid sequences and the use
of insulators may prevent promoter-enhancer interactions from one NOI
expression cassette interacting with another NOI expression cassette in a
modular construct. An insulator may be present between the vector genome
and gag-pol sequences. This therefore limits the likelihood of the
production of a replication-competent retroviral vector and `wild-type`
like RNA transcripts, improving the safety profile of the construct. The
use of insulator elements to improve the expression of stably integrated
multigene vectors is cited in Moriarity et al (Modular assembly of
transposon integratable multigene vectors using RecWay assembly, Nucleic
Acids Res. 2013 April; 41(8):e92).
Vector Titre
[0313] The skilled person will understand that there are a number of
different methods of determining the titre of viral vectors. Titre is
often described as transducing units/mL (TU/mL). Titre may be increased
by increasing the number of vector particles and by increasing the
specific activity of a vector preparation.
Modular Constructs
[0314] The nuclease-secreting viral production system and methods
disclosed herein may incorporate modular nucleic acid constructs (modular
constructs) disclosed in co-pending EP 17210359.0 application number,
entitled RETROVIRAL VECTOR, incorporated by reference in its entirety
herein. A modular construct is a DNA expression construct comprising two
or more nucleic acids used in the production of retroviral vectors. A
modular construct can be a DNA plasmid comprising two or more nucleic
acids used in the production of retroviral vectors. The nucleic acids can
encode for example, gag-pol, rev, env, vector genome. In addition,
modular constructs designed for generation of packaging and producer cell
lines may additionally need to encode transcriptional regulatory proteins
(e.g. TetR, CymR) and/or translational repression proteins (e.g. TRAP)
and selectable markers (e.g Zeocin.TM., hygrornycin, blasticidin,
puromycin, neomycin resistance genes).
[0315] The DNA expression construct can be a DNA plasmid (supercoiled,
nicked or linearised), minicircle DNA (linear or supercoiled), plasmid
DNA containing just the regions of interest by removal of the plasmid
backbone by restriction enzyme digestion and purification, DNA generated
using an enzymatic DNA amplification platform e.g. doggybone DNA
(dbDNA.TM.) where the final DNA used is in a closed ligated form or where
it has been prepared (e.g restriction enzyme digestion) to have open cut
ends.
[0316] As described herein, current methods for retroviral vector
production utilise genetic constructs in which genes essential for
retroviral production are introduced into a production cell on separate
plasmids by transient transfection methods. This can create
batch-to-batch variation and further increases the cost due to the
expensive transfection agents and plasmids. By using such modular
constructs the number of plasmids which are needed in the transfection
process are reduced, thus reducing the burden on labour and material
cost.
[0317] The use of such modular constructs can also aid in the production
of efficient packaging and producer cell lines. In particular,
introducing two or more retroviral vector genes onto one modular
construct will subsequently reduce the number of stable
transfections/transductions, integrations, and selection steps required
in order to create the final packaging/producer cell.
[0318] In particular, it has been surprising to find that bacterial
plasmids are able to perform this function, as it is generally believed
that the large genes involved would not permit multiple genes to be
stably incorporated into a bacterial plasmid.
[0319] In accordance with an aspect of the invention, stable cell lines
(packaging or producer) for producing the retroviral vectors comprise at
least two of the retroviral genes located at the same genetic locus. The
table below lists example combinations of nucleic acids which can be
located at the same locus in stable vector production cells of the
invention disclosed herein and which are expressed from a single modular
construct. The order of each component nucleic acid is also as stated.
The asterisk (*) marks combinations which would be suitable for the
production of EIAV-based retroviral vectors, as Rev is not an essential
component for such vectors. The double asterisk (**) marks combinations
which are associated with a regulatory element or are in reverse and/or
alternating orientations in the vector.
TABLE-US-00008
Number of
expression
cassettes Combination
2 Genome Rev
Genome VSVG*
Genome Gag-Pol*
Rev Genome
Rev VSVG
Rev Gag-Pol**
VSVG Genome*
VSVG Rev
VSVG Gag-Pol*
Gag-Pol Genome*
Gag-Pol Rev**
Gag-Pol VSVG*
3 Genome Rev VSVG
Genome Rev Gag-Pol
Genome VSVG Rev
Genome VSVG Gag-Pol*
Genome Gag-Pol Rev
Genome Gag-Pol VSVG*
Rev Genome VSVG
Rev Genome Gag-Pol
Rev VSVG Genome
Rev VSVG Gag-Pol
Rev Gag-Pol Genome
Rev Gag-Pol VSVG
VSVG Genome Rev
VSVG Genome Gag-Pol*
VSVG Rev Genome
VSVG Rev Gag-Pol
VSVG Gag-Pol Genome*
VSVG Gag-Pol Rev
Gag-Pol Genome Rev
Gag-Pol Genome VSVG*
Gag-Pol Rev Genome
Gag-Pol Rev VSVG
Gag-Pol VSVG Genome*
Gag-Pol VSVG Rev
4 Genome Rev VSVG Gag-Pol
Genome Rev Gag-Pol VSVG
Genome VSVG Rev Gag-Pol
Genome VSVG Gag-Pol Rev
Genome Gag-Pol Rev VSVG
Genome Gag-Pol VSVG Rev
Rev Genome VSVG Gag-Pol
Rev Genome Gag-Pol VSVG
Rev VSVG Genome Gag-Pol
Rev VSVG Gag-Pol Genome
Rev Gag-Pol Genome VSVG
Rev Gag-Pol VSVG Genome
VSVG Genome Rev Gag-Pol
VSVG Genome Gag-Pol Rev
VSVG Rev Genome Gag-Pol
VSVG Rev Gag-Pol Genome
VSVG Gag-Pol Genome Rev
VSVG Gag-Pol Rev Genome
Gag-Pol Genome Rev VSVG
Gag-Pol Genome VSVG Rev
Gag-Pol Rev Genome VSVG
Gag-Pol Rev VSVG Genome
Gag-Pol VSVG Genome Rev
Gag-Pol VSVG Rev Genome
[0320] When using such modular constructs, the cell or vector of the
invention does not contain an origin of replication sequence derived from
a PAC, BAC, YAC, cosmid or fosmid. PAC, BAC, YAC, cosmid and fosmids are
artificially generated nucleic acid vectors designed to hold large
quantities of DNA. Thus, their core sequences are well known and defined
in the art.
Viral Vector Production Systems and Cells
[0321] Generally speaking, a "viral vector production system" or "vector
production system" or "production system" is to be understood as a system
comprising the necessary components for viral vector production.
[0322] In one embodiment of the invention, the viral vector production
system is a retroviral vector production system which comprises nucleic
acid sequences encoding Gag and Gag/Pol proteins, and Env protein thereof
and the vector genome sequence. The production system may optionally
comprise a nucleic acid sequence encoding the Rev protein, or functional
substitute thereof.
[0323] In another embodiment, the retroviral vector is derived from a
lentivirus. In another embodiment, the retroviral vector is derived from
HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna lentivirus. Another
aspect of the invention relates to a set of DNA constructs for use in the
retroviral vector production system of the invention comprising the
modular constructs of the invention. In one embodiment of the invention,
the set of DNA constructs additionally comprises a DNA construct encoding
Rev protein or a functional substitute thereof.
[0324] Another aspect of the invention relates to a retroviral vector
production cell comprising the nucleic acid sequence, such as expression
cassettes and/or some or all modular constructs, encoding the viral
vector components.
[0325] In another embodiment of the invention, viral vector production
system is an AAV viral vector production system or an adenoviral vector
production system.
[0326] A "viral vector production cell", "vector production cell", or
"production cell" is to be understood as a cell that is capable of
producing a viral vector or viral vector particle. Retroviral vector
production cells may be "producer cells" or "packaging cells". One or
more DNA constructs of the viral vector system may be either stably
integrated or episomally maintained within the viral vector production
cell. Alternatively, all the DNA components of the viral vector system
may be transiently transfected into the viral vector production cell. In
yet another alternative, a production cell stably expressing some of the
components may be transiently transfected with the remaining components
required for vector production.
[0327] As used herein, the term "packaging cell" refers to a cell which
contains the elements necessary for production of retroviral vector
particles but which lacks the vector genome. Optionally, such packaging
cells contain one or more expression cassettes which are capable of
expressing viral structural proteins (such as gag, gag/poi and env).
[0328] Producer cells/packaging cells can be of any suitable cell type.
Producer cells are generally mammalian cells but can be, for example,
insect cells.
[0329] As used herein, the term "producer/production cell" or "vector
producing/production cell" refers to a cell which contains all the
elements necessary for production of retroviral vector particles. The
producer cell may be either a stable producer cell line or derived
transiently or may be a stable packaging cell wherein the retroviral
genome is transiently expressed.
[0330] The vector production cells may be cells cultured in vitro such as
a tissue culture cell line. Suitable cell lines include, but are not
limited to, mammalian cells such as murine fibroblast derived cell lines
or human cell lines. Preferably the vector production cells are derived
from a human cell line.
Cells and Production Methods
[0331] The invention relates to a process for producing viral vectors
comprising introducing the nucleic acid sequences described herein into a
cell (e.g. a production cell) and culturing the cell under conditions
suitable for the production of the viral vectors.
[0332] In a further aspect, the invention disclosed herein provides a
replication defective retroviral vector produced by any method of the
invention.
[0333] Suitable production cells are those cells which are capable of
producing viral vectors or viral vector particles when cultured under
appropriate conditions. They are generally eukaryotic cells such as
mammalian or human cells, for example HEK293T, HEK293, CAP, CAP-T, CHO
cells, or PER.C6 cells but can be, for example, insect cells such as SF9
cells.
[0334] Methods for introducing nucleic acids into production cells are
well known in the art and have been described previously.
[0335] Stable cells may be packaging or producer cells. To generate
producer cells from packaging cells the vector genome DNA construct may
be introduced stably or transiently.
[0336] Packaging/producer cells can be generated by transducing a suitable
cell line with a retroviral vector which expresses one of the components
of the packaging/producer cell, i.e. a genome, the gag-poi components and
an envelope as described in WO 2004/022761.
[0337] Alternatively, the nucleic acid can be transfected into cells and
then integration into the production cell genome occurs infrequently and
randomly. The transfection methods may be performed using methods well
known in the art. For example, the transfection process may be performed
using calcium phosphate or commercially available formulations such as
Lipofectamine.TM. 2000 CD (Invitrogen, CA), FuGENE.RTM. HD or
polyethylenimine (PEI). Alternatively modular constructs of the invention
may be introduced into the production cell via electroporation. The
skilled person will be aware of methods to encourage integration of the
nucleic acids into production cells. For example, linearising a nucleic
acid construct can help if it is naturally circular. Less random
integration methodologies may involve the nucleic acid construct
comprising of areas of shared homology with the endogenous chromosomes of
the mammalian host cell to guide integration to a selected site within
the endogenous genome. Furthermore, if recombination sites are present on
the construct then these can be used for targeted recombination. For
example, the nucleic acid construct may contain a loxP site which allows
for targeted integration when combined with Cre recombinase (i.e. using
the Cre/lox system derived from P1 bacteriophage). Alternatively or
additionally, the recombination site is an att site (e.g. from A phage),
wherein the att site permits site-directed integration in the presence of
a lambda integrase. This would allow the retroviral genes to be targeted
to a locus within the host cellular genome which allows for high and/or
stable expression.
[0338] Other methods of targeted integration are well known in the art.
For example, methods of inducing targeted cleavage of genomic DNA can be
used to encourage targeted recombination at a selected chromosomal locus.
These methods often involve the use of methods or systems to induce a
double strand break (DSB) e.g. a nick in the endogenous genome to induce
repair of the break by physiological mechanisms such as non-homologous
end joining (NHEJ). Cleavage can occur through the use of site-specific
nucleases such as engineered zinc finger nucleases (ZFN),
transcription-activator like effector nucleases (TALENs), using
CRISPR/Cas9 systems with an engineered crRNA/tracr RNA (`single guide
RNA`) to guide specific cleavage, and/or using nucleases based on the
Argonaute system (e.g., from T. thermophilus). Such gene-editing-type
nucleases are not suitable for use in the vector production cells for the
purposes of non-specifically degrading residual nucleic acid during
vector production because they are site-specific enzymes targeted to the
nuclease and not secreted as are the non-specific nucleases of the
invention disclosed herein.
[0339] Packaging/producer cell lines can be generated by integration of
nucleic acids using methods of retroviral transduction or nucleic acid
transfection, or a combination thereof. Methods for generating retroviral
vectors from production cells and in particular the processing of
retroviral vectors are described in WO 2009/153563.
[0340] A production cell may optionally comprise the RNA-binding protein
(e.g. tryptophan RNA-binding attenuation protein, TRAP) and/or the Tet
Repressor (TetR) protein or alternative regulatory proteins (e.g. CymR).
[0341] Production of retroviral vector from production cells can be via
transfection methods, from production from stable cell lines which can
include induction steps (e.g. doxycycline induction) or via a combination
of both. The transfection methods may be performed using methods well
known in the art, and examples have been described previously.
[0342] Production cells, either packaging or producer cell lines or those
transiently transfected with the retroviral vector encoding components
are cultured to increase cell and virus numbers and/or virus titres.
Culturing a cell is performed to enable it to metabolize, and/or grow
and/or divide and/or produce viral vectors of interest according to the
invention. This can be accomplished by methods well known to persons
skilled in the art, and includes but is not limited to providing
nutrients for the cell, for instance in the appropriate culture media.
The methods may comprise growth adhering to surfaces, growth in
suspension, or combinations thereof. Culturing can be done for instance
in tissue culture flasks, tissue culture multi-well plates, dishes,
roller bottles, wave bags or in bioreactors, using batch, fed-batch,
continuous systems and the like. In order to achieve large scale
production of viral vector through cell culture it is preferred in the
art to have cells capable of growing in suspension. Suitable conditions
for culturing cells are known (see e.g. Tissue Culture, Academic Press,
Kruse and Paterson, editors (1973), and R. I. Freshney, Culture of animal
cells: A manual of basic technique, fourth edition (Wiley-Liss Inc.,
2000, ISBN 0-471-34889-9).
[0343] In one embodiment, cells are initially `bulked up` in tissue
culture flasks or bioreactors and subsequently grown in multi-layered
culture vessels or large bioreactors (greater than 50 L) to generate the
vector producing cells of the invention disclosed herein.
[0344] In another embodiment, cells are grown in an adherent mode to
generate the vector producing cells of the invention.
[0345] In yet another embodiment, cells are grown in a suspension mode to
generate the vector producing cells of the invention.
Use
[0346] Another aspect of the invention relates to the use of the viral
vector of the invention or a cell or tissue transduced with the viral
vector of the invention in medicine.
[0347] Another aspect of the invention relates to the use of the viral
vector of the invention, a production cell of the invention or a cell or
tissue transduced with the viral vector of the invention for the
preparation of a medicament to deliver a nucleotide of interest to a
target site in need of the same. Such uses of the viral vector or
transduced cell of the invention may be for therapeutic or diagnostic
purposes, as described previously.
[0348] Another aspect of the invention relates to a cell transduced by the
viral vector of the invention.
[0349] A "cell transduced by a viral vector particle" is to be understood
as a cell, in particular a target cell, into which the nucleic acid
carried by the viral vector particle has been transferred.
Pharmaceutical Compositions
[0350] Another aspect of the invention relates to a pharmaceutical
composition comprising the viral vector of the invention or a cell or
tissue transduced with the viral vector of the invention, in combination
with a pharmaceutically acceptable carrier, diluent or excipient.
[0351] The invention disclosed herein also provides a pharmaceutical
composition for treating an individual by gene therapy, wherein the
composition comprises a therapeutically effective amount of a viral
vector. The pharmaceutical composition may be for human or animal usage.
