U.S. patent application number 11/187319 was filed with the patent office on 2006-12-07 for novel method for the protection and purification of adenoviral vectors.
This patent application is currently assigned to INTROGEN THERAPEUTICS INC.. Invention is credited to Peter Clarke, Hai Pham, Shuyuan Zhang.
Application Number | 20060275781 11/187319 |
Document ID | / |
Family ID | 36336919 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060275781 |
Kind Code |
A1 |
Pham; Hai ; et al. |
December 7, 2006 |
Novel method for the protection and purification of adenoviral
vectors
Abstract
The present invention relates to improved methods for producing
adenovirus compositions wherein host cells are grown in a
bioreactor and purified by size partitioning purification to
provide purified adenovirus compositions.
Inventors: |
Pham; Hai; (Houston, TX)
; Zhang; Shuyuan; (Sugar Land, TX) ; Clarke;
Peter; (Sugar Land, TX) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
INTROGEN THERAPEUTICS INC.
Austin
TX
|
Family ID: |
36336919 |
Appl. No.: |
11/187319 |
Filed: |
July 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60624627 |
Nov 3, 2004 |
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/5 |
Current CPC
Class: |
C12N 7/00 20130101; A61K
48/00 20130101; C12N 2710/10351 20130101; C12Q 1/70 20130101; C12N
15/86 20130101; C12N 15/1017 20130101; C12N 2710/10343
20130101 |
Class at
Publication: |
435/006 ;
435/005 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method for removing contaminants from a virus-containing
composition comprising obtaining an aqueous composition comprising
a selected virus and undesirable contaminants and subjecting the
aqueous composition to size partitioning purification using a size
partitioning membrane having partitioning pores that retain virus
and permit the passage of contaminants therethrough to remove
contaminants and thereby provide a purified virus composition.
2. The method of claim 1, wherein the virus is adenovirus,
lentivirus, adenoassociated virus, retrovirus, or herpes virus.
3. A method of producing purified adenovirus composition
comprising: a) growing host cells in a medium; b) providing
nutrients to said host cells; c) infecting said host cells with an
adenovirus; d) lysing said host cells to provide a cell lysate
comprising adenovirus; and e) purifying adenovirus from said lysate
by size partitioning purification utilizing a size partitioning
membrane to provide a purified adenovirus composition.
4. The method of claim 1 or 3 wherein the size partitioning
membrane is a dialysis membrane.
5. The membrane of claim 1 or 3 wherein the size partitioning
membrane is a porous filter
6. The method of claim 1 or 3, wherein the size partitioning
membrane is in a tangential flow filtration device.
7. The method of claim 1 or 3 wherein the size partitioning
membrane has a pore size of less than about 0.08 microns.
8. The method of claim 5 whrein the size partitioning membrane has
a pore size of less than about 0.08 microns and greater than about
0.0001 microns.
9. The method of claim 5 wherein the size partitioning membrane has
a pore size of less than about 0.05 microns and greater than about
0.0001 microns.
10. The method of claim 5 wherein the size partitioning membrane
has a pore size of less than about 0.02 microns and greater than
about 0.0001 microns.
11. The method of claim 5 wherein the size partitioning membrane
has a pore size of less than about 0.01 microns and greater than
about 0.0001 microns.
12. The method of claim 1 or 3, wherein the virus is purified to a
pharmaceutically acceptable degree without the use of ion exchange
chromatography.
13. The method of claim 1 or 3, wherein said medium is a serum-free
medium and said host cells are capable of growing in serum-free
media.
14. The method of claim 13 wherein said host cells have been
adapted for growth in serum-free media by a sequential decrease in
the fetal bovine serum content of the growth media.
15. The method of claim 3, wherein said host cells are 293
cells.
16. The method of claim 3 wherein the lysis step is carried out by
a process that includes hypotonic solution, hypertonic solution,
impinging jet, microfluidization, solid shear, detergent, liquid
shear, high pressure extrusion, autolysis or sonication.
17. The method of claim 16 wherein the cells are lysed by detergent
lysis.
18. The method of claim 16 wherein the cells are lysed by detergent
Thesit.RTM., NP-40.RTM., Tween-20.RTM., Brij-58.RTM., Triton
X-100.RTM. or octyl glucoside.
19. The method of claim 17 wherein said detergent is present in the
lysis solution at a concentration of about 1% (w/v).
20. The method of claim 3, wherein the host cells are grown at
least part of the time in a perfusion chamber, a bioreactor, a
flexible bed platform or by fed batch.
21. The method of claim 1 wherein the purified adenovirus
composition has a purity of less than 10 nanograms of contaminating
DNA per 1 milliliter dose.
22. The method of claim 3, wherein said adenovirus comprises an
adenoviral vector encoding an exogenous gene construct.
23. The method of claim 21, wherein said gene construct is
operatively linked to a promoter.
24. The method of claim 23, wherein said promoter is SV40 IE, RSV
LTR, .beta.-actin, CMV IE, adenovirus major late, polyoma F9-1, or
tyrosinase.
25. The method of claim 22, wherein said exogenous gene construct
encodes a therapeutic gene.
26. The method of claim 22, wherein said therapeutic gene encodes
antisense ras, antisense myc, antisense raf, antisense erb,
antisense src, antisense fins, antisense jun, antisense trk,
antisense ret, antisense gsp, antisense hst, antisense bcl
antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC,
CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1,
VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, thymidine
kinase or p53.
27. The method of claim 22, wherein said therapeutic gene encodes
p53.
28. The method of claim 22, wherein said adenovirus is a
replication-incompetent adenovirus.
29. The method of claim 22, wherein the adenovirus is lacking at
least a portion of the E1-region.
30. The method of claim 22, wherein the adenovirus is lacking at
least a portion of the E1A and/or E1B region.
31. The method of claim 22, wherein said host cells are capable of
complementing replication.
32. The method of claim 22 wherein the cells are perfused with a
gluconse containing media at a rate to provide a glucose
concentration higher than 0.5 g/L.
33. The method of claim 22 wherein the cells are perfused with a
glucose containing media at a rate to provide a glucose
concentration of between about 0.7 and 1.7 g/L.
34. The method of claim 1 or 3, wherein said cell lysate is treated
with Benzonas.RTM. or Pulmozym.RTM..
35. The method of claim 1 or 3, wherein said cells are grown as a
cell suspension culture.
36. The method of claim 1 or 3, wherein said cells are grown as an
anchorage-dependent culture.
37. The method of claim 3, wherein at least 5.times.10.sup.15 viral
particles are obtained from a single culture preparation.
38. The method of claim 37, wherein at least 1.times.10.sup.16
viral particles are obtained.
39. A purified adenovirus composition produced according to the
method of claims 1 or 3.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Ser. No. 60/624,627 filed Nov. 3, 2004 the disclosure
of which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
cell culture and virus production. More particularly, it concerns
improved methods for the culturing of mammalian cells, infection of
those cells with adenovirus and the production of infectious
adenovirus particles therefrom.