[0352] The composition may comprise a pharmaceutically acceptable carrier,
diluent, excipient or adjuvant. The choice of pharmaceutical carrier,
excipient or diluent can be made with regard to the intended route of
administration and standard pharmaceutical practice. The pharmaceutical
compositions may comprise, or be in addition to, the carrier, excipient
or diluent any suitable binder(s), lubricant(s), suspending agent(s),
coating agent(s), solubilising agent(s) and other carrier agents that may
aid or increase vector entry into the target site (such as for example a
lipid delivery system).
[0353] Where appropriate, the composition can be administered by any one
or more of inhalation; in the form of a suppository or pessary; topically
in the form of a lotion, solution, cream, ointment or dusting powder; by
use of a skin patch; orally in the form of tablets containing excipients
such as starch or lactose, or in capsules or ovules either alone or in
admixture with excipients, or in the form of elixirs, solutions or
suspensions containing flavouring or colouring agents; or they can be
injected parenterally, for example intracavernosally, intravenously,
intramuscularly, intracranially, intraoccularly intraperitoneally, or
subcutaneously. For parenteral administration, the compositions may be
best used in the form of a sterile aqueous solution which may contain
other substances, for example enough salts or monosaccharides to make the
solution isotonic with blood. For buccal or sublingual administration,
the compositions may be administered in the form of tablets or lozenges
which can be formulated in a conventional manner.
[0354] The viral vector of the invention may also be used to transduce
target cells or target tissue ex vivo prior to transfer of said target
cell or tissue into a patient in need of the same. An example of such
target cells may be autologous T cells and an example of such tissue may
be a donor cornea.
[0355] The present invention is useful in the production of gesicles or
exosomes. In this respect, gesicles are typically made by over-expressing
the VSV-G envelope in (HEK293-dereived) cell lines, which produces
vesicles. Exosomes can be induced/expressed endogenously from cells. Both
gesicles or exosomes can be loaded with RNAs and proteins, such as
CRISPR-cas9 or other gene editing components.
Numbered Paragraphs
[0356] Disclosed herein are viral vector production systems secreting
nuclease for degradation of residual nucleic acid during viral vector
production and methods of the same. Such a viral vector production system
comprises a viral vector production cell comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease,
wherein the nuclease is expressed in the production cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production. Another such viral vector production system comprises 1) a
viral vector production cell comprising nucleic acid sequences encoding
viral vector components; and 2) a nuclease helper cell comprising a
nucleic acid sequence encoding a nuclease, wherein the nuclease is
expressed and secreted in co-culture of the production cell of 1) and the
helper cell of 2), thereby degrading residual nucleic acid during viral
vector production.
[0357] Aspects of the present invention will now be described by way of
numbered paragraphs 1-26--presented as numbered paragraphs set 1.
Numbered Paragraphs Set 1:
[0358] 1. A viral vector production system comprising a viral vector
production cell comprising nucleic acid sequences encoding: 1) viral
vector components; and 2) a nuclease, wherein the nuclease is expressed
in the production cell and secreted in cell culture thereby degrading
residual nucleic acid during viral vector production.
[0359] 2. A viral vector production system comprising: 1) a viral vector
production cell comprising nucleic acid sequences encoding viral vector
components; and 2) a nuclease helper cell comprising a nucleic acid
sequence encoding a nuclease, wherein the nuclease is expressed and
secreted in co-culture of the production cell of 1) and the helper cell
of 2), thereby degrading residual nucleic acid during viral vector
production.
[0360] 3. A method of producing a viral vector, the method comprising,
transfecting (sometimes referred to as contacting) a viral vector
production cell with nucleic acid sequences encoding: 1) viral vector
components; and 2) a nuclease, wherein the nuclease is expressed in the
production cell and secreted in cell culture thereby degrading residual
nucleic acid during viral vector production.
[0361] 4. A method of producing a viral vector, the method comprising
contacting 1) a viral vector production cell expressing viral vector
components with 2) a nuclease helper cell expressing a nuclease, wherein
the nuclease is expressed in the helper cell and secreted in co-culture
of the production cell with the helper cell thereby degrading residual
nucleic acid during viral vector production.
[0362] 5. A method of producing a viral vector, the method comprising
contacting 1) a viral vector production cell expressing viral vector
components with 2) a liquid feed from nuclease helper cell expressing a
nuclease, wherein the nuclease is expressed in the cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production.
[0363] 6. The viral vector production system or method of any of claims
1-5, wherein the cell culture comprises a volume of at least about 5
litres of medium.
[0364] 7. The viral vector production system or method of any of
paragraphs 1-6, wherein the cell culture comprises a volume of at least
about 50 litres of medium.
[0365] 8. The viral vector production system or method of any one of
paragraphs 1 to 7, wherein the production cell is a HEK293 cell, or a
derivative thereof.
[0366] 9. The viral vector production system or method of paragraph 8,
wherein the HEK293 production cell is a HEK293T cell.
[0367] 10. The viral vector production system or method of any of
paragraphs 1-9, wherein the viral vector components comprise a nucleotide
of interest (NOI).
[0368] 11. The viral vector production system or method of any of
paragraphs 1-10, wherein the viral vector components are retroviral
vector components.
[0369] 12. A production cell for producing viral vectors comprising
nucleic acid sequences encoding: 1) viral vector components; and 2) a
nuclease, wherein the nuclease is expressed in the viral vector
production cell and secreted in cell culture thereby degrading residual
nucleic acid during viral vector production, and wherein the production
cell is a eukaryotic production cell.
[0370] 13. A production cell for producing viral vectors comprising
nucleic acid sequences encoding: 1) viral vector components; and 2) a
nuclease fusion protein, wherein the nuclease fusion protein comprises an
exonuclease domain fused to an endonuclease domain, and wherein the
nuclease fusion protein is expressed in the viral vector production cell
and secreted in cell culture thereby degrading residual nucleic acid
during viral vector production, and wherein the production cell is a
eukaryotic production cell.
[0371] 14. A cell culture device comprising a viral vector production
system comprising a viral vector production cell comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease,
wherein the nuclease is expressed in the production cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production.
[0372] 15. A modified nuclease having increased cell-retention and/or or
cell-association that is expressed through the secretory pathway of a
eukaryotic cell, wherein the modified nuclease comprises a retention
signal at its C-terminus.
[0373] 16. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein secreted
nuclease activity within the viral vector production culture is at least
about 1 unit per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
[0374] 17. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein secreted
nuclease activity within the viral vector production culture is at least
about 10 units per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
[0375] 18. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein secreted
nuclease activity within the viral vector production culture is at least
about 100 units per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
[0376] 19. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of paragraphs 16-18 wherein the nuclease activity is
supplied by the viral vector production cell.
[0377] 20. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of paragraphs 16-18 wherein the nuclease activity is
supplied by a nuclease helper cell.
[0378] 21. A nuclease helper cell wherein secreted nuclease activity
within the helper cell culture is at least about 10 unit per mL of
equivalent Benzonase.RTM. nuclease activity as determinable by the assay
presented as Assay 1 herein.
[0379] 22. A nuclease helper cell wherein secreted nuclease activity
within the helper cell culture is at least about 100 unit per mL of
equivalent Benzonase.RTM. nuclease activity as determinable by the assay
presented as Assay 1 herein.
[0380] 23. A nuclease helper cell wherein secreted nuclease activity
within the helper cell culture is at least about 2000 units per mL of
equivalent Benzonase.RTM. nuclease activity as determinable by the assay
presented as Assay 1 herein.
[0381] 24. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein the nuclease
expression cassette utilizes a strong promoter, preferably the CMV
promoter.
[0382] 25. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs utilizing a nuclease
helper cell, wherein the nuclease helper cells are added to the main
viral vector production vessel at or after the point of sodium butyrate
supplementation.
[0383] 26. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs, wherein the expression
of nuclease within viral vector production vessels enables the use of
downstream processing without the requirement for further nuclease
treatment.
[0384] These and other aspects of the present invention will now be
described by way of numbered paragraphs 1-82 presented as numbered
paragraphs set 2.
Numbered Paragraphs Set 2:
[0385] 1. A viral vector production system comprising a viral vector
production cell comprising nucleic acid sequences encoding: 1) viral
vector components; and 2) a nuclease, wherein the nuclease is expressed
in the production cell and secreted in cell culture thereby degrading
residual nucleic acid during viral vector production.
[0386] 2. A viral vector production system comprising: 1) a viral vector
production cell comprising nucleic acid sequences encoding viral vector
components; and 2) a nuclease helper cell comprising a nucleic acid
sequence encoding a nuclease, wherein the nuclease is expressed and
secreted in co-culture of the production cell of 1) and the helper cell
of 2), thereby degrading residual nucleic acid during viral vector
production.
[0387] 3. A method of producing a viral vector, the method comprising,
transfecting (sometimes referred to as contacting) a viral vector
production cell with nucleic acid sequences encoding: 1) viral vector
components; and 2) a nuclease, wherein the nuclease is expressed in the
production cell and secreted in cell culture thereby degrading residual
nucleic acid during viral vector production.
[0388] 4. A method of producing a viral vector, the method comprising
contacting 1) a viral vector production cell expressing viral vector
components with 2) a nuclease helper cell expressing a nuclease, wherein
the nuclease is expressed in the helper cell and secreted in co-culture
of the production cell with the helper cell thereby degrading residual
nucleic acid during viral vector production.
[0389] 5. A method of producing a viral vector, the method comprising
contacting 1) a viral vector production cell expressing viral vector
components with 2) a liquid feed from nuclease helper cell expressing a
nuclease, wherein the nuclease is expressed in the cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production.
[0390] 6. In an improved method of producing a viral vector, the
improvement comprising introducing nucleic acid sequences into a viral
vector production cell, wherein the nucleic acid sequences encode: 1)
viral vector components; and 2) a nuclease, wherein the nuclease is
expressed in the production cell and secreted in cell culture thereby
degrading residual nucleic acid during viral vector production.
[0391] 7. In an improved method of producing a viral vector, the
improvement comprising contacting in co-culture a viral vector production
cell expressing viral vector components with a nuclease helper cell
expressing a nuclease, wherein the nuclease is expressed in the
production cell and secreted in cell culture thereby degrading residual
nucleic acid during viral vector production.
[0392] 8. The viral vector production system or method of any of
paragraphs 1-7, wherein cell culture is maintained in a pH range of 6.5
to 7.2.
[0393] 9. The viral vector production system or method of any of
paragraphs 1-7, wherein the extracellular nuclease is selected from the
group consisting of smNucA, VsEndA, VcEndA and BacNucB.
[0394] 10. The viral vector production system or method of any of
paragraphs 1-7, wherein the nuclease is an extracellular nuclease.
[0395] 11. The viral vector production system or method of any of
paragraphs 1-7, wherein the nuclease is a sugar-non-specific nuclease.
[0396] 12. The viral vector production system or method of any of
paragraphs 1-7, wherein the nuclease comprises Serratia marcescens
Nuclease A of SEQ ID NO: 3 or a variant thereof having at least 90%
sequence identity to SEQ ID NO: 3.
[0397] 13. The viral vector production system or method of any of
paragraphs 1-7, wherein the nuclease comprises Vibrio cholerae
Endonuclease I of SEQ ID NO: 1 or a variant thereof having at least 90%
amino acid identity to SEQ ID NO: 1.
[0398] 14. The viral vector production system or method of paragraph 13,
wherein the nuclease is a nuclease variant selected from the group
consisting VcEndA-12glc of SEQ ID NO: 5, VcEndA-123glc of SEQ ID NO: 6,
VcEndA-124glc of SEQ ID NO: 7, VcEndA-134glc of SEQ ID NO: 8,
VcEndA-13glc of SEQ ID NO: 9, VcEndA-14glc of SEQ ID NO: 10, and
VcEndA-1glc of SEQ ID NO: 11.
[0399] 15. The viral vector production system or method of paragraph 13,
wherein the nuclease is nuclease variant VcEndA-1glc of SEQ ID NO: 11.
[0400] 16. The viral vector production system or method of any of
paragraphs 1-7, wherein the nuclease comprises a salt-active nuclease.
[0401] 17. The viral vector production system or method of paragraph 16,
wherein the salt-active nuclease comprises Vibrio salmonicida
Endonuclease I of SEQ ID NO: 2 or a variant thereof having at least 90%
amino acid identity to SEQ ID NO: 2
[0402] 18. The viral vector production system or method of any of
paragraphs 1-7, wherein the nuclease comprises BacNucB of SEQ ID NO: 4,
or a variant thereof having at least 90% amino acid identity to SEQ ID
NO: 4.
[0403] 19. The viral vector production system or method of any of
paragraphs 1-18, wherein the nuclease comprises a native or non-native
N-terminal secretory signal.
[0404] 20. The viral vector production system or method of any of
paragraphs 1-18, wherein the nucleic acid sequences encoding a nuclease
are sequence-optimised to remove potential splice sites and/or unstable
elements.
[0405] 21. The viral vector production system or method of any of
paragraphs 1-18, wherein the nucleic acid sequences encoding a nuclease
are codon-optimised for expression in the production cell.
[0406] 22. The viral vector production system or method of any of
paragraphs 1-21, wherein the production system and/or methods further
comprise one or more additional nucleases.
[0407] 23. The viral vector production system or method of paragraph 22,
wherein the nuclease is fused to at least one of the one or more
additional nucleases.
[0408] 24. The viral vector production system or method of any of
paragraphs 1-23, wherein the cell culture comprises a volume of at least
about 5 litres of medium.
[0409] 25. The viral vector production system or method of any of
paragraphs 1-24, wherein the cells are in suspension or adherent.
[0410] 26. The viral vector production system or method of any of
paragraphs 1-25, wherein the cell culture is serum-free.
[0411] 27. The viral vector production system or method of any of
paragraphs 1-26, wherein the production cell is a eukaryotic cell.
[0412] 28. The viral vector production system or method of paragraph 27,
wherein the production cell is a mammalian cell.
[0413] 29. The viral vector production system or method of paragraph 28,
wherein the production cell is a human production cell.
[0414] 30. The viral vector production system or method of any one of
paragraphs 1-29, wherein the production cell is a HEK293 cell, or a
derivative thereof.
[0415] 31. The viral vector production system or method of paragraph 30,
wherein the HEK293 production cell is a HEK293T cell.
[0416] 32. The viral vector production system or method of any of
paragraphs 1-31, wherein the viral vector components comprise a
nucleotide of interest (NOI).
[0417] 33. The viral vector production system or method of any of
paragraphs 1-32, wherein the viral vector components are retroviral
vector components.
[0418] 34. The viral vector production system or method of paragraph 33,
wherein the retroviral vector components are lentiviral vector
components.
[0419] 35. The viral vector production system or method of paragraph 34,
wherein the viral vector components comprise i) gag-pol; ii) env; iii)
optionally the RNA genome of a retroviral vector; and iv) optional rev,
or a functional substitute thereof.
[0420] 36. The viral vector production system or method of paragraph 35,
wherein at least two of the nucleic acid sequences are modular constructs
encoding the viral vector components located at the same genetic locus.
[0421] 37. The viral vector production system or method of paragraph 35,
wherein at least two of the nucleic acid sequences are modular constructs
encoding the viral vector components in reverse and/or alternating
orientations.
[0422] 38. The viral vector production system or method of paragraph 35,
wherein at least two of the nucleic acid sequences are modular constructs
encoding gag-pol and/or env, wherein the modular constructs are
associated with at least one regulator element.
[0423] 39. The viral vector production system or method of paragraphs
35-38, wherein the env is a VSV-G env.
[0424] 40. The viral vector production system or method of any of
paragraphs 1-39, wherein the nuclease is expressed and secreted from the
cell after transient transfection.
[0425] 41. The viral vector production system or method of any of
paragraphs 1-39, wherein the nuclease is expressed and secreted from the
cell after stable integration of the cell.