[0004] 2. Description of Related Art
[0005] A variety of cancer and genetic diseases currently are being
addressed by gene therapy. Viruses are highly efficient at nucleic
acid delivery to specific cell types, while often avoiding
detection by the infected host's immune system. These features make
certain viruses attractive candidates as gene-delivery vehicles for
use in gene therapies (Robbins and Ghivizzani, 1998; Cristiano et
al., 1998). Modified adenoviruses that are replication incompetent
and therefore non-pathogenic are being used as vehicles to deliver
therapeutic genes for a number of metabolic and oncologic
disorders. These adenoviral vectors may be particularly suitable
for disorders such as cancer that would best be treated by
transient therapeutic gene expression since the DNA is not
integrated into the host genome and the transgene expression is
limited. Adenoviral vector may also be of significant benefit in
gene replacement therapies, wherein a genetic or metabolic defect
or deficiency is remedied by providing for expression of a
replacement gene encoding a product that remedies the defect or
deficiency.
[0006] Adenoviruses can be modified to efficiently deliver a
therapeutic or reporter transgene to a variety of cell types.
Recombinant adenoviruses types 2 and 5 (Ad2 and AdV5,
respectively), which cause respiratory disease in humans, are among
those currently being developed for gene therapy. Both Ad2 and AdV5
belong to a subclass of adenovirus that is not associated with
human malignancies. Recently, the hybrid adenoviral vector AdV5/F35
has been developed and proven of great interest in gene therapies
and related studies (Yotnda et al., 2001).
[0007] Recombinant adenoviruses are capable of providing extremely
high levels of transgene delivery. The efficacy of this system in
delivering a therapeutic transgene in vivo that complements a
genetic imbalance has been demonstrated in animal models of various
disorders (Watanabe, 1986; Tanzawa et al., 1980; Golasten et al.,
1983; Ishibashi et al., 1993; and S. Ishibashi et al., 1994).
Indeed, a recombinant replication defective adenovirus encoding a
cDNA for the cystic fibrosis transmembrane regulator (CFTR) has
been approved for use in at least two human CF clinical trials
(Wilson, 1993). Hurwitz, et al., (1999) have shown the therapeutic
effectiveness of adenoviral mediated gene therapy in a murine model
of cancer (retinoblastoma).
[0008] As the clinical trials progress, the demand for clinical
grade adenoviral vectors is increasing dramatically. The projected
annual demand for a 300 patient clinical trial could reach
approximately 6.times.10.sup.14 PFU.
[0009] Traditionally, adenoviruses are produced in commercially
available tissue culture flasks or "cellfactories." Adenoviral
vector production has generally been performed in culture devices
that supply culture surfaces for attachment of the HEK293 cells,
such as T-flasks. Virus infected cells are harvested and
freeze-thawed to release the viruses from the cells in the form of
crude cell lysate. The produced crude cell lysate (CCL) is then
purified by double CsCl gradient ultracentrifugation. The typically
reported virus yield from 100 single tray cellfactories is about
6.times.10.sup.12 PFU. Clearly, it becomes unfeasible to produce
the required amount of virus using this traditional process. New
scaleable and validatable production and purification processes
have to be developed to meet the increasing demand.
[0010] The purification throughput of CsCl gradient
ultracentrifugation is so limited that it cannot meet the demand
for adenoviral vectors for gene therapy applications. Therefore, in
order to achieve large scale adenoviral vector production,
purification methods other than CsCl gradient ultracentrifugation
have to be developed. Reports on the chromatographic purification
of viruses are very limited, despite the wide application of
chromatography for the purification of recombinant proteins. Size
exclusion, ion exchange and affinity chromatography have been
evaluated for the purification of retroviruses, tick-borne
encephalitis virus, and plant viruses with varying degrees of
success (Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al.,
1978, Smith and Lee, 1978; O'Neil and Balkovic, 1993). Even less
research has been done on the chromatographic purification of
adenovirus. This lack of research activity may be partially
attributable to the existence of the effective, albeit
non-scalable, CsCl gradient ultracentrifugation purification method
for adenoviruses.
[0011] Recently, Huyghe et al. (1996) reported adenoviral vector
purification using ion exchange chromatography in conjunction with
metal chelate affinity chromatography. Virus purity similar to that
from CsCl gradient ultracentrifugation was reported. Unfortunately,
only 23% of virus was recovered after the double column
purification process. Process factors that contribute to this low
virus recovery are the freeze/thaw step utilized by the authors to
lyse cells in order to release the virus from the cells and the two
column purification procedure. Of interest to the present invention
is the disclosure of co-owned U.S. Published Patent Application No.
2004/0106184 A1, the disclosure of which is hereby incorporated by
reference which is directed to methods for passing adenovirus
particle preparations through chromatographic media to provide
purified adenovirus particles.
[0012] For most of the E1 deleted first generation adenoviral
vectors, production is carried out using HEK293 (human embryonal
kidney cells, Invitrogen Corp.) cells which complement the
adenoviral vector E1 deletion in trans. Because of the anchorage
dependency of the HEK293 cells, adenoviral vector production has
generally been performed in culture devices that supply culture
surfaces for attachment of the HEK293 cells, such as T-flasks,
multilayer Cellfactories.TM., and the large scale CellCube.TM.
bioreactor system. Recently, the HEK293 cells have been adapted to
suspension culture in a variety of serum free media allowing
production of adenoviral vectors in suspension bioreactors.
Complete medium exchange at the time of virus infection using
centrifugation is difficult to perform on a large scale. In
addition, the shear stress associated with medium recirculation
required for external filtration devices is likely to have a
detrimental effect on host cells in a protein-free medium.
[0013] Of interest to the present invention are the disclosures of
co-owned U.S. Pat. No. 6,194,191 and co-owned U.S. Pat. No.
6,726,907 the disclosures of which are hereby incorporated by
reference, which are directed to improved Ad-p53 production methods
with cells grown in serum-free conditions, and in particular in
serum-free suspension culture. Also of interest to the present
invention is the disclosure of WO 00/32754 based on U.S. Ser. No.
09/203,078, the disclosure of which is hereby incorporated by
reference, which is directed to the use of low-medium perfusion
rates in an attached cell culture system.
[0014] Clearly, there is a demand for improved methods of
adenoviral vector production that will recover a high yield of
product to meet the ever increasing demand for such products.
Improved methods for adenoviral vector production can include
improved techniques to make production more efficient, or
optimization of operating conditions to increase adenoviral vector
production.
SUMMARY OF THE INVENTION
[0015] The present invention is related to methods for producing
purified viral compositions including adenovirus compositions of
sufficient purity for therapeutic administration without the
necessity for elaborate purification steps. More specifically, the
invention relates to the discovery that size partitioning
purification techniques may be used to provide adenoviral
preparations of sufficient purity that they may be therapeutically
administered without additional purification steps such as
chromatographic and other methods previously considered necessary.
Without intending to be bound by any particular theory of the
invention it is believed that the steps of processing viral host
cells in a cell suspension culture in a serum free media results in
a viral particle product with a reduced load of contaminants.
Moreover, the contaminants are of a size and nature that they may
be readily separated from viral particles by a simple size
partitioning purification step.
[0016] The ability to produce purified adenoviral preparations
without traditional chromatographic purification steps provides
significant improvements in viral production yields while reducing
expense.