[0426] 42. The viral vector production system or method of paragraph 41,
wherein the expression of the nuclease is inducible or conditional, and
wherein the nucleic acid encoding the nuclease comprises an inducible or
conditional promoter or regulatory element.
[0427] 43. A production cell for producing viral vectors comprising
nucleic acid sequences encoding: 1) viral vector components; and 2) a
nuclease, wherein the nuclease is expressed in the viral vector
production cell and secreted in cell culture thereby degrading residual
nucleic acid during viral vector production, and wherein the production
cell is a eukaryotic production cell.
[0428] 44. The cell according to paragraph 43, wherein the nuclease is an
endonuclease.
[0429] 45. The cell according to paragraph 44, further comprising a
nucleic acid sequence encoding an exonuclease.
[0430] 46. The cell according to paragraph 45, wherein the endonuclease is
fused to the exonuclease.
[0431] 47. The cell according to paragraph 43, wherein the nuclease is an
exonuclease.
[0432] 48. The cell according to paragraph 48, wherein the exonuclease is
fused to the endonuclease.
[0433] 49. The cell of paragraph 43, wherein the cell is a transient
production cell.
[0434] 50. The cell of paragraph 43, wherein the cell is a stable
production cell.
[0435] 51. The cell of paragraph 50, wherein nuclease expression and
secretion is inducible expression and secretion in the stable production
cell.
[0436] 52. A production cell for producing viral vectors comprising
nucleic acid sequences encoding: 1) viral vector components; and 2) a
nuclease fusion protein, wherein the nuclease fusion protein comprises an
exonuclease domain fused to an endonuclease domain, and wherein the
nuclease fusion protein is expressed in the viral vector production cell
and secreted in cell culture thereby degrading residual nucleic acid
during viral vector production, and wherein the production cell is a
eukaryotic production cell.
[0437] 53. The cell of paragraph 52, wherein the endonuclease is VcEndA or
a variant thereof.
[0438] 54. The cell of paragraph 52, wherein the endonuclease is
VcEndA-1glc of SEQ ID NO: 11.
[0439] 55. A cell culture device comprising a viral vector production
system comprising a viral vector production cell comprising nucleic acid
sequences encoding: 1) viral vector components; and 2) a nuclease,
wherein the nuclease is expressed in the production cell and secreted in
cell culture thereby degrading residual nucleic acid during viral vector
production.
[0440] 56. The cell culture device of paragraph 46, which is a stir-tank
bioreactor or a wave-bag or iCELLis.RTM. bioreactor.
[0441] 57. A variant of a secreted nuclease capable of degrading residual
nucleic acid during viral vector production, said variant comprising the
amino acid sequence of SEQ ID NO: 11.
[0442] 58. A modified nuclease having increased cell-retention and/or or
cell-association that is expressed through the secretory pathway of a
eukaryotic cell, wherein the modified nuclease comprises a retention
signal at its C-terminus.
[0443] 59. The modified nuclease of paragraph 58, wherein the modified
nuclease localizes to the endoplasmic reticulum (ER) and/or the golgi
compartments thereby resulting in increased cell retention compared to
cell retention of a corresponding unmodified nuclease.
[0444] 60. The modified nuclease of paragraph 58, wherein the modified
nuclease is bound to ER receptors of the ER retention-defective
complementation group.
[0445] 61. The modified nuclease of paragraph 58, wherein the retention
signal is at its C-terminus of consensus [KRHQSA]-[DENQ]-E-L or KKXX
[0446] 62. The modified nuclease of paragraph 58, wherein the retention
signal is at its C-terminus of consensus KDEL.
[0447] 63. A eukaryotic cell expressing the modified nuclease of paragraph
58.
[0448] 64. A viral vector production system comprising the cell of
paragraph 63.
[0449] 65. A production cell for producing viral vectors comprising a
viral production cell as defined in any one of paragraphs 1 to 42.
[0450] 66. A production cell according to paragraph 43 or any paragraph
dependent thereon comprising a viral production cell as defined in any
one of paragraphs 1 to 42.
[0451] 67. A production cell according to paragraph 52 or any paragraph
dependent thereon comprising a viral production cell as defined in any
one of paragraphs 1 to 42.
[0452] 68. A cell culture device comprising a production cell as defined
in any one of paragraphs 1 to 42.
[0453] 69. A cell culture device comprising a production cell as defined
in paragraph 43 or any paragraph dependent thereon.
[0454] 70. A cell culture device comprising a production cell as defined
in paragraph 52 or any paragraph dependent thereon.
[0455] 71. The viral vector production system or method according to any
one of paragraphs 1 to 42 comprising a nuclease as defined in any one of
paragraphs 57-62.
[0456] 72. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein secreted
nuclease activity within the viral vector production culture is at least
about 1 unit per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
[0457] 73. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein secreted
nuclease activity within the viral vector production culture is at least
about 10 units per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
[0458] 74. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein secreted
nuclease activity within the viral vector production culture is at least
about 100 units per mL of equivalent Benzonase.RTM. nuclease activity as
determinable by the assay presented as Assay 1 herein.
[0459] 75. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of paragraphs 72-74 wherein the nuclease activity is
supplied by the viral vector production cell
[0460] 76. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of paragraphs 72-74 wherein the nuclease activity is
supplied by a nuclease helper cell
[0461] 77. A nuclease helper cell wherein secreted nuclease activity
within the helper cell culture is at least about 10 unit per mL of
equivalent Benzonase.RTM. nuclease activity as determinable by the assay
presented as Assay 1 herein.
[0462] 78. A nuclease helper cell wherein secreted nuclease activity
within the helper cell culture is at least about 100 unit per mL of
equivalent Benzonase.RTM. nuclease activity as determinable by the assay
presented as Assay 1 herein.
[0463] 79. A nuclease helper cell wherein secreted nuclease activity
within the helper cell culture is at least about 2000 units per mL of
equivalent Benzonase.RTM. nuclease activity as determinable by the assay
presented as Assay 1 herein.
[0464] 80. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs wherein the nuclease
expression cassette utilizes a strong promoter such as CMV.
[0465] 81. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs utilizing a nuclease
helper cell, wherein the nuclease helper cells are added to the main
viral vector production vessel at or after the point of sodium butyrate
supplementation.
[0466] 82. The viral vector production system, method, production cell,
cell culture device, variant of a secreted nuclease, modified nuclease or
eukaryotic cell of any of the previous paragraphs, wherein the expression
of nuclease within viral vector production vessels enables the use of
downstream processing without the requirement for further nuclease
treatment.
[0467] Various preferred features and embodiments of the invention will
now be described by way of non-limiting examples.
EXAMPLES
Example 1: Expression Constructs Encoding Widely Divergent Nucleases for
the Reduction of Residual DNA During Viral Vector Production Cell Culture
[0468] Expression plasmids were constructed for Serratia marcescens
Endonuclease A (SmNucA), VsEndA and BacNucB according to FIG. 3. All
constructs contained the SV40 promoter and polyadenylation signal. ORFs
were codon-optimised (Homo sapiens) and were 6.times.Histidine tagged at
their C-terminus (H6). SmNucA expression plasmids encoded wild type
smNucA with its own bacterial secretory sequence or had its bacterial
secretory sequence replaced with that of human Albumin or VSV-G, or
additionally included N.times.S/T sequon mutations at the stated
positions. VsEndA or VcEndA expression plasmids encoded wild type
nuclease with its own bacterial secretory sequence or had its bacterial
secretory sequence replaced with that of human Albumin, or additionally
included N.times.S/T sequon mutations at T121V, N130D, S135R. BacNucB
expression plasmids encoded wild type BacNucB with its own bacterial
secretory sequence or additionally contained y>N mutations at the
stated positions, resulting in N.times.S/T sequons.
Example 2: Demonstration of Expression and Secretion of a Nuclease from
HEK293T Cells Correlated with Reduced Residual DNA in Culture Media
[0469] HEK293T cells were transfected with a fixed amount of plasmid DNA
(total .mu.g) using different ratios of stuffer DNA (pBluescript) and
pSV40-smNucAH6, which encodes SmNucA fused with a C-terminal His-tag to
allow for protein detection. Cell lysates and culture media were analysed
by immunoblotting to the His-tag (an endogenous His-tagged protein
TRAPH6' was used as a loading control), which demonstrated that SmNucAH6
was expressed and secreted in the cultures in a dose-dependent manner.
Clarified culture supernatants were analysed for residual DNA by
PicoGreen assay, which demonstrated that reduction in DNA detection only
occurred in the presence of SmNucAH6 in the media. Results are shown in
FIG. 4.
Example 3: C-Terminal Histidine Tagged and Untagged Secreted Nucleases
During Production of Lentiviral Vector Production
[0470] HIV-1 based lentiviral vectors were produced by transient
co-transfection of secreted nuclease plasmids into either adherent or
suspension HEK293T cells at 10% input of total pDNA. Lentiviral vector
harvests were left `untreated` or were treated with Benzonase.RTM. for 1
hour prior to clarification. Secreted nuclease cultures were not treated
with Benzonase.RTM.. Filtered culture media was analysed by PicoGreen
assay. Secreted nucleases produced equivalent or better DNA reduction
than Benzonase.RTM. in culture media. Results are shown in FIG. 5.
Example 4: Eukaryotic ER Signal Peptides can Replace Bacterial Secretory
Peptide of smNucA
[0471] HIV-1 based lentiviral vectors (HIV-CMV-GFP) were produced by
transient co-transfection of secreted nuclease plasmids into adherent
HEK293T cells at 1% input of total pDNA. The SmNucA ORF contained either
its native bacterial secretory signal (Native) or the human Albumin ER
signal peptide (Hu Albumin ER-SP) or the VSV-G ER signal peptide (VSV-G
ER-SP). None of the lentiviral vector harvests were treated with
Benzonase.RTM.. Filtered culture media was analysed by PicoGreen assay
(black bars) and lentiviral vectors were titrated by transduction of
HEK293T cells followed by flow cytometry. Results are shown in FIG. 6.
Example 5: Divergent Secreted Nucleases During Generation of Lentiviral
Vectors in Adherent HEK293T Cell Cultures During Transient Transfection
of Vector Components
[0472] HIV-1 based lentiviral vectors (HIV-CMV-GFP) were produced by
transient co-transfection of either SmNucAH6 or VsEndAH6 nuclease
plasmids into adherent HEK293 Ts at 1% input of total pdNA. Lentiviral
vector harvests were left `untreated` or were treated with Benzonase.RTM.
for 1 hour prior to vector harvest. Secreted nuclease cultures were not
treated with Benzonase.RTM.. Filtered culture media was analysed by
PicoGreen assay (black bars) and LVs titrated by transduction of HEK293
Ts followed by flow cytometry. Results are shown in FIG. 7.
Example 6: Divergent Secreted Nucleases During Generation of Lentiviral
Vectors in Adherent Stable Producer Cell Line Cultures
[0473] HIV-1 based lentiviral vectors (HIV-CMV-GFP) were produced by
transient transfection of a producer cell line with either SmNucAH6 or
VsEndAH6 nuclease plasmids at 1% input of total pDNA (pBluescript was
added as standard stuffer). Approximately 18 hours post-transfection,
cultures were induced by addition of doxycycline (1000 ng/ml final
concentration), which resulted in expression of packaging components
driven by the CMV-tetO promoter (GFP genome constitutively expressed).
Filtered culture media was analysed by PicoGreen assay (black bars) and
LVs titrated by transduction of HEK293 Ts followed by flow cytometry.
Results are shown in FIG. 8.
Example 7: Divergent Secreted Nucleases During Generation of Lentiviral
Vectors in Suspension Cell Cultures
[0474] HIV-1 based lentiviral vectors (HIV-CMV-GFP) were produced in
HEK293T cells in suspension. The cells were transiently co-transfection
with either SmNucAH6 or VsEndAH6 nuclease plasmids at 5% input of total
pDNA. Lentiviral vector harvests were left `untreated` or were treated
with Benzonase.RTM. for 1 hour prior to vector harvest. Secreted nuclease
cultures were not treated with Benzonase.RTM.. Filtered culture media was
analysed by PicoGreen assay (black bars) and purified total DNA was
analysed for production cell DNA by qPCR against 18S DNA (light grey
bars). Lentiviral vector supernatant was titrated by transduction of
HEK293 Ts followed by flow cytometry (dark grey bars). Concentrated
vector supernatant was subjected to SDS-PAGE and immunoblotting using
anti-His Tag antibody to detect secreted nuclease proteins. Results are
shown in FIG. 9.
Example 8: Clearance of Residual Plasmid DNA in Lentiviral Vector
Production Suspension, Serum-Free Cell Cultures Using BacNucB and BacNucB
Variants
[0475] Bacillus species BacNucB protein (SEQ ID No: 4) does not comprise
any N.times.S/T sequons and therefore cannot be N-glycosylated when
targeted to the secretory pathway in eukaryotic cells. It was therefore
unknown if BacNucB could be used effectively in the clearance of residual
DNA from vector production cultures, as lack of N-glycosylation might
hinder secretion levels. The work flow outlined in FIG. 2 was applied to
the primary sequence of BacNucB (SEQ ID NO: 4). The sequence was analysed
and TMHMM (CBS) was used to determine that there were no TM
(transmembrane) domains on the C-terminal side of the signal peptide
(which is predicted to be a TM). Analysis of BacNucB by SignalP (CBS)
confirmed that the bacterial secretory peptide is predicted to function
as an ER signal peptide, cleavage of which is predicted to generate the
complete mature nuclease. Further analysis by YAPIN (Centre for
integrative Bioinformatics VU (IBIVU)) predicted regions of
alpha-helices, beta-strands and coils which pinpoints regions where
N.times.S/T motifs could be introduced into the BacNucB protein sequence
without disrupting smaller folds. As such, the following 4 variants of
BacNucB were identified: P43N (sequon=NAS) is variant `N1`, G61N
(sequon=NHS) is `N2`, V65N (sequon=NCT) is variant `N3`, and D133N
(sequon=NGT) is variant `N4`. The primary sequence of BacNucB with the
`N` mutations at 43, 61, 65, and 133 were submitted to NetNGlyc (CBS) for
analysis. All four N.times.S/t sequons had scores above the threshold
(0.5), with N65 and N133 variants achieving the highest probability of
improved functionality. Results are shown in FIG. 10.
[0476] Based on the above analysis, four variants of BacNucB (N1, N2, N3,
and N4) were generated by inserting N.times.S/T sequons into its primary
amino acid sequence by mutating the Y residue to an N, at four
Y.times.S/T sites within the native protein sequence. This was done at
positions that are predicted not to participate in secondary folds in
order to maximize the likelihood that potential N-glycosylation would not
affect protein folding.
[0477] All BacNucB expression plasmids were His-tagged at their C-termini
for ease of detection. Suspension, serum-free HEK293T cells were
transfected with HIV-CMV-GFP vector components and nuclease plasmids at
5% input of total pDNA. Purified total DNA from vector harvests was
analysed for residual plasmid by qPCR against KanR sequences (black
bars). Lentiviral vector supernatant was titrated by transduction of
HEK293T cells followed by flow cytometry (grey bars). Concentrated vector
supernatant was subjected to SDS-PAGE and immunoblotting using
anti-HisTag antibody to detect secreted nuclease proteins. The data
demonstrates that residual plasmid DNA is only modestly cleared by
BacNucBH6 but that two of the N-glycan variants showed enhanced clearance
of residual plasmid DNA. Note that the expression plasmid encoding
VsEndAH6 was superior to all expression plasmids encoding BacNucB in
terms of residual DNA clearance.