[0017] Specifically, the invention provides a method for removing
contaminants from a virus-containing composition comprising
obtaining an aqueous composition comprising a selected virus and
undesirable contaminants and subjecting the aqueous composition to
size partitioning purification using a size partitioning membrane
having partitioning pores that retain virus and permit the passage
of contaminants therethrough to remove contaminants and thereby
provide a purified virus composition. Of course, the size of the
partitioning pores will preferably be selected on the basis of the
size of the virus that is to be retained, in which case one will
select a membrane having a pore or inclusion size sufficiently
smaller than the virus so as to retain the virus and yet permit the
passage of contaminants. Similarly, if the pore or inclusion size
is too small, some undesirable contaminants may be retained.
Therefore, an optimal pore size is one that retains the most virus
yet permits the passage of the most contaminants. Generally, the
size of the virus and corresponding proposed preferred pore sizes
will be as in Table 1 below: TABLE-US-00001 TABLE 1 Virus Average
Particle Size Preferred Pore Size Range Adenovirus 80 nm
.ltoreq.0.05 .mu.m AAV 20 nm .ltoreq.0.01 .mu.m Retroviruses 100 nm
.ltoreq.0.05 .mu.m Herpes virus 100 nm .ltoreq.0.05 .mu.m
Lentivirus 100 nm .ltoreq.0.05 .mu.m
[0018] In particular embodiments, the invention provides a method
of producing purified adenovirus composition comprising the steps
of a) growing host cells in a medium; b) providing nutrients to
said host cells; c) infecting said host cells with an adenovirus;
d) lysing said host cells to provide a cell lysate comprising
adenovirus; and e) purifying adenovirus from said lysate by size
partitioning purification utilizing a size partitioning membrane to
provide a purified adenovirus composition.
[0019] The methods of the invention may be used when the virus is
adenovirus, lentivirus, adenoassociated virus, retrovirus or herpes
virus.
[0020] Particularly preferred methods of the invention are those in
which the size partitioning membrane is in a tangential flow
filtration device.
[0021] According to one aspect of the invention the size
partitioning membrane is a porous filter. More specifically, the
size partitioning membrane may be a dialysis membrane. The size
partitioning membrane preferably has a pore size of less than about
0.08 microns and greater than about 0.0001 microns. Size
partitioning membranes having pore sizes less than 0.05 microns and
greater than 0.0001 microns and those having pore sizes less than
0.02 microns and greater than 0.0001 microns are particularly
preferred. For viruses such as adeno-associtated virus (AAV) a pore
size of less than 0.01 microns but greater than 0.0001 microns is
preferred.
[0022] According to one aspect of the invention, the size
partitioning purification could be carried out by gel filtration
purification. Such a method is not preferred, however, because get
filtration size partitioning effects a dramatic increase in volume
and dilutes the viral preparation. Such diluted preparations must
then be reconcentrated which is costly and undesirable.
[0023] According to one aspect of the invention virus may be
purified to a pharmaceutically acceptable degree without the use of
additional purification steps such as ion exchange chromatography.
By pharmaceutically acceptable degree is meant substantially free
of animal derived components and free of other protein impurities
as seen on an SDS-PAGE gel so as to not impact on the human
clinical use of the product. As another aspect of the invention,
the purified adenovirus composition has a purity of less than 10
nanograms of contaminating DNA per 1 milliliter dose.
[0024] According to a preferred aspect of the invention at least
5.times.10.sup.15 viral particles and more preferably
1.times.10.sup.16 viral particles are obtained from a single
culture preparation.
[0025] The host cells are preferably capable of growing in
serum-free media and are grown in a serum-free medium. According to
this method, the host cells may be adapted for growth in serum-free
media by a sequential decrease in the fetal bovine serum content of
the growth media. Preferred host cells are HEK293 cells. The host
cells may be grown at least part of the time in a perfusion
chamber, a bioreactor, a flexible bed platform or by fed batch.
According to one method, the cells are perfused with a glucose
containing media at a rate to provide a glucose concentration
higher than 0.5 g/L with perfusion at a rate to provide a glucose
concentration of between about 0.7 and 1.7 g/L being particularly
preferred. The cells may be grown as a cell suspension culture or
alternatively as an anchorage-dependent culture.
[0026] Lysis of the host cells may be carried out by a process that
includes hypotonic solution, hypertonic solution, impinging jet,
microfluidization, solid shear, detergent, liquid shear, high
pressure extrusion, autolysis or sonication. Suitable detergents
include those commercially available as Thesit.RTM., NP-40.RTM.,
Tween-20.RTM., Brij-58.RTM., Triton X-100.RTM. and octyl glucoside.
According to one aspect of the invention the detergent is present
in the lysis solution at a concentration of about 1% (w/v). The
cell lysate may then be treated with a nuclease such as those
available commercially as Benzonase.RTM. or Pulmozym.RTM..
[0027] According to one aspect of the invention the viral particles
are intended for use in gene therapy. Accordingly, the viral
particle is an adenovirus which comprises an adenoviral vector
encoding an exogenous gene construct. According to a further aspect
of the invention the gene construct is operatively linked to a
promoter. Suitable promoters include those selected from the group
consisting of SV40 IE, RSV LTR, .beta.-actin, CMV IE, adenovirus
major late, polyoma F9-1, or tyrosinase.
[0028] The exogenous gene construct can encode a therapeutic gene.
Such genes are known to those of skill in the art and include, but
are not limited to, those which encode antisense ras, antisense
myc, antisense raf, antisense erb, antisense src, antisense fms,
antisense jun, antisense trk, antisense ret, antisense gsp,
antisense hst, antisense bcl antisense abl, Rb, CFTR, p16, p21,
p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2,
WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 L-12,
GM-CSF G-CSF, thymidine kinase and p53.
[0029] Preferred viral vectors include adenoviral vectors and
particularly those in which the adenovirus is a
replication-incompetent adenovirus. Such replication incompetent
adenoviral vectors include those in which the adenovirus is lacking
at least a portion of the E1-region with those lacking at least a
portion of the E1A and/or E1B region being particularly preferred.
According to one method, a replication incompetent adenovirus is
produced in host cells which are capable of complementing
replication. The present invention describes a new process for the
production and purification of adenovirus. This new production
process offers not only scalability and validatability but also
excellent virus purity.
[0030] In preferred embodiments of the present invention, the
adenovirus comprises an adenoviral vector encoding an exogenous
gene construct. In certain such embodiments, the gene construct is
operatively linked to a promoter. In particular embodiments, the
promoter is SV40 IE, RSV LTR, .beta.-actin or CMV IE, adenovirus
major late, polyoma F9-1, or tyrosinase. In particular embodiments
of the present invention, the adenovirus is a
replication-incompetent adenovirus. In other embodiments, the
adenovirus is lacking at least a portion of the E1-region. In
certain aspects, the adenovirus is lacking at least a portion of
the E1A and/or E1B region. In other embodiments, the host cells are
capable of complementing replication. In particularly preferred
embodiments, the host cells are HEK293 cells.