Example 9: Clearance of Residual Plasmid DNA in Lentiviral Vector
Production in Suspension, Serum-Free Cell Cultures Using VcEndA Variants
[0478] VcEndA and VsEndA share 68% identity at their amino acid sequence
(secreted forms). VcEndA has been shown to have optimal activity between
150 and 200 mM salt (i.e., close to salt concentration in typical
eukaryotic cell cultures) and is more active at pH6.5 and pH7 than
VsEndA. Analysis of VcEndA (as per the workflow shown in FIG. 2) reveals
three of its four N.times.S/T sequons are not present in VsEndA. glc'
VcEndA variants were generated (as shown in FIG. 3) by ablating these
sequons at all three positions. Additional VcEndA variants were generated
with the human Albumin ER signal peptide, as well as optional C-terminal
His-tags. (Key: wt=VcEndA, 1=VcEndA-1glc, 2=AlbVcEndA, 3=AlbVcEndA-1glyc;
4, 5, 6, 7=His-tagged versions of wt, 1, 2, 3 respectively. HIV-CMV-GFP
vector was made in suspension, serum-free cultures co-transfected with
nuclease plasmids at 5% total input pDNA. Lentiviral vector supernatant
was titrated by transduction of HEK 293T cells followed by flow cytometry
(bars). Concentrated vector supernatant (VcEndA variants 4-7) were
subjected to SDS-PAGE/immunoblotting using anti-HisTag antibody to detect
secreted nuclease proteins, as well as VSV-G (lentiriviral vector
virions). Post-production cell lysates were also probed for nuclease
expression. Residual DNA from vector supernatants was visualized by
agarose-electrophoresis. VcEndA (wt) is poorly secreted into the culture
media but the `1glc` variants are efficiently secreted, leading to
efficient clearance of residual DNA. Results are shown in FIG. 12.
Example 10: Degradation of Residual DNA from Lentiviral Vector Production
Bioreactors at 0.5 L Scale
[0479] Suspension, serum-free adapted HEK293T cells were seeded into six
0.5 litre (L) bioreactors and triplicate bioreactors transfected with
HIV-1 based lentiviral vector components (GFP expressing genome) together
with either pBluescript (referred as STD--Benzonase.RTM. in FIG. 13) or
pSV40-VsEndAH6 at 5% total plasmid input. Twenty hours post-transfection,
sodium butyrate (NaBut) was added to all bioreactors at a final
concentration of 10 mM. Four hours after NaBut induction, samples were
taken (Bioreactor [4 hr post-NaBut]). One hour prior to vector harvest,
the pBluescript-co-transfected bioreactors were inoculated with
Benzonase.RTM. at 5 U/ml final concentration. The
pSV40-VsEndAH6-co-transfected bioreactors were left untreated. The bulk
harvests from the triplicate bioreactors were then pooled to generated
.about.1 L of harvest, which was subject to 0.45 .mu.m filtration, and
samples taken for analysis (CL Harvest). This pooling was done in order
to be able to perform a downstream process at a scale that allowed use of
equipment/flow rates reflective of typical large scale downstream
process. Material was subjected to ion-exchange (IEX) chromatography,
collecting samples (IEX flow through, IEX washes, IEX elutate, and IEX
column clean with 2M salt) before further processing using
dia-/ultra-filtration using hollow fibre cartridges. The first step
allowed buffer exchange (HFF-pre-Benzonase.RTM.) and then 400 U/ml
Benzonase.RTM. was added to both the `standard Benzonase.RTM.` processed
vector and the VsEndAH6 treated vector, as a second nuclease step.
Finally, buffer exchange occurred to remove Benzonase.RTM. (HFF-post
Benzonase.RTM.). Samples were titrated to generate lentiviral vector
titers (GFP/FACS; TU/ml) and residual DNA purified and subjected to
plasmid copy number analysis by qPCR against KanR sequences. Total TUs
and total kanR copies were generated for each step by factoring total
volumes at each step. Results are shown in FIG. 13. The data show that
secreted VsEndAAH6 reduces KanR detection by 10-fold in the bioreactor as
early as 24 hours post-transfection, whilst lentiviral vector titers
remain unaffected in the bioreactor, and during downstream processing.
Whilst Benzonase.RTM.-treated harvest appeared to reduce KanR detection
to a similar, albeit slightly higher level compared to VsEndAH6, KanR
detection through the downstream process was lower in the VsEndAH6
material. This suggests that whiles similar numbers of KanR copies were
detected in CL Harvest comparing both approaches, there was a qualitative
difference between the two conditions i.e., residual DNA in the VsEndAH6
material is likely to have been smaller in size (see FIG. 14 for evidence
of this), and easier to remove during the downstream process.
Example 11: Degradation of Residual DNA from Lentiviral Vector Production
Bioreactors at 5 L Scale
[0480] HIV-1 based lentiviral vectors expressing GFP were produced in a
similar manner as described for 0.5 L bioreactors (Example 10) however in
single bioreactors at 5 L scale. SAN-HQ was also tested in parallel to
Benzonase.RTM. and the secreted nuclease approach (co-transfected
pSV40-VsEndAH6 at 5% of total plasmid input). FIG. 14A shows total
lentiviral vector TUs and detectable plasmid (KanR) at each process step,
and FIG. 14B shows concentrated samples from upstream & downstream
process steps were analysed by gel electrophoresis (Ethidium bromide
stain for DNA). As is shown in FIG. 13, expression plasmid-expressed
secreted nuclease achieves comparable or better reduction in plasmid DNA
compared to commercial nuclease (such as Benzonase.RTM.) but
additionally, total DNA (which will include production cell DNA) is
digested to smaller fragments which appear to be more efficiently removed
by the final nuclease step on the HFF cartridge.
Example 12: Degradation of Residual DNA within Lentiviral Vector
Production Cultures Using a tetR-Regulated Nuclease Expression Plasmid
[0481] HIV-1 based vectors encoding GFP were produced by transient
transfection of serum-free, suspension-adapted HEK293T.tetR cells with
vector components and pCMV-TO-VsEndAH6 at either 1% or 5% input of total
plasmid DNA (FIG. 15). Cultures receiving no nuclease plasmid were
co-transfected with pBluescript. Approximately 20 hours
post-transfection, all cultures were induced with sodium butyrate and
1000 ng/mL doxycycline (lanes 1, 3-6; to induce VsEndAH6 expression where
present), except for a replicate set of cultures transfected with 5%
pCMV-TO-VsEndAH6, which only received sodium butyrate (no dox; lane 2).
Control cultures were either left untreated (No Nuc) or treated with
Benzonase.RTM. or SAN at 5 U/mL final concentration 1 hour prior to
harvest. All cultures received 2 mM MgCl.sub.2 final concentration 1 hour
prior to harvest. All conditions were carried out in triplicate. At
harvest, production cells were removed by centrifugation, and
supernatants filtered (0.22 .mu.m) before titration by transduction of
HEK293T cells followed by flow cytometry. Supernatants from each of the
triplicate cultures were pooled, and .about.2 mL concentrated to 0.12 mL
by centrifugation using 3K cut-off Amicon-15 devices, and 50% of this
material loaded onto a 2% agarose gel for residual DNA electrophoresis
(Ethidium Bromide). The data show that tetR-regulated nuclease expression
can be employed to allow for temporal control of nuclease expression;
doxycycline addition 20 hour post-transfection leads to sufficiently
active levels of secreted nuclease as indicated by improved clearance of
residual DNA compared to the commercial nucleases. The titres of
lentiviral vectors produced in the presence of secreted nuclease were all
above 1.times.10.sup.6 TU/mL, demonstrating minimal impact on the output
of vector.
Example 13: Degradation of Residual DNA within Lentiviral Vector
Production Cultures by Co-Culturing with Helper Cells Expressing Secreted
Nuclease
[0482] HIV-1 based vectors encoding GFP were produced by transient
transfection of serum-free, suspension-adapted HEK293T-1.65S cells with
vector components. In parallel and at the same scale, serum-free
suspension-adapted HEK293T.tetR cells were transfected with
tetR-regulated nuclease plasmids pCMV-TO-VsEndAH6 or pCMV-TO-smNucAH6.
Approximately 20 hours post-transfection, cultures were inoculated with
10% of the helper Cell cultures, together with sodium butyrate and 1000
ng/mL dox, to induce nuclease expression from the helper cells. Control
cultures were either left untreated (No Nuc) or treated with
Benzonase.RTM. or SAN at 5 U/mL final concentration 1 hour prior to
harvest. All cultures received 2 mM MgCl.sub.2 final concentration 1 hour
prior to harvest. All conditions were carried out in triplicate. At
harvest, production cells were removed by centrifugation, and
supernatants filtered (0.22 .mu.m) before titration by transduction of
HEK293T cells followed by flow cytometry. Supernatants from each of the
triplicate cultures were pooled, and -2 mL concentrated to 0.12 mL by
centrifugation using 3K cut-off Amicon-15 devices, and 50% of this
material loaded onto a 2% agarose gel for residual DNA electrophoresis
(Ethidium Bromide). Results are shown in FIG. 16. The data show that
nuclease expressing helper cells can be co-cultured with vector
production cells allowing sufficiently active levels of secreted nuclease
as indicated by improved clearance of residual DNA compared to the
commercial nucleases. The titres of lentiviral vectors produced in the
presence of secreted nuclease were all above 1.times.10.sup.6 TU/mL,
demonstrating no impact impact on the output of vector.
Example 14: AAV-Based and Adenovirus-Based Vector Production Systems
Expressing Secreted Nuclease for Degradation of Residual Nucleic Acid
During Vector Production Requiring Freeze-Thaw Step
[0483] A. Work flow of use of secreted nuclease in production of AAV
vectors by transient transfection: [0484] 1. Seeding of production cells
(e.g. HEK293) into culture vessel (adherent or suspension) [0485] 2.
Transient co-transfection of vector components with nuclease plasmid:
[0486] i. Multi-plasmid transfection of vector components pAAV
genome:pRepCap:pHelper (e.g. 1:1:1 ratio), plus pNuclease cotransfected
as a subfraction of total pDNA [0487] ii. Transfection via reagents such
as Lipofectamine or PEI [0488] 3. Incubation of transfected cell
cultures for several (e.g. 2-to-4) days [0489] 4. Cell lysis: [0490] i.
Direct freeze-thawing of production cells in cultures media, or [0491]
ii. Concentration of production cells, followed by chemical lysis or
physical lysis e.g. microfluidisation/freeze-thaw [0492] iii. Limited
incubation period to allow secreted nuclease to digest contaminating DNA
[0493] 5. Filtration and optional freezing of bulk substance [0494] 6.
Downstream purification/concentration e.g chromatography (ion-exchange),
dia/ultra-filtration and/or Size exclusion. [0495] B. Work flow of use
of secreted nuclease in production of Adenoviral vectors by amplification
in Adenovirus E1-expressing cells: [0496] 1. Seeding of production cells
(e.g. HEK293) into culture vessel (adherent or suspension) [0497] 2.
Inoculation of Adenovirus vector seed stock at MOI=1 [0498] 3. Incubate
cultures for 24-72 hours or until .about.50% visible cytopathic effect
(cpe) [0499] 4. Cell lysis: [0500] i. Direct freeze-thawing of
production cells in cultures media, or [0501] ii. Concentration of
production cells, followed by chemical lysis or physical lysis e.g.
microfluidisation/freeze-thaw [0502] iii. Limited incubation period to
allow secreted nuclease to digest contaminating DNA [0503] 5.
Filtration and optional freezing of bulk substance [0504] 6. Optionally,
successive scaling-up of steps 1-5 [0505] 7. Downstream
purification/concentration e.g chromatography (ion-exchange),
dia/ultra-filtration and/or Size exclusion.
Example 15: Generation and Evaluation of VcEndA Variants Containing
N.times.S/T Sequon Ablation Mutations to Degrade Residual DNA in
Lentiviral Vector Production Serum-Free, Suspension Cultures
[0506] HIV-1 based vectors encoding GFP were produced from serum-free,
suspension-adapted HEK293T-1.655 cells with different secreted nuclease
encoding plasmids (0.5% or 5% total input pDNA) co-transfected with LV
component pDNA. The secreted nucleases tested were all His-tagged at
their C-termini for ease of detection by immunoblotting. All the
sequon-mutated variants displayed in FIG. 17B were tested alongside wild
type VcEndA (1234-glc), SmNucA and VsEndA.
[0507] End-of-production cell lysates and concentrated supernatants were
subjected to SDS-PAGE and Western blot, using anti-sera against His6-Tag
(nucleases), tubulin (cell control) or VSVG (secretion control i.e. VSVG
on LV virions)--see FIGS. 18A&B. These immunoblots demonstrated that the
secreted nucleases (in all their glycosylated forms) were expressed in
cells and secreted to some level during LV production. The wild type
VcEndAH6-1234glc nuclease (and the VcEndAH6-123glc variant) were poorly
secreted, demonstrating that the N.times.S/T sequon ablation approach can
be used to generate novel, and improved secretion nuclease variants. To
summarise, the 1glc and 12glc variants were predominantly singly
glycosylated, whereas all the other forms of VcEndAH6 were doubly
glycosylated. Through deduction, it can be concluded that if present,
sequons at 102NCT (#1), 130NRS (#3) and 133NFS (#4) are utilized, except
for VcEndAH6 (wt) and VcEndAH6-134glc, where predominantly only two of
these sequons are actually utilized. LV titres were all above
1.times.10.sup.7 TU/mL, demonstrating that high titre vector can be
produced in the presence of secreted nucleases (FIG. 19A).
[0508] Culture supernatants were also subjected to processing resulting in
concentrated samples that were analysed for residual DNA content by
visualization via gel electrophoresis (FIG. 19B). This analysis revealed
a general correlation with residual DNA degradation and improved
secretion. However, when the 130NRS sequon (#3) was present within
VcEndAH6, DNA degradation was much less efficient, even if the nuclease
was efficiently secreted; this indicates that the NRS sequon within
VcEndA is preferentially utilised for N-glycosylation but that this
modification results in attenuation of nuclease activity.
Example 16: Degradation of Residual DNA within Lentiviral Vector
Production Serum-Free, Suspension Cultures Using Secreted Nucleases at
Different Set pH Conditions
[0509] HIV-1 based vectors encoding GFP were produced from serum-free,
suspension-adapted HEK293T-1.65S cells with different secreted nuclease
encoding plasmids (5% total input pDNA) co-transfected with LV component
pDNA, with cultures set at two different pH levels post-transfection;
pH7.2 (slightly alkaline) and pH6.6 (slightly acidic)--see FIGS. 20-22.
Duplicate 12 mL cultures were set up using an AMBR-15 system, and pH
levels (FIG. 20A) and cell viability (FIG. 20B) measured each day of the
production run. On the day of transfection (day 2), pH set points were
either 6.6.+-.0.15 or 7.2.+-.0.15 after the addition of
pDNA/lipofectamine transfection mix, and these pH set points were
maintained until LV harvest on day 4. The secreted nuclease plasmids used
were pSV40-VcEndAH6-1glc (SEQ ID NO: 11, with C-terminal His6 Tag),
pSV40-VsEndAH6 (SEQ ID NO: 2, with C-terminal His6 Tag), and
pSV40-SmNucAH6 (SEQ ID NO: 3, with C-terminal His6 Tag). The remaining 12
transfections/cultures were spiked with 5% pBluescript and did not
receive any secreted nuclease. These remaining 12 cultures were treated
with either Benzonase.RTM. (four cultures), SAN (four cultures) or
untreated (four cultures) for 1 hour prior to LV harvest; as per the
pSecNuc transfected cultures (four in each case) these were paired into
pH6.6 and pH7.2 set point test conditions.
[0510] End-of-production cell lysates and concentrated supernatants were
subjected to SDS-PAGE and Western blot, using anti-sera against His6-Tag
(nucleases), tubulin (cell control) or VSVG (secretion control i.e. VSVG
on LV virions)--see FIGS. 21A&B. These immunoblots demonstrated that the
secreted nucleases (in all their glycosylated forms) were expressed in
cells and efficiently secreted during LV production.
[0511] LV-CMV-GFP supernatants were titrated on HEK293T cells, which
indicated that all conditions enabled LV production over 1.times.10.sup.6
TU/mL, with general similarity in production titres at both pH set points
(FIG. 22A). Culture supernatants were also subjected to processing
resulting in concentrated samples that were analysed for residual DNA
content by visualization via gel electrophoresis (FIG. 22B). All cultures
treated with secreted nuclease or Benzonase.RTM./SAN displayed lower
amounts of residual DNA compared to untreated, except that the secreted
nucleases generally outperformed the commercial nucleases. VsEndAH6
displayed better residual DNA clearance properties at pH7.2 compared to
pH6.6. However, both VcEndAH6-1glc and SmNucAH6 appeared to be efficient
at clearing residual DNA at both pH set points; note that VcEndAH6-1glc
achieved this whilst appearing to provide minimal impact on LV titres.
Example 17: Degradation of Residual DNA within Lentiviral Vector
Production Serum-Free, Suspension Cultures at Different Set pH
Conditions: Comparing Activity of Two Different N-Glycan-Mutants of
VcEndA
[0512] Example 15 identified three VcEndA variants with improved secretion
and residual DNA clearance compared to wild type VcEndA, and had
minimal/no impact on LV titres. These were VcEndAH6-1glc, -14glc and
-124glc. Example 16 tested the VcEndAH6-1glc variant alongside VsEndAH6
and SmNucAH6 in stir tank mini-bioreactors under alkaline or acid
culturing conditions, demonstrating that VsEndAH6 works less efficiently
than VcEndAH6-1glc at acidic pH. Since VcEndAH6-1glc harbours mutated
N.times.S/T sequons with altered residues based on VsEndA, it was unclear
as to whether the improved activity of VcEndAH6-1glc under acidic
conditions might be further improved if other N.times.S/T sequons were
`re-instated`. A similar experiment as outlined in Example 16 was
performed (production of LV-CMV-GFP vector and secreted nucleases in stir
tank mini-bioreactors [AMBR-15] at pH6.6 or 7.2), and VcEndAH6-124glc,
VcEndAH6-1glc and VsEndAH6 were compared against commercial nucleases
(FIGS. 23 & 24). Set points for pH and cell viability were recorded
(FIGS. 24 A&B), and LVs were titrated demonstrating that these nucleases
had no impact on production titres (FIG. 24A). Culture supernatants were
also subjected to processing resulting in concentrated samples that were
analysed for residual DNA content by visualization via gel
electrophoresis (FIG. 24B). This analysis revealed very similar levels of
residual DNA clearance by VcEndAH6-1glc and VcEndAH6-124glc, with both
variants out-performing VsEndAH6 as well as showing enhanced activity
under acidic conditions. These data demonstrate that the VcEndAH6-1glc
variant possess the full characteristics of activity at acidic pH despite
the modifications introduced into its primary sequence, perhaps
indicating that the altered residues do not contribute to nuclease
activity that distinguishes VcEndA from VsEndA.
Example 18: Use of Secreted and Cell-Retained Nucleases to Degrade
Residual DNA During Production of AAV-Based Vectors
[0513] For production of viral vectors whereby the vector virions remain
associated with production cells--such as AAVs and AdVs--it is
anticipated that production cells may be lysed directly in situ within
the bioreactor by addition of cell-disrupting agent such as detergent. In
this case, secreted nuclease within the culture media will be present to
degrade contaminating DNA. However, if production cells are partitioned
away from the culture media (e.g. by precipitation or centrifugation or
media change) then it is desirable for co-expressed nuclease to remain
associated with cells in order to be present and active subsequently to
cell lysis. This example shows how the C-terminal appendage of nuclease
with the known ER-retention signal `KDEL` improves residual DNA clearance
from cell lysate during scAAV2-GFP production. FIG. 25 shows (in
non-limiting terms) how an ER-retention signal can be appended to a
nuclease secreted into the ER lumen resulting in its binding to an `ER
retention-defective [ERD] complementation group` protein receptor, such
that is it continually cycled back to the ER lumen. To test this
approach, VcEndAH6 [wt] (shown to be poorly secreted in HEK293T based
cells; see FIG. 18AB) and VcEndAH6-1glc were engineered to contain a
C-terminal `KDEL` sequence. FIG. 26 displays the results of scAAV2-GFP
vector production by co-transfection of suspension, serum-free HEK293T
cells with the indicated nuclease expression plasmids (5% of total input)
together with AAV2 vector component plasmids (Genome:RepCap2:Helper ratio
was 1:1:1). The SmNucAH6 nuclease was also tested. Two controls were
included in which no nuclease expression plasmid was included (i.e. 5%
pBluescript: `AAV-NEG`). After two days, cells were pelleted and
separated from supernatant, and three freeze-thaw events were carried out
in the presence of a detergent-based lysis buffer. Magnesium Chloride was
added to all conditions (2 mM final), and SAN added to one of the
controls to allow comparison to a commercial nuclease (effectively 75
U/0.5 mL cell pellet). Debris was cleared by low-speed centrifugation,
and the resulting lysate filtered, before crude vector-containing
supernatant was titrated on HEK293T cells FIG. 26A and residual DNA
analysed by agarose gel electrophoresis (ethidium bromide) FIG. 26B. This
example demonstrates that high titre scAAV2-GFP can be produced in the
presence of co-expressed nuclease, albeit that SmNucAH6 reduced titres by
3-4 fold at this level of plasmid input. The other nucleases tested had
no impact on vector production at the 5% input level. Analysis of
residual DNA clearance indicated that actually, a very good level of DNA
digestion was achievable with VcEndAH6-1glc and smNucAH6 without the KDEL
sequence. The levels of these two secreted nuclease associated with cell
lysates (see FIG. 18B) appear to be sufficient to allow efficient DNA
clearance, and indicates that these are fully active proteins `on their
way` to being secreted from the cell. Nevertheless, the KDEL appendage to
VcEndAH6 [wt] and VcEndAH6-1glc appeared to allow improved DNA clearance
compared to the non-KDEL variants. As observed with lentiviral vector
production, VcEndAH6-1glc was much more efficient than the VcEndAH6 [wt]
version, and the VcEndAH6-1glc-KDEL variant was capable of reducing
residual DNA levels close to background levels when comparing
densitometry profiles (FIG. 27) of the gel image in FIG. 26B. The
VcEndAH6-1glc-KDEL variant out-performed the other secreted nucleases
tested as well as SAN in eliminating the small, `resistant` forms of DNA
typically observed in gel analysis. The example demonstrates the utility
of secreted nucleases in clearing residual DNA in a cell-associated
fashion but that this can be improved upon by the use of ER-retention
signals.
Example 19: Optimisation of Transfection Parameters to Generate Nuclease
Helper Cells by Transient Transfection
[0514] One of the principal ways of applying the Secreted nuclease
technology in a preferred embodiment will be to employ nuclease helper
cells generated separately, and applied to the main viral vector
production culture once viral vector particles are being produced. One
approach is to transiently transfect cells--ideally the same base cell
line that will be used for transient transfection by viral vector
encoding plasmids--in parallel during the GMP manufacturing campaign.
Conceivably, a GMP bank of transiently transfected nuclease helper cells
could be generated (characterized and validated before freezing), which
could then be revived and applied to the main viral vector production
culture. The perceived advantage of the use of transiently transfected
nuclease helper cells over stable, tet-regulated nuclease helper cells is
the lack of requirement of a chemical inducer such as tetracycline or
doxycycline. However the helper cell approach is applied (transient or
stable), in a preferred embodiment an important parameter will be the
total number of helper cells added to the main viral vector production
culture in order to achieve a desirable (efficacious) level of secreted
nuclease activity in the main viral vector production culture.
[0515] The approach of co-transfection of secreted nuclease plasmids
together with viral vector plasmids (see previous Examples) has revealed
a general range of percentage input levels (relative to the total amount
of plasmid DNA being transfected) of 1-5%, depending on the strength of
promoter being employed. However, in this approach the viral vector
plasmids act as `carrier` DNA for each other and also for the secreted
nuclease plasmid. For a helper cell approach to be compatible with GMP
production in a preferred embodiment only the secreted nuclease plasmid
should be transfected into cells (without any `steer` DNA), and moreover,
it will be desirable to achieve maximal expression/secretion of the
secreted nuclease from the helper cells in order to be able to apply low
numbers of helper cells to the main viral vector production culture to
achieve maximal clearance of residual DNA. To evaluate this, serum-free
suspension HEK293T cells were transiently transfected with VcEndAH6-1glc
or SmNucAH6 encoding plasmids driven by either the weaker SV40 promoter
or stronger CMV promoter at a range of input amounts (100-to-900 ng per
mL final culture). Transfections were carried out in two groups: with or
without sodium butyrate induction (at 10 mM final concentration). The
sodium butyrate induction was performed within 20 hours post-transfection
(a typical regime for sodium butyrate induction of Lentiviral vector
production) in order to mimic the effect of adding helper cells into the
main viral vector production culture at (or beyond) the time of sodium
butyrate induction (sodium butyrate is known to up-regulate expression of
genes driven by promoters such as CMV in HEK293-based cells). A
co-transfection control was included for each secreted nuclease plasmid,
whereby pSecNuc (at 5% total; 62ng per mL final culture) was mixed-in
with pBlueScript (at 95% total); this was done to be able to compare
secreted nuclease expression of helper cell cultures with the
co-transfection approach exemplified in other Examples herein. Two days
post-transfection, the supernatants from the transfected helper cells
were harvested and analysed by immunoblotting to the secreted nucleases
(anti-HisTag); cell lysates were also analysed by immunoblotting to the
secreted nucleases and GAPDH. Secreted nuclease band intensity was
quantified and normalized to GAPDH bands in order to assess the level of
secreted nuclease expressed in cells (FIG. 28A), and this compared to
immunoblots of secreted nuclease in the supernatants (FIG. 28B).
[0516] The data reveals that (as expected) choice of promoter strength
correlates with expression levels of secreted nuclease in the cells, with
the CMV-promoter driven cassettes achieving a higher level of expression
than SV40-promoter counterparts. It was identified that transfection
inputs of 700 ng plasmid DNA (per mL of cell culture, containing
8.times.10.sup.5 viable cells) achieved close-to-maximal levels of
secreted nuclease expression. The effect of sodium butyrate induction was
generally observed with the CMV-promoter driven cassettes; the boost to
VcEndAH6-1glc expression was more obvious with VcEndAH6-1glc compared to
SmNucAH6. This is in line with previous work indicating SmNucAH6
expression may be in some way `self-limiting`, building a further body of
evidence that the VcEndAH6-1glc nuclease is a superior choice in
application of the invention. When comparing the level of VcEndAH6-1glc
expression achieved using pSV40-VcEndAH6-1glc (used mainly for the
`co-transfection` approach exemplified in other Examples herein) with the
pCMV-VcEndAH6-1glc construct at 700 ng/mL input levels, the latter was
able to achieve at least 10-fold more expression than the former.
Example 20: Quantitation of Nuclease Activity Output of Nuclease Helper
Cells when Spiked into Viral Vector Production Cultures
[0517] Example 19 demonstrated that nuclease helper cell cultures can be
generated with extremely high levels of nuclease secretion and nuclease
activity. Clearly this invention describes a number of ways of generating
nuclease helper cells, and conceivably the nuclease output activity of a
given helper cell culture may vary. Thus, in order to achieve
desirable/efficacious levels of nuclease activity with viral vector
production cultures by spiking-in helper cells, the number of helper
cells being supplemented would vary depending on the nuclease activity
output of the helper cells being employed. However, the most desirable
approach will be one in which as few helper cells as possible are needed
when adding to the main viral vector production culture so as to minimize
impact on the vector production process (e.g. effects on viral vector
production cells such as dilution effects and/or competition for growth
metabolites). In order to achieve this in a preferred embodiment, maximal
expression of the secreted nuclease will be desirable or, in some cases,
necessary; this was exemplified in Example 19. To evaluate the efficiency
of DNA clearance within viral vector production cultures by spiking-in
helper cells, an initial experiment was performed whereby nuclease helper
cells were generated by transient transfection of serum-free, suspension
HEK293T cells with 750ng plasmid DNA per mL culture, and were spiked into
lentiviral vector production cultures at different percentage inputs
relative to the total numbers of cells in the main lentiviral vector
production vessel; these were 10%, 5%, 2.5% and 1%. In this initial
experiment the SV40-promoter driven nuclease plasmids for VcEndAH6-1glc
and SmNucAH6 were used. Cells from the main viral vector production and
helper cultures were transfected with the relevant components in
parallel, and then were counted .about.20 hours post-transfection before
the appropriate volume of helper cell culture suspension (representing
the above percentages) was added to replicate viral vector production
cultures split-out from the original vector production culture. Normal
vector production was then continued from this point (in this case,
sodium butyrate induction occurred concomitantly with helper cell
addition), and vector harvests taken for titration and analysis of
nuclease secretion by immunoblot and for residual DNA content (FIG. 29).
Vector titres produced with or without helper cells were
>1.times.10.sup.7 TU/mL, and were minimally impacted at 10% and 5%
helper cell inputs (<2-fold), perhaps indicating that addition of
greater than 10% of helper cells into viral vector production cultures
may not be desirable (FIG. 29A). The levels of secreted nuclease achieved
in the helper cell cultures were extremely high (as denoted by burn-out
of bands on the immunoblots), and the resulting levels of secreted
nuclease in the vector production cultures correlated well with the input
percentages (FIG. 29B). Again, a greater level of VcEndAH6-1glc protein
level was achieved compared to SmNucAH6. Surprisingly, extremely good
levels of residual DNA clearance were observed for all secreted nuclease
conditions, even at the lowest input level of 1% (FIG. 29C). This
indicated that the nuclease helper cell approach can be used using
secreted nuclease expression cassettes using promoters of modest activity
but importantly in a preferred embodiment, if a stronger promoter is used
then even fewer nuclease helper cells may be added to the viral vector
production culture, potentially avoiding any observable impact on vector
titres.
[0518] A further evaluation of the nuclease helper cell approach was
undertaken by focusing on the use of the pCMV-VcEndAH6-1glc plasmid, and
reducing the number of helper cells added to the main viral vector
production culture. helper cell and lentiviral vector production cells
were transfected with the respective plasmids in parallel, and then
different proportions of the helper cells were spiked into the vector
production cultures -20 hours post-transfection at the sodium butyrate
induction point. Similarly as before, nuclease helper cells were
generated by transient transfection of serum-free, suspension HEK293T
cells with 750ng plasmid DNA per mL culture. Since it was previously
demonstrated that the CMV-promoter driven cassette yielded >10-fold
more VcEndAH6-1glc protein than the SV40-promoter, the range of cell
input of the helper cells was taken down as far as just 0.1% (relative to
the number of cells within the vector production culture). After
considering viable cell counting, this equated to 30 .mu.L of helper cell
culture added to 40 mL of vector production culture. In this experiment
additional controls included standard Benzonase/SAN (5 U/mL) treatment 1
hr prior to harvest, and the 5% `co-transfection` control
(pSV40-VcEndAH6-1glc mixed with LV components) was included to directly
compare the helper cell approach with the `co-transfection` approach. As
before, vector production continued as normal, and vector harvests were
taken for titration and analysis of residual DNA content (FIG. 30).
Vector titres produced with or without helper cells were
>1.times.10.sup.7 TU/mL, and were minimally impacted at 5% and 1%
helper cell inputs (<3-fold) (FIG. 30A).
[0519] The assessment of residual DNA clearance in concentrated (44-fold)
vector production cultures revealed extremely good clearance of residual
DNA by secreted VcEndAH6-1glc, which appeared to be the same or better
than that of the Benzonase/SAN treated vector samples (FIG. 30B).