[0031] In a preferred embodiment of the invention it is
contemplated that the exogenous gene construct encodes a
therapeutic gene. For example, the therapeutic gene may encode
antisense ras, antisense myc, antisense raf, antisense erb,
antisense src, antisense fins, antisense jun, antisense trk
antisense ret, antisense gsp, antisense hst, antisense bcl
antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC,
CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1,
VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF G-CSF, thymidine
kinase or p53.
[0032] In certain aspects of the present invention, the cells may
be harvested and lysed ex situ using a hypotonic solution,
hypertonic solution, freeze-thaw, sonication, impinging jet,
microfluidization or a detergent. In other aspects, the cells are
harvested and lysed in situ using a hypotonic solution, hypertonic
solution, or a detergent. As used herein the term "in situ" refers
to the cells being located within the tissue culture apparatus for
example CellCube.TM. and "ex situ" refers to the cells being
removed from the tissue culture apparatus.
[0033] In particular embodiments, the cells are lysed and harvested
using detergent. In preferred embodiments the detergent may be
Thesit.RTM., NP-40.RTM., Tween-20.RTM., Brij-58.RTM., Triton
X.RTM.-100 or octyl glucoside. In other aspects of the present
invention lysis is achieved through autolysis of infected cells. In
more particular embodiments the detergent is present in the lysis
solution at a concentration of about 1% (w/v). In certain other
aspects of the present invention the cell lysate is treated with
Benzonase.RTM., or Pulmozyme.RTM.
[0034] In particular embodiments, the method further comprises a
concentration step employing membrane filtration. In particular
embodiments, the filtration is tangential flow filtration. In
preferred embodiments, the filtration may utilize a 100 to 1000K
NMWC, regenerated cellulose, or polyether sulfone membrane.
[0035] The present invention also provides an adenovirus produced
according to a process comprising the steps of growing host cells
in media, infecting the host cells with an adenovirus, harvesting
and lysing the host cells to produce a crude cell lysate,
concentrating the crude cell lysate, exchanging buffer of crude
cell lysate, and reducing the concentration of contaminating
nucleic acids in the crude cell lysate.
[0036] In yet another embodiment, the present invention provides a
method for the purification of an adenovirus comprising the steps
of growing host cells in serum-free media; infecting said host
cells with an adenovirus; harvesting and lysing said host cells to
produce a crude cell lysate; concentrating said crude cell lysate;
exchanging buffer of crude cell lysate; and reducing the
concentration of contaminating nucleic acids in said crude cell
lysate. In preferred embodiments, the cells may be grown
independently as a cell suspension culture or as an
anchorage-dependent culture.
[0037] In particular embodiments, the host cells are adapted for
growth in serum-free media. In more preferred embodiments, the
adaptation for growth in serum-free media comprises a sequential
decrease in the fetal bovine serum content of the growth media.
More particularly, the serum-free media comprises a fetal bovine
serum content of less than 0.03% v/v.
[0038] Also contemplated by the present invention is an adenovirus
produced according to a process comprising the steps of growing
host cells in serum-free media; infecting said host cells with an
adenovirus; harvesting and lysing said host cells to produce a
crude cell lysate; concentrating said crude cell lysate; exchanging
buffer of crude cell lysate; and reducing the concentration of
contaminating nucleic acids in said crude cell lysate.
[0039] The present invention further provides a 293 host cell
adapted for growth in serum-free media. In certain aspects, the
adaptation for growth in serum-free media comprises a sequential
decrease in the fetal bovine serum content of the growth media. In
particular embodiments, the cell is adapted for growth in
suspension culture. In particular embodiments, the cells of the
present invention are designated IT293SF cells. These cells were
deposited with the American Tissue Culture Collection (ATCC) in
order to meet the requirements of the Budapest Treaty on the
international recognition of deposits of microorganisms for the
purposes of patent procedure. The cells were deposited by Dr.
Shuyuan Zhang on behalf of Introgen Therapeutics, Inc. (Houston,
Tex.), on Nov. 17, 1997. IT293SF cell line is derived from an
adaptation of 293 cell line into serum free suspension culture as
described herein. The cells may be cultured in IS 293 serum-free
media (Irvine Scientific. Santa Ana, Calif.) supplemented with 100
mg/L heparin and 0.1% Puronic F-68, and are permissive to human
adenovirus infection.
[0040] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
[0041] Other embodiments of the present invention pertain to
methods for producing an adenovirus, including: (1) preparing an
adenovirus preparation, including the steps of growing host cells
in media in a bioreactor and initiating virus infection by diluting
the host cells with fresh media and adenovirus; and (2) isolating
adenovirus from the adenovirus preparation. Any bioreactor known to
those of skill in the art that is capable of supporting host cell
growth is contemplated for use in the present invention. A detailed
discussion of various types of bioreactors is presented below in
other parts of the specification.
[0042] According to one aspect of the present invention serum-free
media is preferred for use in conjunction with the bioreactor, as
long as the media is capable of supporting cell growth in the
bioreactor. In other embodiments, the media is protein-free media.
In some embodiments, the media is CD293 media medium (Invitrogen
Corp..TM.). In the embodiments of the present invention, the host
cells may be grown in an anchorage-dependent culture or a
non-anchorage-dependent (suspension) culture.
[0043] In the embodiments of the present invention that pertain to
methods of producing an adenovirus which require a bioreactor, any
bioreactor known to those of skill in the art is contemplated by
the present invention. In certain embodiments, for example, the
bioreactor comprises a bioreactor that uses axial rocking of a
planar platform to induce wave motions inside of the bioreactor. In
some embodiments, wave motions are induced inside of a sterilized
polyethylene bag wherein the host cells are located. In further
embodiments, the bioreactor is a disposable bioreactor. Any size of
bioreactor is contemplated by the present invention. For example,
the bioreactor may be a 10 L, a 20 L up to 200 L or larger
bioreactor. In addition, the bioreactor may be a
commercially-available bioreactor. For example, the bioreactor may
be a Wave Bioreactor.RTM. (Wave Biotech, LLC, Bedminster, N.J.).
According to one aspect of the invention a 20 L Wave
Bioreactor.RTM. with an 8 L working volume may be used to culture
adenoviral vectors transformed with the native p53 gene. The
culture may be harvested on day 2 post infection using
Tween.RTM.-20 to produce a yield of 2.3.times.10.sup.11 viral
particles/mL or 230,000 viral particles/cell. At such yields a 200
L bioreactor would be expected to yield approaching
2.times.10.sup.16 VP.
[0044] In the embodiments of the present invention that pertain to
methods of producing an adenovirus, it is contemplated that the
operating conditions of the cell culture may be monitored or
measured by any technique known to those of skill in the art.
Examples of such conditions which may be monitored include pH of
the media and dissolved oxygen tension of the media.
[0045] In the embodiments of the present invention that pertain to
methods of producing an adenovirus, it is contemplated that the
operating conditions of the cell culture may be monitored or
measured by any technique known to those of skill in the art.
Examples of such conditions which may be monitored include pH of
the media and dissolved oxygen tension of the media.