Remarkably, when just 0.1% of the total number of cells within the vector
production culture comprised helper cells, the level of apparent residual
DNA clearance was just as effective compared to the high input levels of
helper cells. Interestingly, a `nuclease-resistant` band
(`Feed-specific`) was observed for all concentrated vector supernatants
but not in the concentrated helper cell supernatant. This indicated that
this band was specific to lentiviral vector-containing supernatant,
suggesting that it could be (cellular) RNA--possibly residing within
virions--protected from the RNAse activity of VcEndAH6-1glc. Looking back
through many of the other Examples of use of secreted nucleases during
lentiviral vector production, a similar band can sometimes be observed.
In this Example, the fold-concentration of vector supernatants was
greater than previous Examples, perhaps increasing the sensitivity of
detection of this band in this experiment. However, such a band is not
observed in purified vector material (See Examples 11 and 21), indicating
that this material unlikely to be within virions and can be removed by
further processing such as buffer-exchange. Given the extremely efficient
removal of all apparent Ethidium bromide stained contaminants by
VcEndAH6-1glc in 5 L bioreactor production scales demonstrated in Example
21, it seems likely that the occasional appearance of this band in
agarose gel analysis could be due to some variable and unidentified
contaminant--possibly free cellular/ribosomal RNA--which is generally not
detected within downstream-processed vector.
[0520] To be able to link the level of secreted nuclease achieved in both
helper cell and viral vector production cell cultures to unit-defined
nuclease activity, the culture supernatants were evaluated for nuclease
activity by DNAse Alert assay, and also by immunoblot to VcEndAH6-1glc
(anti-HisTag). A commercial Benzonase.RTM. enzymes stock of known
nuclease activity was used as standard curves within the assay. FIG. 31
displays this relationship, and enables this invention to describe
desirable secreted nuclease expression ranges in terms of objective
nuclease activity units. The nuclease activity (FIG. 31A) and immunoblot
(FIG. 31B) data correlated very well, not only between both assay types
but also for the proportion of nuclease helper cells spiked into the
vector production cultures. Importantly, the DNAse Alert assay was
validated as a highly accurate method because the control vector cultures
treated with 5 U/mL Benzonase.RTM. were calculated to contain an average
of 4.5 U/mL. The data show that for vector production cultures comprising
5% nuclease helper cells the activity of nuclease within the culture
media was .about.100 Benzonase.RTM. unit equivalents per mL. The nuclease
activity of the cultures comprising 1%, 0.5% and 0.1% nuclease helper
cells were 15.2, 8.8 and 2.1 Benzonase.RTM. unit equivalents per mL, in
good agreement with the proportional reduction in helper cells added to
the vector production culture. In this experiment, the `co-transfected`
vector production culture achieved 26.3 Benzonase.RTM. unit equivalents
per mL. Importantly, whilst the 0.1% nuclease helper cell-containing
vector production culture `only` achieved 2.1 Benzonase.RTM. unit
equivalents per mL compared to the `standard` vector cultures treated
with 5 U/mL of Benzonase.RTM. or SAN, the clearance of residual DNA was
at least as good as these standard treatments. This indicates that the
use of secreted nuclease holds advantage over use of commercial nucleases
not only from the point of view of achieving higher levels of nuclease
activity (at much reduced cost) but also that supplying a tonic level of
nuclease through-out vector production (even at lower levels) provides a
more efficacious mode of residual DNA clearance. Using the nuclease
activities measured within the vector production cultures, it is possible
to back-calculate the amount of secreted nuclease activity within the
helper culture; this was .about.2000 Benzonase.RTM. unit equivalents per
mL.
Example 21: Use of VcEndAH6-1Glc to Degrade Residual DNA with Serum-Free,
Suspension Bioreactors to a Level that Negates the Use of Further
Nuclease Treatment in the Downstream Process
[0521] Examples 10 and 11 show that the application of secreted nuclease
to lentiviral vector production cultures in mid-to-large scale suspension
bioreactors, aids in the clearance of residual DNA. The use of VsEndAH6
at 5 L scale suggested that this specific nuclease may be limited in
activity in cultures at lower pH because maximal clearance of residual
DNA in downstream process material was still aided by the use of
commercial nuclease added on the hollow fibre cartridge. This pH
sensitivity was later verified and led to the development of the
`pH-tolerant` VcEndAH6-1glc nuclease (Examples 15-17). To assess if the
VcEndAH6-1glc nuclease applied in the upstream phase could enable the
retraction of the commercial nuclease treatment during the same
downstream process (during dia-/ultra-filtration), lentiviral vector
encoding GFP was produced in the following manner. Suspension, serum-free
adapted HEK293T cells were seeded into two 5 L bioreactors transfected
with HIV-1 based lentiviral vector components (GFP expressing genome)
together with either pBluescript (control; for standard Benzonase.RTM.
treatment) or pSV40-VcEndAH6-1glc at 5% total plasmid input. Twenty hours
post-transfection, sodium butyrate was added to all bioreactors at a
final concentration of 10 mM. One hour prior to vector harvest, `In Bio`
samples were taken (cells removed and supernatant filtered), and then the
control bioreactor was inoculated with Benzonase.RTM. at 5 U/ml final
concentration together with 2 mM MgCl.sub.2. The pSV40-VcEndAH6-1glc
co-transfected bioreactor received only 2 mM MgCl.sub.2. Both bioreactors
were incubated for 1 hour under standard growth conditions prior to
harvest, where upon 5 L of the culture was clarified (10 .mu.m>0.45
.mu.m) and samples taken (CLH). Approximately 2.5 L of the harvests were
then subject to downstream processing. Material was subjected to
ion-exchange (IEX) chromatography, generating .about.215 mL IEX Eluate,
before further processing by dia-/ultra-filtration using hollow fibre
cartridges reduced this volume to 65-70 mL. The first step allowed buffer
exchange out of salt `HFF-Pre` (Pre-Benzonase.RTM. treatment)--where
samples were taken--and then 400 U/ml Benzonase.RTM. was added to both
the standard (`+Benzonase.RTM.`) processed vector and the VcEndAH6-1Igc
treated vector; this was done to assess any further benefit of an on-HFF
nuclease step, should the secreted-nuclease vector material require it.
Finally, buffer exchange occurred to remove Benzonase.RTM. (`HFF Post`).
FIG. 32A shows the pH profiles of the two bioreactors during the
production run, demonstrating that the target pH of 6.7 was achieved. The
clarified harvest vector (CLH) samples were titrated by FACS assay and
revealed titres of 1.7.times.10.sup.7 and 1.0.times.10.sup.7 TU/mL for
standard and VcEndAH6-1glc vectors, respectively. Samples from the whole
production process were concentrated by centrifugation on 3K cut-off
centrifugal devices before samples were loaded onto agarose gels
(Ethidium bromide) for residual DNA analysis (FIG. 32B). The residual DNA
analysis demonstrates excellent clearance of residual DNA using
VcEndAH6-1glc, and that the use of this nuclease in upstream production
essentially allows removal of the vast majority of contaminating residual
DNA at the dia-/ultra-filtration step--no further use of commercial
nuclease is required. To expand, whilst there is detectable residual DNA
within the VcEndAH6-1glc vector eluate from the IEX column, this is
apparently in a form that could be extracted from the vector material
during buffer exchange. Given that the dia-/ultra-filtration hollow fibre
pore size was 0.2 .mu.m, this indicated that the residual DNA that
co-eluted with the vector was smaller (or was within smaller protein
complexes) than the vector particles. Note also the presence of the
`Feed-specific` band (see Example 20) in unprocessed samples, which is
certainly lost at or before the dia-/ultra-filtration step. Residual DNA
could still be observed within the `HFF post` control (2.times. treated
Benzonase.RTM.) vector material. This represents for the first time the
ability to remove residual DNA from lentiviral vector production using a
nuclease approach that is not dependent on use of commercial, recombinant
nuclease.
Example 22: The Use of the KDEL Retention Signal to Enrich
VcEndAH6-1Glc-KDEL in Cell Fractions
[0522] Example 18 demonstrated the use of secreted and ER-retained
nucleases to clear residual DNA during upstream production of AAV
vectors. FIG. 33 displays SDS-PAGE analysis of harvest cell lysates
resulting from transfection of serum-free, suspension HEK293T cells with
either pSV40-VcEndAH6-1glc or pSV40-VcEndAH6-1glc-KDEL. The analysis
supports the previous work indicating that nuclease expression can be
directed such that the nuclease is retained/enriched within cells by use
of the ER-retention signal appended at the C-terminus of the nuclease
protein.
Materials and Methods
Nuclease Expression Constructs
[0523] Nuclease open-reading frames were codon and sequence optimized for
high expression in Homo sapiens by GeneArt. smNucA (NCBI Reference
Sequence: WP_047571650.1), VsEndA (GenBank: CAQ78235.1), VcEndA (NCBI
Reference Sequence: WP_000972597.1), BacNucB (NCBI Reference Sequence:
WP_003182220.1). All nucleases were evaluated by designing gene
expression cassettes that used the SV40 promoter and SV40 polyadenylation
signal (FIG. 3A-C). Typically, entire expression cassettes were
synthesized as one fragment encoding the
promoter-5'utr-nuclease-3'utr-polyA and received within GeneArt's pMK
backbone. The 3'utr regions were designed to harbor an alternative
C-terminus fused to a 6.times.His-tag, such that His-tagged variants of
each nuclease could be generated by simple restriction enzyme digestion
and re-ligation of the plasmid. Construction of ER signal peptide
variants and N.times.S/T sequon deletion/insertion variants were
typically carried out by cloning of re-derived, synthetic fragments
(GeneArt) into existing nuclease plasmids described above. Inducible
nuclease expression cassettes (FIG. 3D) were cloned by inserting the
nuclease ORF into a re-derived version pf pcDNA5/TO via HindIII/NotI
using standard cloning techniques. Broadly, digestions were carried out
at 37'C for 1-2 hours with enzyme (5-10 units per microgram of pDNA),
followed by a 10 minutes `Rapid` dephosphorylation reaction for backbone
fragments (Roche) and then run on a 1% agarose gel for 1 hour at 100V.
The relevant bands were extracted using the QIAQuick gel extraction kit
(QIAGEN). Ligations were carried out with backbone:insert ratios of 1:3
using the `Rapid` DNA ligation kit (Roche) in 20 .mu.L volumes under
recommended conditions. Approximately 25-50 .mu.L Turbo (NEB) or Stb12
(Thermo) competent cells were transformed with 2-3 .mu.L of ligation
reactions. Bacteria were grown on LBKAN agarose plates, and in LBKAN
media for minipreps. After restriction enzyme analysis and sequence
confirmation plasmid DNA was prepared by Plus Mega kit (QIAGEN).
Adherent Cell Culture, Transfection and Lentiviral Vector Production
[0524] HEK293T cells were used for vector production and titration. The
cells were maintained in complete media (Dulbecco's Modified Eagle Medium
(DMEM) (Sigma) supplemented with 10% heat-inactivated (FBS)(Gibco), 2 mM
L-glutamine (Sigma) and 1% Non-essential amino acids (NEAA) (Sigma)), at
37.degree. C. in 5% CO.sup.2.
[0525] The standard scale production of HIV-1 vectors in adherent mode was
in 10 cm dishes under the following conditions (all conditions were
scaled by area when performed in other formats): HEK293T cells were
seeded at 3.5.times.10.sup.5 cell per ml in complete media and
approximately 24 hrs later the cells were transfected using the following
mass ratios of plasmids per 10 cm plate: 4.5 pg Genome, 1.5 pg Gag-Pol,
1.1 pg Rev, 0.7 pg VSV-G (i.e. total of 7.8 pg/plate). Where appropriate,
nuclease expression plasmid was spiked into this vector mix at the
indicated % input of total pDNA (typical range from 0.001 to 10%).
[0526] Transfection was mediated by mixing DNA with Lipofectamine 2000CD
in Opti-MEM as per manufacturer's protocol (Life Technologies). Sodium
butyrate (Sigma) was added -18 hrs later to 10 mM final concentration for
5-6 h, before 10 ml fresh serum-free media replaced the transfection
media. Typically, vector supernatant was harvested 20-24 h later, and
then filtered (0.22 .mu.m) and frozen at -20/-80.degree. C. As a positive
control for nuclease treatment, typically Benzonase.RTM. was added to the
harvests at 5 U/mL for 1 hour prior to filtration.
[0527] HEK293T.GFP.PrCL (stable cell line) was developed in-house using
the established HEK293T.tetR14 adherent cell line. All the packaging
components and the HIV-GFP genome were previously stably transfected into
HEK293T.TetR cells along with selection markers. Rev and VSV-G were
placed under the selection of Zeocin (Zeo), Gag-Pol was under the
selection of Blasticidin (Bsr) and the GFP genome was under the selection
of Hygromycin (Hyg). For evaluation of the secreted nuclease approach
within HIV-1 based vector producer cell lines, nuclease-expression
plasmids were transfected into cultures at the indicated % input of total
(where pBlueScript was added as stuff to a total of 7.8 .mu.g/10 cm plate
or equivalent scaled amount/area). To induce vector production,
doxycycline was added from the Sodium butyrate induction step at a final
concentration of 1 pg/mL.
Suspension Cell Culture, Transfection and Lentiviral Vector Production
[0528] HEK293T.TetR14S (stably integrated codon-optimised tetR) and
HEK293T.1-65s suspension cells were grown in Freestyle+0.1% CLC (Gibco)
at 37.degree. C. in 5% CO.sup.2, in a shaking incubator (25 mm orbit set
at 190 RPM). All vector production using suspension was carried out in a
125 ml shake flasks at a working volume of 25 ml or in AMBR-15
bioreactors at a working volume of 12 mL. HEK293 Ts cells were seeded at
8.times.10.sup.5 cells per ml in serum-free media and were incubated at
37.degree. C. in 5% CO2, shaking, through-out vector production. For
AMBR-15 bioreactor production, the cultures were set to pH7.2.
Approximately 24 hrs after seeding the cells were transfected with a mix
of vector component plasmids encoding genome (GFP), gagpol, rev and VSVG.
Where appropriate, nuclease expression plasmid was spiked into this
vector mix at the indicated % input of total pDNA (typical range from 0.1
to 10%).
[0529] Transfection was mediated by mixing DNA with Lipofectamine 2000CD
in Opti-MEM as per manufacturer's protocol (Life Technologies). For
AMBR-15 bioreactor production, the cultures were set to pH6.6 or pH7.2
immediately post-transfection. Sodium butyrate (Sigma) was added
.about.18 hrs later to 10 mM final concentration. Typically, vector
supernatant was harvested 20-24 h later, and then filtered (0.22 .mu.m)
and frozen at -20/-80.degree. C. As a positive control for nuclease
treatment, typically Benzonase.RTM. or SAN was added to the harvests at 5
U/mL for 1 hour prior to filtration. For AMBR-15 bioreactor production,
pH and cell viability was monitored through-out the process.
Production of Lentiviral Vectors in 0.5 or 5 L Stir-Tank Bioreactors and
Downstream Processing
[0530] The suspension adapted HEK293T cell line, clone 1.65s and
supplemented serum-free media were used for the bioreactors studies.
Cells cultured from the MCB were revived and scaled-up in Erlenmeyer
flasks, and incubated in a pre-equilibrated 5% CO.sub.2 in air incubator
at 37.degree. C. with a humidified atmosphere. The cells were kept in
suspension on an orbital shaker platform.
[0531] Bioreactors were actively managed throughout for dissolved oxygen
and pH; metabolite and by-product levels were also monitored. Cell
viability was monitored, and antifoam was used if appropriate. Bioreactor
cultures were co-transfected with four plasmids; pHIV-CMV-GFP (genome),
pSynGPK (gagpol), pOB-Rev and pOB-VSVG, using a transfection reagent.
Where indicated, pNuclease was typically spiked into pDNA mixes at 5% of
total. The bioreactor cultures were induced 20 hours post transfection
with 10 mM of sodium butyrate. Vector harvests were taken 24 hours
post-induction, with optional treatment with 5 U/mL or 25 U/mL commercial
nucleases (Benzonase.RTM. or SAN-HQ) for 1 hour prior to harvest were
indicated.
[0532] Vector supernatants were pre-clarified (10 .mu.m) and then further
clarified by 0.22-0.45 .mu.m filtration. At least 1 L clarified vector
harvests were processed by ion-exchange chromatography using Sarto-Q
cartridges, typically including low concentration salt wash and
pre-elution wash buffers, before elution in >1M NaCl-containing buffer
and immediate dilution of the eluate to preserve vector virion activity.
Eluate was then quickly subjected to Hollow Fibre dia/ultrafiltration to
first buffer exchange into low salt conditions, before 400 U/mL
commercial nuclease and 2 mM magnesium chloride was added as a second
nuclease treatment polishing step. Finally, a final buffer exchange was
performed to remove the commercial nuclease.
Generation of Nuclease Helper Cells by Transient Transfection Achieving
Maximal Nuclease Expression
[0533] The suspension adapted, serum-free HEK293T cells were seeded at
8.times.10.sup.5 cell per mL in parallel to the main viral vector
production cultures and grown overnight in Freestyle+0.1% CLC (Gibco) at
37.degree. C. in 5% CO.sub.2. The next day cells were transfected with
>750ng pCMV-VcEndAH6-1glc per mL of culture using Lipofectamine 2000CD
(vector production cultures were transfected with vector components). At
the point of sodium butyrate induction (10 mM final), nuclease helper
cells were inoculated at the target cell input levels to achieve the
stated percentage of total cell count in the main vector production
vessel. Vector production then continued as described above.
Lentiviral Vector Titration Assays
[0534] For lentiviral vector titration, HEK293T cells were seeded at
1.2.times.10.sup.4 cells/well in 96-well plates. GFP-encoding viral
vectors were used to transduce the cells in complete media containing 8
mg/ml polybrene and 1.times. Penicillin Streptomycin for approximately
5-6 hrs after which fresh media was added. The transduced cells were
incubated for 2 days at 37.degree. C. in 5% CO.sup.2. Cultures were then
prepared for flow cytometry by FACSVerse (BD Biosciences) percent GFP
expression was measured and vector titres were estimated using predicted
cell count following the 2-day incubation.
AAV Vector Production
[0535] For AAV vector production, HEK293T-1.65S serum-free, suspension
adapted cells were seeded into culture vessels at 8.times.10.sup.5
cells/mL -20 hours prior to transfection. At transfection,
self-complementary (sc) AAV-GFP vector production was initiated by
transfecting cells with equal masses of pscAAV-CMV-GFP (genome), pRepCap2
(encoding all necessary viral packaging components, including
assembly-activating protein [AAP]) and pHelper (Adeno E2A, E4 and VA RNA
functions), together with either nuclease encoding plasmids or
pBluescript (negative control) at 5% mass of total pDNA. Transfection was
mediated by mixing DNA with Lipofectamine 2000CD in serum-free media as
per manufacturer's protocol (Life Technologies). After two days, cells
were harvested by centrifugation at low speed and cells lysed by three
freeze-thaw cycles in the presence of detergent lysis buffer. Next, the
lysate was pipetted up-and-down in 8 cell pellet volumes of serum-free
DMEM. Magnesium Chloride was added to lysate at a final concentration of
2 mM, and incubated for 1 hour at 37.degree. C. For the `commercial`
positive control, salt active nuclease (SAN-HQ; Articzymes) was added at
25 U per 0.5 mL effective cell pellet volume of a replicate Negative
control lysate and incubated at 1 hour at 37.degree. C. in the presence
of 2 mM MgCl.sub.2. Negative control lysate was incubated without
nuclease. Treated cell lysates were centrifuged at low speed to remove
debris, and then supernatants filtered (0.45 .mu.m) prior to storage at
-20.degree. C..ltoreq.prior to further analysis.
AAV Vector Titration Assays
[0536] For AAV vector titration, HEK293T cells were seeded at
1.2.times.10.sup.4 cells/well in 96-well plates. GFP-encoding viral
vectors were used to transduce the cells in serum-free media containing
1.times. Penicillin Streptomycin for approximately 5 hrs after which
fresh, serum-containing media was added. The transduced cells were
incubated for 3 days at 37.degree. C. in 5% CO.sup.2. Cultures were then
prepared for flow cytometry by Attune N.times.T (Thermofisher); percent
GFP expression was measured and vector titres were estimated using cell
counts and vector dilutions.
Residual DNA Detection by PicoGreen.RTM. Assay
[0537] Quant-iT.TM. PicoGreen.RTM. dsDNA Assay Kit (Life Technologies) was
used to assay the residual DNA in harvested vector material. Lambda stock
DNA was serially diluted in Tris and EDTA (TE) buffer to prepare a DNA
standard. TE buffer was also used to dilute harvested vector samples 1:10
before adding the PicoGreen assay reagent (prepared as per the
manufacturer's protocol) and incubating the samples, in the dark, for 2
to 5 mins. The fluorescence of the PicoGreen reagent, in the samples, was
measured with top read using an EM Microplate Reader (Gemini).
Residual DNA Detection by Quantitative PCR Assay
[0538] DNA was extracted from vector samples using a QIAamp DNA Mini Kit
(QIAGEN). During the extraction process, mock samples, containing
nuclease free water (NFW)(Gibco) were used as in process controls (IPCs).
[0539] Extracted DNA was subjected to 18S quantitative Polymerase Chain
Reaction (PCR) (qPCR); the reaction amplified a short fragment of cell
derived 18S DNA. The mix included SYBR.RTM. Green PCR Mastermix (Life
Technologies), the 18S primers (prepared in 10 mM Tris-HCl, pH7) (Sigma)
and NFW.
TABLE-US-00009
The Forward primer:
5' ACCGCAGCTAGGAATAATGG 3'
The Reverse primer:
5' CTCAGTTCCGAAAACCAACA 3'
[0540] To generate a DNA standard, linearised 18S plasmid was serially
diluted from 1.times.10.sup.6 copies/5 .mu.l to 1.times.10.sup.2 copies/5
.mu.l. Samples were diluted 1:5 before the reaction mix was added. The
reaction was carried out under standard chemistry qPCR conditions using
QuantStudio.TM. 6 Flex System (Life Technologies). The qPCR run was set
up and analysed the using QuantStudio.TM. 6 Flex System Software (Life
Technologies).
[0541] The controls set up include a no template control (NTC), to control
for PRC reaction mix contamination, a IPC sample, to control for DNA
contamination during the extraction process and finally, to monitor PCR
inhibition, a spiked PCR inhibition control (SPIC) was included by
spiking 1.times.104 copies of 18S standard DNA into the IPC sample.
[0542] Extracted DNA was subjected to KanR quantitative Polymerase Chain
Reaction (PCR) (qPCR); the reaction amplified a short fragment of plasmid
derived KanR DNA. The mix included TaqMan.RTM. Universal PCR Master Mix
(Life Technologies), the KanR primer and probe set (prepared in 10 mM
Tris-HCl, pH7) (Sigma) and NFW.
TABLE-US-00010
The Forward primer:
5' AGATGGATTGCACGCAGGTT 3'
The Reverse primer:
5' TGCCCAGTCATAGCCGAATAG 3'
Probe:
5' (FAM) CTCCACCCAAGCGGCCGGA (TAMRA) 3'
[0543] To generate a DNA standard, linearised KanR+ plasmid was serially
diluted from 1.times.10.sup.6 copies/5 .mu.l to 1.times.10.sup.2 copies/5
.mu.l. Samples were diluted 1:5 before the reaction mix was added. The
reaction was carried out under standard chemistry qPCR conditions using
QuantStudio.TM. 6 Flex System (Life Technologies). The qPCR run was set
up and analysed the using QuantStudio.TM. 6 Flex System Software (Life
Technologies).
[0544] The controls set up include a no template control (NTC), to control
for PRC reaction mix contamination, a IPC sample, to control for DNA
contamination during the extraction process and finally, to monitor PCR
inhibition, a spiked PCR inhibition control (SPIC) was included by
spiking 1.times.104 copies of KanR standard DNA into the IPC sample.
Visualisation of Residual DNA Detection by Gel Electrophoresis
[0545] LV crude vector supernatants were filter-clarified and optionally
treated with Proteinase-K at 37.degree. C. for 1 hour. Clarified samples
were centrifuged in Amicon Ultra-15 3K cut-off filter units to
concentrated DNA by up to 150-fold. Samples were subjected to standard
electrophoresis on 2% agarose/TBE gels (DNA visualised by Ethidium
bromide) and 1 kbp/100 bp ladders run in parallel.
Quantification of Nuclease Activity by DNAse Alert.TM. Assay (Referred to
Herein as "Assay 1")
[0546] The DNAse Alert.TM. assay (IDT) can be, and was, used to quantify
nuclease activity within viral vector culture media. The basis of the
assay is the use of fluorescently labelled and quenched nucleotide probes
that emit a fluorescence signal only when degraded by a nuclease. The kit
was used under manufacturers recommendations, using a standard curve
composed of serially diluted Benzonase.RTM. from 5 U/mL to 0.08 U/mL. The
viral vector culture supernatant containing secreted nuclease should be
serially diluted such that the fluorescence activity falls within the
upper and lower limits of the standard curve. The unit definition of
Benzonase.RTM. (at the time of this work) is: "One unit will digest
sonicated salmon sperm DNA to acid-soluble oligonucleotides equivalent to
a .DELTA.A260 of 1.0 in 30 min at pH 8.0 at 37.degree. C. (reaction
volume 2.625 ml)". It is not expected that this unit definition will
change in the future, and so commercially obtainable Benzonase.RTM. that
has been QC checked against this standard unit activity can be used in
the DNAse Alert assay to verify secreted nuclease activity achieved in
the invention. Should for any reason suppliers of Benzonase.RTM. alter
the unit definition then it is expected that the relationship between
`old` and `new` unit definitions will be known, and therefore also
applied to secreted nuclease activity reported and claimed within this
invention.
[0547] All publications mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of the
described methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments.
Sequence CWU
1
1
261231PRTVibrio cholerae 1Met Met Ile Phe Arg Phe Val Thr Thr Leu Ala Ala
Ser Leu Pro Leu1 5 10
15Leu Thr Phe Ala Ala Pro Ile Ser Phe Ser His Ala Lys Asn Glu Ala
20 25 30Val Lys Ile Tyr Arg Asp His
Pro Val Ser Phe Tyr Cys Gly Cys Glu 35 40
45Ile Arg Trp Gln Gly Lys Lys Gly Ile Pro Asp Leu Glu Ser Cys
Gly 50 55 60Tyr Gln Val Arg Lys Asn
Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65 70
75 80His Val Val Pro Ala Trp Gln Phe Gly His Gln
Leu Gln Cys Trp Gln 85 90
95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr Ser Pro Glu Phe Asn Gln
100 105 110Met Glu Ala Asp Leu His
Asn Leu Thr Pro Ala Ile Gly Glu Val Asn 115 120
125Gly Asn Arg Ser Asn Phe Ser Phe Ser Gln Trp Asn Gly Ile
Asp Gly 130 135 140Val Thr Tyr Gly Gln
Cys Glu Met Gln Val Asn Phe Lys Glu Arg Thr145 150
155 160Ala Met Pro Pro Glu Arg Ala Arg Gly Ala
Ile Ala Arg Thr Tyr Leu 165 170
175Tyr Met Ser Glu Gln Tyr Gly Leu Arg Leu Ser Lys Ala Gln Asn Gln
180 185 190Leu Met Gln Ala Trp
Asn Asn Gln Tyr Pro Val Ser Glu Trp Glu Cys 195
200 205Val Arg Asp Gln Lys Ile Glu Lys Val Gln Gly Asn
Ser Asn Arg Phe 210 215 220Val Arg Glu
Gln Cys Pro Asn225 2302234PRTVibrio salmonicida 2Met Lys
Leu Ile Arg Leu Val Ile Ser Leu Ile Ala Val Ser Phe Thr1 5
10 15Val Asn Val Met Ala Ala Pro Pro
Ser Ser Phe Ser Lys Ala Lys Lys 20 25
30Glu Ala Val Lys Ile Tyr Leu Asp Tyr Pro Thr Ser Phe Tyr Cys
Gly 35 40 45Cys Asp Ile Thr Trp
Lys Asn Lys Lys Lys Gly Ile Pro Glu Leu Glu 50 55
60Ser Cys Gly Tyr Gln Val Arg Lys Gln Glu Lys Arg Ala Ser
Arg Ile65 70 75 80Glu
Trp Glu His Val Val Pro Ala Trp Gln Phe Gly His Gln Arg Gln
85 90 95Cys Trp Gln Lys Gly Gly Arg
Lys Asn Cys Thr Arg Asn Asp Lys Gln 100 105
110Phe Lys Ser Met Glu Ala Asp Leu His Asn Leu Val Pro Ala
Ile Gly 115 120 125Glu Val Asn Gly
Asp Arg Ser Asn Phe Arg Phe Ser Gln Trp Asn Gly 130