[0046] Some embodiments of the present invention pertaining to
methods of producing an adenovirus also involve processing and
treating the media by any method known to those of skill in the
art. For example, in certain embodiments of the present invention,
the methods for producing an adenovirus involve perfusing the media
through a filter. The filter may be a filter that is internal to
the bioreactor system, or the filter may be incorporated so that it
is external to the bioreactor. In certain embodiments, the filter
is a floating flat filter. The floating flat filter may be used to
remove spent media from the bioreactor. Any method known to those
of skill in the art may be used to monitor and maintain media
volume. In some embodiments, culture volume is maintained by a load
cell used to trigger fresh media addition.
[0047] In embodiments of the present invention, media may or may
not be perfused into the culture of host cells. In some embodiments
of the present invention, media is perfused beginning on day 3 of
host cell growth. One of skill in the art would be familiar with
the wide range of techniques and apparatus available for perfusing
media into a cell culture system.
[0048] In embodiments of the present invention that pertain to
methods of producing an adenovirus, the step of diluting host cells
with fresh media may be combined with the adenovirus infection
step. This is based on the inventors' discovery that these two
steps can be efficiently combined to provide for excellent yields
of adenoviral vectors. The invention contemplates use of any method
of dilution known to those of skill in the art. In certain
embodiments, the host cells are diluted 2-fold to 50-fold with
fresh media and adenovirus. In other embodiments, the host cells
are diluted 10-fold with fresh media and adenovirus.
[0049] In the embodiments of the present invention that pertain to
methods of producing an adenovirus, the initiating of virus
infection of the host cells may be accomplished by any method known
to those of skill in the art. For example, in embodiments of the
present invention that involve use of bioreactors, the virus
infection may take place in a second bioreactor. For example, virus
infection of host cells may be accomplished by adding 20-100
vp/host cell. In certain other embodiments, virus infection
involves adding about 50 vp/host cell. Virus infection may be
allowed to proceed for any duration of time. One of skill in the
art would be familiar with techniques pertaining to monitoring the
progress of virus infection. In certain embodiments of the present
invention, virus infection is allowed to proceed for about 4
days.
[0050] In certain other embodiments of the present invention, the
isolating of the adenovirus from the adenovirus preparation occurs
at about 4 days after viral infection is completed.
[0051] In the embodiments of the present invention that involve
production of adenovirus, use of host cells is contemplated. Any
cell type can be used as a host cell, as long as the cell is
capable of supporting replication of adenovirus. One of skill in
the art would be familiar with the wide range of host cells that
can be used in the production of adenovirus from host cells. For
example, in some embodiments of the present invention, the host
cells complement the growth of the replication-deficient
adenovirus. The replication-deficient adenovirus may be an
adenovirus that lacks at least a portion of the E1-region, or it
may be an adenovirus that lacks at least a portion of the E1A
and/or E1B region. The host cells, for example, may be 293, HEK293,
PER.C6, 911, and IT293SF cells. In certain embodiments of the
present invention, the host cells are HEK293 cells.
[0052] In some embodiments of the present invention, the adenovirus
is a recombinant adenovirus. For example, the recombinant
adenovirus may encode a recombinant gene that is operatively linked
to a promoter. Any promoter known to those of skill in the art can
be used, as long as the promoter is capable of functioning as a
promoter. For or example, in certain embodiments the promoter is an
SV40 EI, RSV LTR, .beta.-actin, CMV-IE, adenovirus major late,
polyoma F9-1, or tyrosinase promoter.
[0053] In embodiments of the present invention where the adenovirus
is an adenovirus encoding a recombinant gene, any recombinant gene,
particularly a therapeutic gene, is contemplated by the present
invention. For example, the recombinant gene may be selected from
the group consisting of antisense ras, antisense myc, antisense
raf, antisense erb, antisense src, antisense fms, antisense jun,
antisense trk, antisense ret, antisense gsp, antisense hst,
antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73,
C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I,
MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF,
thymidine kinase, mda7, fus, interferon .alpha., interferon .beta.,
interferon .gamma., ADP (adenoviral death protein), or p53. In some
embodiments, the recombinant gene is a p53 gene. In other
embodiments, the recombinant gene is a mda-7 gene.
[0054] In some embodiments of the present invention, the
recombinant gene is antisense ras, antisense myc, antisense raf
antisense erb, antisense src, antisense fms, antisense jun,
antisense trk, antisense ret, antisense gsp, antisense hst,
antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73,
C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I,
MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF,
thymidine kinase, mda7, fus, interferon .alpha., interferon .beta.,
interferon .gamma., ADP, p53, ABLI, BLC1, BLC6, CBFA1, CBL, CSFIR,
ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN,
KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML,
RET, SRC, TAL1, TCL3, YES, MADH4, RB1, TP53, WT1, TNF, BDNF, CNTF,
NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV, ApoE, Rap1A,
cytosine deaminase, Fab, ScFv, BRCA2, zac1, ATM, HIC-1, DPC-4,
FHIT, PTEN, ING1, NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-1, Rb,
zac1, DBCCR-1, rks-3, COX-1, TFPI, PGS, Dp, E2F, ras, myc, neu,
raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, VEGF, FGF,
thrombospondin, BAI-1, GDAIF, or MCC.
[0055] In further embodiments of the present invention, the
recombinant gene is a gene encoding an ACP desaturase, an ACP
hydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcohol
dehydrogenase, an amylase, an amyloglucosidase, a catalase, a
cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, an
esterase, a DNA polymerase, an RNA polymerase, a hyaluron synthase,
a galactosidase, a glucanase, a glucose oxidase, a GTPase, a
helicase, a hemicellulase, a hyaluronidase, an integrase, an
invertase, an isomerase, a kinase, a lactase, a lipase, a
lipoxygenase, a lyase, a lysozyme, a pectinesterase, a peroxidase,
a phosphatase, a phospholipase, a phosphorylase, a
polygalacturonase, a proteinase, a peptidease, a pullanase, a
recombinase, a reverse transcriptase, a topoisomerase, a xylanase,
a reporter gene, an interleukin, or a cytokine.
[0056] In other embodiments of the present invention, the
recombinant gene is a gene encoding carbamoyl synthetase I,
ornithine transcarbamylase, arginosuccinate synthetase,
arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase,
phenylalanine hydroxylase, alpha-1 antitrypsin,
glucose-6-phosphatase, low-density-lipoprotein receptor,
porphobilinogen deaminase, factor VIII, factor IX, cystathione
beta.-synthase, branched chain ketoacid decarboxylase, albumin,
isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl
malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,
beta.-glucosidase, pyruvate carboxylase, hepatic phosphorylase,
phosphorylase kinase, glycine decarboxylase, H-protein, T-protein,
Menkes disease copper-transporting ATPase, Wilson's disease
copper-transporting ATPase, cytosine deaminase,
hypoxanthine-guanine phosphoribosyltransferase,
galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,
glucocerbrosidase, sphingomyelinase, .alpha.-L-iduronidase,
glucose-6-phosphate dehydrogenase, HSV thymidine kinase, or human
thymidine kinase. Alternatively, the recombinant gene may encode
growth hormone, prolactin, placental lactogen, luteinizing hormone,
follicle-stimulating hormone, chorionic gonadotropin,
thyroid-stimulating hormone, leptin, adrenocorticotropin,
angiotensin I, angiotensin II, .beta.-endorphin, .beta.-melanocyte
stimulating hormone, cholecystokinin, endothelin I, galanin,
gastric inhibitory peptide, glucagon, insulin, lipotropins,
neurophysins, somatostatin, calcitonin, calcitonin gene related
peptide, .beta.-calcitonin gene related peptide, hypercalcemia of
malignancy factor, parathyroid hormone-related protein, parathyroid
hormone-related protein, glucagon-like peptide, pancreastatin,
pancreatic peptide, peptide YY, PHM, secretin, vasoactive
intestinal peptide, oxytocin, vasopressin, vasotocin,
enkephalinamide, metorphinamide, alpha melanocyte stimulating
hormone, atrial natriuretic factor, amylin, amyloid P component,
corticotropin releasing hormone, growth hormone releasing factor,
luteinizing hormone-releasing hormone, neuropeptide Y, substance K,
substance P, or thyrotropin releasing hormone.
[0057] Certain of the embodiments of the present invention pertain
to methods of producing an adenovirus that involve isolating the
adenovirus from an adenovirus preparation. Any method of isolating
the adenovirus from the adenovirus preparation known to those of
skill in the art is contemplated by the present invention. In
certain embodiments of the present invention, the host cells are
harvested following infection but prior to lysis by the adenovirus,
and lysing the host cells is performed by freeze-thaw, autolysis,
or detergent lysis. In certain other embodiments of the present
invention, the methods of producing adenovirus involve reducing the
concentration of contaminating nucleic acids in the adenovirus
preparation.
[0058] In some embodiments of the invention, the adenovirus that is
isolated is placed into a pharmaceutically acceptable composition.
One of skill in the art would be familiar with the extensive
methods and techniques employed in preparing pharmaceutically
acceptable compositions. Any pharmaceutical composition into which
adenovirus can be formulated is contemplated by the present
invention. For example, certain embodiments of the invention
pertain to pharmaceutical preparation of adenovirus for oral
administration, topical administration, or intravenous
administration.
[0059] Some embodiments of the present invention involve analysis
of virus production. For example, virus production may be analyzed
using HPLC. Any technique for analyzing virus production known to
those of skill is contemplated by the present invention.
[0060] In some embodiments of the invention, the methods for
producing an adenovirus disclosed above and elsewhere in this
specification concern methods for isolating and purifying an
adenovirus that involve obtaining a purified adenovirus composition
having one or more of the following properties: (1) a virus titer
of between 1.times.10.sup.9 and about 1.times.10.sup.13 pfu/ml; (2)
a virus particle concentration between about 1.times.10.sup.10 and
about 2.times.10.sup.13 particles/ml; (3) a particle:pfu ratio
between about 10 and about 60; (4) having less than 50 ng BSA per
1.times.10.sup.12 viral particles; (5) between about 50 pg and 1 ng
of contaminating human DNA per 1.times.10.sup.12 viral particles;
(6) a single HPLC elution peak consisting essentially of 97% to
100% of the area under the peak. In certain embodiments, the
adenovirus composition prepared in accordance with the steps
discussed above includes between 5.times.10.sup.14 and
1.times.10.sup.18 viral particles. In other embodiments, the
composition is a pharmaceutically-acceptable composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0062] FIG. 1 depicts a production and purification flow chart for
adenovirus using tangential flow filtration (TFF) diafiltration
alone and TFF diafiltration in conjunction with chromatographic
purification;
[0063] FIG. 2 (Scanned image) depicts analysis of tangential flow
filtration (TFF) purified, virus, lanes 1-5 and virus purified by
conventional methods utilizing a chromatography column;
[0064] FIG. 3 depicts a diagram of a perfusion bioreactor
system;
[0065] FIG. 4 depicts the cell growth and viability versus days in
culture;
[0066] FIG. 5 depicts the glucose and lactate concentrations (g/L)
in perfusion culture versus days in culture; and
[0067] FIG. 6 depicts a comparison of gene expression of viral
products produced by CellCube and Wave bioreactor processes.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0068] It has been shown that adenoviral vectors can successfully
be used in eukaryotic gene expression and vaccine development.
Recently, animal studies have demonstrated that recombinant
adenovirus could be used for gene therapy. Successful studies in
administering recombinant adenovirus to different tissues have
proven the effectiveness of adenoviral vectors in therapy. This
success has led to the use of such vectors in human clinical
trials. There now is an increased demand for the production of
adenoviral vectors to be used in various therapies. The techniques
currently available are insufficient to meet such a demand. The
present invention provides methods for the production of large
amounts of adenovirus for use in such therapies.
[0069] Therefore, the present invention is designed to take
advantage of improvements in large scale culturing systems and
purification for the purpose of producing and purifying adenoviral
vectors. The various components for such a system, and methods of
producing adenovirus therewith, are set forth in detail below.
[0070] A. Adenovirus
[0071] Adenoviruses comprise linear double stranded DNA, with a
genome ranging from 30 to 35 kb in size (Reddy et al., 1998;
Morrison et al., 1997; Chillon et al., 1999). There are over 50
serotypes of human adenovirus, and over 80 related forms which are
divided into six families based on immunological, molecular, and
functional criteria (Wadell et al, 1980). Physically, adenovirus is
a medium-sized icosahedral virus containing a double-stranded,
linear DNA genome which, for adenovirus type 5, is 35,935 base
pairs (Chroboczek et al., 1992). Adenoviruses require entry into
the host cell and transport of the viral genome to the nucleus for
infection of the cell and replication of the virus. Salient
features of the adenovirus genome; are an early region (E1, E2, E3
and E4 genes), an intermediate region (pIX gene, Iva2 gene), a late
region (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP),
inverted-terminal-repeats (ITRs) and a .psi. sequence (Zheng, et
al., 1999; Robbins et al., 1998; Graham and Prevec, 1995). The
early genes E1, E2, E3 and E4 are expressed from the virus after
infection and encode polypeptides that regulate viral gene
expression, cellular gene expression, viral replication, and
inhibition of cellular apoptosis. Further on during viral
infection, the MLP is activated, resulting in the expression of the
late (L) genes, encoding polypeptides required for adenovirus
encapsidation. The intermediate region encodes components of the
adenoviral capsid. Adenoviral inverted terminal repeats (ITRs;
100-200 bp in length), are cis elements, function as origins of
replication and are necessary for viral DNA replication. The .psi.
sequence is required for the packaging of the adenoviral
genome.
[0072] The mechanism of infection by adenoviruses, particularly
adenovirus serotypes 2 and 5, has been extensively studied. A host
cell surface protein designated CAR (Coxsackie Adenoviral Receptor)
has been identified as the primary binding receptor for these
adenoviruses. The endogenous cellular function of CAR has not yet
been elucidated. Interaction between the fiber knob and CAR is
sufficient for binding of the adenovirus to the cell surface.
However, subsequent interactions between the penton base and
additional cell surface proteins, members of the .alpha..sub.v
integrin family, are necessary for efficient viral internalization.