135 140Ser Lys Gly Ala Phe Tyr Gly Gln Cys Ala Phe Lys
Val Asp Phe Lys145 150 155
160Gly Arg Val Ala Glu Pro Pro Ala Gln Ser Arg Gly Ala Ile Ala Arg
165 170 175Thr Tyr Leu Tyr Met
Asn Asn Glu Tyr Lys Phe Asn Leu Ser Lys Ala 180
185 190Gln Arg Gln Leu Met Glu Ala Trp Asn Lys Gln Tyr
Pro Val Ser Thr 195 200 205Trp Glu
Cys Thr Arg Asp Glu Arg Ile Ala Lys Ile Gln Gly Asn His 210
215 220Asn Gln Phe Val Tyr Lys Ala Cys Thr Lys225
2303266PRTSerratia marcescens 3Met Arg Phe Asn Asn Lys Met
Leu Ala Leu Ala Ala Leu Leu Phe Ala1 5 10
15Ala Gln Ala Ser Ala Asp Thr Leu Glu Ser Ile Asp Asn
Cys Ala Val 20 25 30Gly Cys
Pro Thr Gly Gly Ser Ser Asn Val Ser Ile Val Arg His Ala 35
40 45Tyr Thr Leu Asn Asn Asn Ser Thr Thr Lys
Phe Ala Asn Trp Val Ala 50 55 60Tyr
His Ile Thr Lys Asp Thr Pro Ala Ser Gly Lys Thr Arg Asn Trp65
70 75 80Lys Thr Asp Pro Ala Leu
Asn Pro Ala Asp Thr Leu Ala Pro Ala Asp 85
90 95Tyr Thr Gly Ala Asn Ala Ala Leu Lys Val Asp Arg
Gly His Gln Ala 100 105 110Pro
Leu Ala Ser Leu Ala Gly Val Ser Asp Trp Glu Ser Leu Asn Tyr 115
120 125Leu Ser Asn Ile Thr Pro Gln Lys Ser
Asp Leu Asn Gln Gly Ala Trp 130 135
140Ala Arg Leu Glu Asp Gln Glu Arg Lys Leu Ile Asp Arg Ala Asp Ile145
150 155 160Ser Ser Val Tyr
Thr Val Thr Gly Pro Leu Tyr Glu Arg Asp Met Gly 165
170 175Lys Leu Pro Gly Thr Gln Lys Ala His Thr
Ile Pro Ser Ala Tyr Trp 180 185
190Lys Val Ile Phe Ile Asn Asn Ser Pro Ala Val Asn His Tyr Ala Ala
195 200 205Phe Leu Phe Asp Gln Asn Thr
Pro Lys Gly Ala Asp Phe Cys Gln Phe 210 215
220Arg Val Thr Val Asp Glu Ile Glu Lys Arg Thr Gly Leu Ile Ile
Trp225 230 235 240Ala Gly
Leu Pro Asp Asp Val Gln Ala Ser Leu Lys Ser Lys Pro Gly
245 250 255Val Leu Pro Glu Leu Met Gly
Cys Lys Asn 260 2654142PRTBacillus
licheniformis 4Met Ile Lys Lys Trp Ala Val His Leu Leu Phe Ser Ala Leu
Val Leu1 5 10 15Leu Gly
Leu Ser Gly Gly Ala Ala Tyr Ser Pro Gln His Ala Glu Gly 20
25 30Ala Ala Arg Tyr Asp Asp Ile Leu Tyr
Phe Pro Ala Ser Arg Tyr Pro 35 40
45Glu Thr Gly Ala His Ile Ser Asp Ala Ile Lys Ala Gly His Ser Asp 50
55 60Val Cys Thr Ile Glu Arg Ser Gly Ala
Asp Lys Arg Arg Gln Glu Ser65 70 75
80Leu Lys Gly Ile Pro Thr Lys Pro Gly Phe Asp Arg Asp Glu
Trp Pro 85 90 95Met Ala
Met Cys Glu Glu Gly Gly Lys Gly Ala Ser Val Arg Tyr Val 100
105 110Ser Ser Ser Asp Asn Arg Gly Ala Gly
Ser Trp Val Gly Asn Arg Leu 115 120
125Ser Gly Phe Ala Asp Gly Thr Arg Ile Leu Phe Ile Val Gln 130
135 1405231PRTVibrio cholerae 5Met Met Ile Phe
Arg Phe Val Thr Thr Leu Ala Ala Ser Leu Pro Leu1 5
10 15Leu Thr Phe Ala Ala Pro Ile Ser Phe Ser
His Ala Lys Asn Glu Ala 20 25
30Val Lys Ile Tyr Arg Asp His Pro Val Ser Phe Tyr Cys Gly Cys Glu
35 40 45Ile Arg Trp Gln Gly Lys Lys Gly
Ile Pro Asp Leu Glu Ser Cys Gly 50 55
60Tyr Gln Val Arg Lys Asn Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65
70 75 80His Val Val Pro Ala
Trp Gln Phe Gly His Gln Leu Gln Cys Trp Gln 85
90 95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr Ser
Pro Glu Phe Asn Gln 100 105
110Met Glu Ala Asp Leu His Asn Leu Thr Pro Ala Ile Gly Glu Val Asn
115 120 125Gly Asp Arg Ser Asn Phe Arg
Phe Ser Gln Trp Asn Gly Ile Asp Gly 130 135
140Val Thr Tyr Gly Gln Cys Glu Met Gln Val Asn Phe Lys Glu Arg
Thr145 150 155 160Ala Met
Pro Pro Glu Arg Ala Arg Gly Ala Ile Ala Arg Thr Tyr Leu
165 170 175Tyr Met Ser Glu Gln Tyr Gly
Leu Arg Leu Ser Lys Ala Gln Asn Gln 180 185
190Leu Met Gln Ala Trp Asn Asn Gln Tyr Pro Val Ser Glu Trp
Glu Cys 195 200 205Val Arg Asp Gln
Lys Ile Glu Lys Val Gln Gly Asn Ser Asn Arg Phe 210
215 220Val Arg Glu Gln Cys Pro Asn225
2306231PRTVibrio cholerae 6Met Met Ile Phe Arg Phe Val Thr Thr Leu Ala
Ala Ser Leu Pro Leu1 5 10
15Leu Thr Phe Ala Ala Pro Ile Ser Phe Ser His Ala Lys Asn Glu Ala
20 25 30Val Lys Ile Tyr Arg Asp His
Pro Val Ser Phe Tyr Cys Gly Cys Glu 35 40
45Ile Arg Trp Gln Gly Lys Lys Gly Ile Pro Asp Leu Glu Ser Cys
Gly 50 55 60Tyr Gln Val Arg Lys Asn
Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65 70
75 80His Val Val Pro Ala Trp Gln Phe Gly His Gln
Leu Gln Cys Trp Gln 85 90
95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr Ser Pro Glu Phe Asn Gln
100 105 110Met Glu Ala Asp Leu His
Asn Leu Thr Pro Ala Ile Gly Glu Val Asn 115 120
125Gly Asn Arg Ser Asn Phe Arg Phe Ser Gln Trp Asn Gly Ile
Asp Gly 130 135 140Val Thr Tyr Gly Gln
Cys Glu Met Gln Val Asn Phe Lys Glu Arg Thr145 150
155 160Ala Met Pro Pro Glu Arg Ala Arg Gly Ala
Ile Ala Arg Thr Tyr Leu 165 170
175Tyr Met Ser Glu Gln Tyr Gly Leu Arg Leu Ser Lys Ala Gln Asn Gln
180 185 190Leu Met Gln Ala Trp
Asn Asn Gln Tyr Pro Val Ser Glu Trp Glu Cys 195
200 205Val Arg Asp Gln Lys Ile Glu Lys Val Gln Gly Asn
Ser Asn Arg Phe 210 215 220Val Arg Glu
Gln Cys Pro Asn225 2307231PRTVibrio cholerae 7Met Met Ile
Phe Arg Phe Val Thr Thr Leu Ala Ala Ser Leu Pro Leu1 5
10 15Leu Thr Phe Ala Ala Pro Ile Ser Phe
Ser His Ala Lys Asn Glu Ala 20 25
30Val Lys Ile Tyr Arg Asp His Pro Val Ser Phe Tyr Cys Gly Cys Glu
35 40 45Ile Arg Trp Gln Gly Lys Lys
Gly Ile Pro Asp Leu Glu Ser Cys Gly 50 55
60Tyr Gln Val Arg Lys Asn Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65
70 75 80His Val Val Pro
Ala Trp Gln Phe Gly His Gln Leu Gln Cys Trp Gln 85
90 95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr
Ser Pro Glu Phe Asn Gln 100 105
110Met Glu Ala Asp Leu His Asn Leu Thr Pro Ala Ile Gly Glu Val Asn
115 120 125Gly Asp Arg Ser Asn Phe Ser
Phe Ser Gln Trp Asn Gly Ile Asp Gly 130 135
140Val Thr Tyr Gly Gln Cys Glu Met Gln Val Asn Phe Lys Glu Arg
Thr145 150 155 160Ala Met
Pro Pro Glu Arg Ala Arg Gly Ala Ile Ala Arg Thr Tyr Leu
165 170 175Tyr Met Ser Glu Gln Tyr Gly
Leu Arg Leu Ser Lys Ala Gln Asn Gln 180 185
190Leu Met Gln Ala Trp Asn Asn Gln Tyr Pro Val Ser Glu Trp
Glu Cys 195 200 205Val Arg Asp Gln
Lys Ile Glu Lys Val Gln Gly Asn Ser Asn Arg Phe 210
215 220Val Arg Glu Gln Cys Pro Asn225
2308231PRTVibrio cholerae 8Met Met Ile Phe Arg Phe Val Thr Thr Leu Ala
Ala Ser Leu Pro Leu1 5 10
15Leu Thr Phe Ala Ala Pro Ile Ser Phe Ser His Ala Lys Asn Glu Ala
20 25 30Val Lys Ile Tyr Arg Asp His
Pro Val Ser Phe Tyr Cys Gly Cys Glu 35 40
45Ile Arg Trp Gln Gly Lys Lys Gly Ile Pro Asp Leu Glu Ser Cys
Gly 50 55 60Tyr Gln Val Arg Lys Asn
Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65 70
75 80His Val Val Pro Ala Trp Gln Phe Gly His Gln
Leu Gln Cys Trp Gln 85 90
95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr Ser Pro Glu Phe Asn Gln
100 105 110Met Glu Ala Asp Leu His
Asn Leu Val Pro Ala Ile Gly Glu Val Asn 115 120
125Gly Asn Arg Ser Asn Phe Ser Phe Ser Gln Trp Asn Gly Ile
Asp Gly 130 135 140Val Thr Tyr Gly Gln
Cys Glu Met Gln Val Asn Phe Lys Glu Arg Thr145 150
155 160Ala Met Pro Pro Glu Arg Ala Arg Gly Ala
Ile Ala Arg Thr Tyr Leu 165 170
175Tyr Met Ser Glu Gln Tyr Gly Leu Arg Leu Ser Lys Ala Gln Asn Gln
180 185 190Leu Met Gln Ala Trp
Asn Asn Gln Tyr Pro Val Ser Glu Trp Glu Cys 195
200 205Val Arg Asp Gln Lys Ile Glu Lys Val Gln Gly Asn
Ser Asn Arg Phe 210 215 220Val Arg Glu
Gln Cys Pro Asn225 2309231PRTVibrio cholerae 9Met Met Ile
Phe Arg Phe Val Thr Thr Leu Ala Ala Ser Leu Pro Leu1 5
10 15Leu Thr Phe Ala Ala Pro Ile Ser Phe
Ser His Ala Lys Asn Glu Ala 20 25
30Val Lys Ile Tyr Arg Asp His Pro Val Ser Phe Tyr Cys Gly Cys Glu
35 40 45Ile Arg Trp Gln Gly Lys Lys
Gly Ile Pro Asp Leu Glu Ser Cys Gly 50 55
60Tyr Gln Val Arg Lys Asn Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65
70 75 80His Val Val Pro
Ala Trp Gln Phe Gly His Gln Leu Gln Cys Trp Gln 85
90 95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr
Ser Pro Glu Phe Asn Gln 100 105
110Met Glu Ala Asp Leu His Asn Leu Val Pro Ala Ile Gly Glu Val Asn
115 120 125Gly Asn Arg Ser Asn Phe Arg
Phe Ser Gln Trp Asn Gly Ile Asp Gly 130 135
140Val Thr Tyr Gly Gln Cys Glu Met Gln Val Asn Phe Lys Glu Arg
Thr145 150 155 160Ala Met
Pro Pro Glu Arg Ala Arg Gly Ala Ile Ala Arg Thr Tyr Leu
165 170 175Tyr Met Ser Glu Gln Tyr Gly
Leu Arg Leu Ser Lys Ala Gln Asn Gln 180 185
190Leu Met Gln Ala Trp Asn Asn Gln Tyr Pro Val Ser Glu Trp
Glu Cys 195 200 205Val Arg Asp Gln
Lys Ile Glu Lys Val Gln Gly Asn Ser Asn Arg Phe 210
215 220Val Arg Glu Gln Cys Pro Asn225
23010231PRTVibrio cholerae 10Met Met Ile Phe Arg Phe Val Thr Thr Leu Ala
Ala Ser Leu Pro Leu1 5 10
15Leu Thr Phe Ala Ala Pro Ile Ser Phe Ser His Ala Lys Asn Glu Ala
20 25 30Val Lys Ile Tyr Arg Asp His
Pro Val Ser Phe Tyr Cys Gly Cys Glu 35 40
45Ile Arg Trp Gln Gly Lys Lys Gly Ile Pro Asp Leu Glu Ser Cys
Gly 50 55 60Tyr Gln Val Arg Lys Asn
Glu Asn Arg Ala Ser Arg Ile Glu Trp Glu65 70
75 80His Val Val Pro Ala Trp Gln Phe Gly His Gln
Leu Gln Cys Trp Gln 85 90
95Gln Gly Gly Arg Lys Asn Cys Thr Arg Thr Ser Pro Glu Phe Asn Gln
100 105 110Met Glu Ala Asp Leu His
Asn Leu Val Pro Ala Ile Gly Glu Val Asn 115 120
125Gly Asp Arg Ser Asn Phe Ser Phe Ser Gln Trp Asn Gly Ile
Asp Gly 130 135 140Val Thr Tyr Gly Gln
Cys Glu Met Gln Val Asn Phe Lys Glu Arg Thr145 150
155 160Ala Met Pro Pro Glu Arg Ala Arg Gly Ala
Ile Ala Arg Thr Tyr Leu 165 170
175Tyr Met Ser Glu Gln Tyr Gly Leu Arg Leu Ser Lys Ala Gln Asn Gln
180 185 190Leu Met Gln Ala Trp
Asn Asn Gln Tyr Pro Val Ser Glu Trp Glu Cys 195
200 205Val Arg Asp Gln Lys Ile Glu Lys Val Gln Gly Asn
Ser Asn Arg Phe 210 215 220Val Arg Glu
Gln Cys Pro Asn225 23011231PRTVibrio cholerae 11Met Met
Ile Phe Arg Phe Val Thr Thr Leu Ala Ala Ser Leu Pro Leu1 5
10 15Leu Thr Phe Ala Ala Pro Ile Ser
Phe Ser His Ala Lys Asn Glu Ala 20 25
30Val Lys Ile Tyr Arg Asp His Pro Val Ser Phe Tyr Cys Gly Cys
Glu 35 40 45Ile Arg Trp Gln Gly
Lys Lys Gly Ile Pro Asp Leu Glu Ser Cys Gly 50 55
60Tyr Gln Val Arg Lys Asn Glu Asn Arg Ala Ser Arg Ile Glu
Trp Glu65 70 75 80His
Val Val Pro Ala Trp Gln Phe Gly His Gln Leu Gln Cys Trp Gln
85 90 95Gln Gly Gly Arg Lys Asn Cys
Thr Arg Thr Ser Pro Glu Phe Asn Gln 100 105
110Met Glu Ala Asp Leu His Asn Leu Val Pro Ala Ile Gly Glu
Val Asn 115 120 125Gly Asp Arg Ser
Asn Phe Arg Phe Ser Gln Trp Asn Gly Ile Asp Gly 130
135 140Val Thr Tyr Gly Gln Cys Glu Met Gln Val Asn Phe
Lys Glu Arg Thr145 150 155
160Ala Met Pro Pro Glu Arg Ala Arg Gly Ala Ile Ala Arg Thr Tyr Leu
165 170 175Tyr Met Ser Glu Gln
Tyr Gly Leu Arg Leu Ser Lys Ala Gln Asn Gln 180
185 190Leu Met Gln Ala Trp Asn Asn Gln Tyr Pro Val Ser
Glu Trp Glu Cys 195 200 205Val Arg
Asp Gln Lys Ile Glu Lys Val Gln Gly Asn Ser Asn Arg Phe 210
215 220Val Arg Glu Gln Cys Pro Asn225
2301221PRTArtificial Sequencesignal peptide 12Met Trp Trp Arg Leu Trp
Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp1 5
10 15Pro Met Val Trp Ala 201320PRTMus
musculus 13Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val
Pro1 5 10 15Gly Ser Thr
Gly 201422PRTHomo sapiens 14Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp1 5 10
15Leu Arg Gly Ala Arg Cys 201516PRTHomo sapiens
15Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala1
5 10 151623PRTHomo sapiens
16Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1
5 10 15Ala Val Phe Val Ser Pro
Ser 201716PRTHomo sapiens 17Met Lys Trp Val Thr Phe Ile Ser
Leu Leu Phe Ser Ser Ala Tyr Ser1 5 10
151816PRTVesicular stomatitis virus 18Met Lys Cys Leu Leu
Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys1 5
10 15194PRTArtificial Sequenceretention
signalmisc_feature(1)..(1)Xaa is Lys, Arg, His, Gln, Ser,
Alamisc_feature(2)..(2)Xaa is Asp, Glu, Asn or Gln 19Xaa Xaa Glu
Leu1204PRTArtificial Sequenceretention signalmisc_feature(3)..(4)Xaa can
be any naturally occurring amino acid 20Lys Lys Xaa Xaa1214PRTArtificial
Sequenceretention signal 21Lys Asp Glu Leu12220DNAArtificial
Sequenceprimer 22accgcagcta ggaataatgg
202320DNAArtificial Sequenceprimer 23ctcagttccg aaaaccaaca
202420DNAArtificial
Sequenceprimer 24agatggattg cacgcaggtt
202521DNAArtificial Sequenceprimer 25tgcccagtca tagccgaata g
212619DNAArtificial
Sequenceprobe 26ctccacccaa gcggccgga
19
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