Disassembly of the adenovirus begins during internalization; the
fiber proteins remain on the cell surface bound to CAR. The
remainder of the adenovirus is dissembled in a stepwise manner as
the viral particle is transported through the cytoplasm to a pore
complex at the nuclear membrane. The viral DNA is extruded through
the nuclear membrane into the nucleus where viral DNA is
replicated, viral proteins are expressed, and new viral particles
are assembled. Specific steps in this mechanism of adenoviral
infection may be potential targets to modulate viral infection and
gene expression.
[0073] In certain embodiments of the present invention, the
adenovirus used in the methods for producing an adenovirus may be a
replication-deficient adenovirus. For example, the adenovirus may
be a replication-deficient adenovirus lacking at least a portion of
the E1 region. In certain embodiments, the adenovirus may be
lacking at least a portion of the E1A and/or E1B region. In other
embodiments, the adenovirus is a recombinant adenovirus (discussed
further below).
[0074] B. Host Cells
[0075] Various embodiments of the present invention involve methods
for producing an adenovirus. A "host cell" is defined as a cell
that is capable of supporting replication of adenovirus. Any cell
type for use as a host cell is contemplated by the present
invention, as longs as the cell is capable of supporting
replication of adenovirus. For example, the host cells may be
HEK293, PER.C6, 911, or IT293SF cells. One of skill in the art
would be familiar with the wide range of host cells that are
available for use in methods for producing an adenovirus.
[0076] In certain embodiments, the generation and propagation of
the adenoviral vectors depend on a unique helper cell line,
designated 293, which was transformed from human embryonic kidney
cells by Adenovirus serotype 5 (Ad5) DNA fragments and
constitutively expresses E1 proteins (Graham et al., 1977). Since
the E3 region is dispensable from the Ad genome (Jones and Shenk,
1978), the current Ad vectors, with the help of 293 cells, carry
foreign DNA in either the E1, the E3 or both regions (Graham and
Prevec, 1991; Bett et al., 1994).
[0077] The host cells used in the various embodiments of the
present invention may be derived, for example, from mammalian cells
such as human embryonic kidney cells or primate cells. Other cell
types might include, but are not limited to Vero cells, CHO cells
or any eukaryotic cells for which tissue culture techniques are
established as long as the cells are adenovirus permissive. The
term "adenovirus permissive" means that the adenovirus or
adenoviral vector is able to complete the entire intracellular
virus life cycle within the cellular environment.
[0078] The host cell may be derived from an existing cell line,
e.g., from a 293 cell line, or developed de novo. Such host cells
express the adenoviral genes necessary to complement in trans
deletions in an adenoviral genome or which supports replication of
an otherwise defective adenoviral vector, such as the E1, E2, E4,
E5 and late functions. A particular portion of the adenovirus
genome, the E1 region, has already been used to generate
complementing cell lines. Whether integrated or episomal, portions
of the adenovirus genome lacking a viral origin of replication,
when introduced into a cell line, will not replicate even when the
cell is superinfected with wild-type adenovirus. In addition,
because the transcription of the major late unit is after viral DNA
replication, the late functions of adenovirus cannot be expressed
sufficiently from a cell line. Thus, the E2 regions, which overlap
with late functions (L1-5), will be provided by helper viruses and
not by the cell line. Typically, a cell line according to the
present invention will express E1 and/or E4.
[0079] Recombinant host cells, which are host cells that express
part of the adenoviral genome, are also contemplated for use as
host cells in the present invention. As used herein, the term
"recombinant" cell is intended, to refer to a cell into which a
gene, such as a gene from the adenoviral genome or from another
cell, has been introduced. Therefore, recombinant cells are
distinguishable from naturally-occurring cells which do not contain
a recombinantly-introduced gene. Recombinant cells are thus cells
having a gene or genes introduced through "the hand of man."
[0080] Recombinant host cells lines are capable of supporting
replication of adenovirus recombinant vectors and helper viruses
having defects in certain adenoviral genes, i.e., are "permissive"
for growth of these viruses and vectors. The recombinant cell also
is referred to as a helper cell because of the ability to
complement defects in, and support replication of,
replication-incompetent adenoviral vectors.
[0081] Examples of other useful mammalian cell lines that may be
used with a replication competent virus or converted into
complementing host cells for use with replication deficient virus
are Vero and HeLa cells and cell lines of Chinese hamster ovary,
W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cells.
[0082] Two methodologies have been used to adapt 293 cells into
suspension cultures. Graham adapted 293 A cells into suspension
culture (293N3S cells) by 3 serial passages in nude mice (Graham,
1987). The suspension 293N3S cells were found to be capable of
supporting E1.sup.- adenoviral vectors. However, Garnier et al.
(1994) observed that the 293N35 cells had a relatively long initial
lag phase in suspension, a low growth rate, and a strong tendency
to clump.
[0083] The second method that has been used is a gradual adaptation
of 293A cells into suspension growth (Cold Spring Harbor
Laboratories, 293S cells). Garnier et al. (1994) reported the use
of 293S cells for production of recombinant proteins from
adenoviral vectors. The authors found that 293S cells were much
less clumpy in calcium-free media and a fresh medium exchange at
the time of virus infection could significantly increase the
protein production. It was found that glucose was the limiting
factor in culture without medium exchange.
[0084] 1. Growth in Selection Media
[0085] In certain embodiments, it may be useful to employ selection
systems that preclude growth of undesirable cells. This may be
accomplished by virtue of permanently transforming a cell line with
a selectable marker or by transducing or infecting a cell line with
a viral vector that encodes a selectable marker. In either
situation, culture of the transformed/transduced cell with an
appropriate drug or selective compound will result in the
enhancement, in the cell population, of those cells carrying the
marker.
[0086] Examples of markers include, but are not limited to, HSV
thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase
and adenine phosphoribosyltransferase genes, in tk-, hgprt- or
aprt-cells, respectively. Also, anti-metabolite resistance can be
used as the basis of selection for dhfr, that confers resistance to
methotrexate; gpt, that confers resistance to mycophenolic acid;
neo, that confers resistance to the aminoglycoside G418; and hygro,
that confers resistance to hygromycin.
[0087] 2. Growth in Serum Weaning
[0088] Serum weaning adaptation of anchorage-dependent cells into
serum-free suspension cultures have been used for the production of
recombinant proteins (Berg, 1993) and viral vaccines (Perrin,
1995). There have been few reports on the adaptation of 293A cells
into serum-free suspension cultures until recently. Gilbert
reported the adaptation of 293A cells into serum-free suspension
cultures for adenovirus and recombinant protein production
(Gilbert, 1996). Similar adaptation method had been used for the
adaptation of A549 cells into serum-free suspension culture for
adenovirus production (Morris et al., 1996). Cell-specific virus
yields in the adapted suspension cells, however, are about
5-10-fold lower than those achieved in the parental attached
cells.
[0089] Using the similar serum weaning procedure, the inventors
have successfully adapted the 293A cells into serum-free suspension
culture (293SF cells). In this procedure, the 293 cells were
adapted to a commercially available 293 media by sequentially
lowering down the FBS concentration in T-flasks. Briefly, the
initial serum concentration in the media was approximately 10% FBS
DMEM media in T-75 flask and the cells were adapted to serum-free
IS 293 media in T-flasks by lowering down the FBS concentration in
the media sequentially. After 6 passages in T-75 flasks the FBS %
was estimated to be about 0.019% and the 293 cells. The cells were
subcultured two more times in the T flasks before they were
transferred to spinner flasks. The results described herein below
show that cells grow satisfactorily in the serum-free medium (IS293
medium, Irvine Scientific, Santa Ana, Calif.). Average doubling
time of the cells was 20-35 hours achieving stationary cell
concentrations in the order of 3-5.times.10.sup.6 cells/ml without
medium exchange.
[0090] 3. Adaptation of Cells for Suspension Culture
[0091] Two methodologies have been used to adapt 293 cells, into
suspension cultures. Graham adapted 293A cells into suspension
culture (293N3S cells) by 3 serial passages in nude mice (Graham,
1987). The suspension 293N3S cells were found to be capable of
supporting E1.sup.- adenoviral vectors. However, Garnier et al.
(1994) observed that the 293N35 cells had a relatively long initial
lag phase in suspension, a low growth rate, and a strong tendency
to clump.
[0092] The second method that has been used is a gradual adaptation
of 293A cells into suspension growth (Cold Spring Harbor
Laboratories, 293S cells). Garnier et al. (1994) reported the use
of 293S cells for production of recombinant proteins from
adenoviral vectors. The authors found that 293S cells were much
less clumpy in calcium-free media and a fresh medium exchange at
the time of virus infection could significantly increase the
protein production. It was found that glucose was the limiting
factor in culture without medium exchange.
[0093] In the present invention, the 293 cells adapted for growth
in serum-free conditions were adapted into a suspension culture.
The cells were transferred in a serum-free 250 mL spinner
suspension culture (100 mL working volume) for the suspension
culture at an initial cell density of between about 1.18E+5 vc/mL
and about 5.22E+5 vc/mL. The media may be supplemented with heparin
to prevent aggregation of cells. This cell culture systems allows
for some increase of cell density whilst cell viability is
maintained. Once these cells are growing in culture, they cells are
subcultured in the spinner flasks approximately 7 more passages. It
may be noted that the doubling time of the cells is progressively
reduced until at the end of the successive passages the doubling
time is about 1.3 day, i.e. comparable to 1.2 day of the cells in
10% FBS media in the attached cell culture. In the serum-free IS
293 media supplemented with heparin almost all the cells existed as
individual cells not forming aggregates of cells in the suspension
culture.
[0094] C. Cell Culture Systems
[0095] The ability to produce infectious viral vectors is
increasingly important to the pharmaceutical industry, especially
in the context of gene therapy. Over the last decade, advances in
biotechnology have led to the production of a number of important
viral vectors that have potential uses as therapies, vaccines and
protein production machines. The use of viral vectors in mammalian
cultures has advantages over proteins produced in bacterial or
other lower life form hosts in their ability to
post-translationally process complex protein structures such as
disulfide-dependent folding and glycosylation.
[0096] Development of cell culture for production of virus vectors
has been greatly aided by the development in molecular biology of
techniques for design and construction of vector systems highly
efficient in mammalian cell cultures, a battery of useful selection
markers, gene amplification schemes and a more comprehensive
understanding of the biochemical and cellular mechanisms involved
in procuring the final biologically-active molecule from the
introduced vector.
[0097] Frequently, factors which affect the downstream (in this
case, beyond the cell lysis) side of manufacturing scale-up were
not considered before selecting the cell line as the host for the
expression system. Also, development of bioreactor systems capable
of sustaining very high density cultures for prolonged periods of
time have not lived up to the increasing demand for increased
production at lower costs.
[0098] The present invention will take advantage of the recently
available bioreactor technology. Growing cells according to the
present invention in a bioreactor allows for large scale production
of fully biologically-active cells capable of being infected by the
adenoviral vectors of the present invention. By operating the
system at a low perfusion rate and applying a different scheme for
purification of the infecting particles, the invention provides a
purification strategy that is easily scaleable to produce large
quantities of highly purified product.
[0099] PCT publication No. WO 98/00524, U.S. Pat. No. 6,194,191,
U.S. Published Patent Application No. US-2002-0182723-A1, and U.S.
Provisional Patent Application No. 60/406,591 (filed Aug. 28,
2002), which have described viral production methods, are
specifically herein incorporated by reference for their description
of techniques for culturing, production and purification of
recombinant viral particles.
[0100] Certain embodiments of the present invention pertain to
methods for producing an adenovirus that require the use of a
bioreactor. As used herein, a "bioreactor" refers to any apparatus
that can be used for the purpose of culturing cells. Growing cells
according to the present invention in a bioreactor allows for large
scale production of fully biologically-active cells capable of
being infected by the adenoviral vectors of the present
invention.
[0101] Bioreactors have been widely used for the production of
biological products from both suspension and anchorage dependent
animal cell cultures. The most widely used producer cells for
adenoviral vector production are anchorage dependent human
embryonic kidney cells (293 cells). Bioreactors to be developed for
adenoviral vector production should have the characteristic of high
volume-specific culture surface area in order to achieve high
producer cell density and high virus yield. Microcarrier cell
culture in stirred tank bioreactor provides very high
volume-specific culture surface area and has been used for the
production of viral vaccines (Griffiths, 1986). Furthermore,
stirred tank bioreactors have industrially been proven to be
scaleable. The multiplate Cellcube.TM.. cell culture system
manufactured by Corning-Costar also offers a very high
volume-specific culture surface area. Cells grow on both sides of
the culture plates hermetically sealed together in the shape of a
compact cube. Unlike stirred tank bioreactors, the CellCube.TM..
culture unit is disposable. This is very desirable at the early
stage production of clinical product because of the reduced capital
expenditure, quality control and quality assurance costs associated
with disposable systems. In consideration of the advantages offered
by the different systems, both the stirred tank bioreactor and the
CellCube.TM. system were evaluated for the production of
adenovirus.
[0102] Certain embodiments of the present invention require the use
of a Wave Bioreactor.RTM., particularly for use in methods for
generating adenovirus in serum-free suspension cultures. The Wave
Bioreactor.RTM. is a pre-sterilized disposable bioreactor system
that can be easily retrofitted with a variety of cleanroom
configurations without requiring expensive CIP and SIP process
utilities. The Wave Bioreactor.RTM. can be a Wave Biotech.RTM.
model 20/50EH. The bioreactor can hold any volume of media, but in
a certain embodiment the bioreactor is a 10 L (5 L working volume)
bioreactor. In certain embodiments, the bioreactor can be adjusted
to rock at a particular speed and angle. In certain other
embodiments, the bioreactor may include a device for monitoring
dissolved oxygen tension, such as a disposable dissolved oxygen
tension (DOT) probe. The bioreactor may also include a device for
monitoring temperature in the media. Other embodiments include a
device for measuring and adjusting culture pH, such as a gas mixer
which can adjust CO.sub.2 gas percentage delivered to the media.
The bioreactor may or may not be a disposable bioreactor. According
to a preferred aspect of the invention, the Wave Bioreactor.RTM. is
used with serum-free media and the initial lactate concentration of
the medium is made as low as possible because high lactate
concentration inhibits virus production. Further, an adequate
glucose concentration should be maintained as glucose limitation
can also inhibit virus production. As used herein, "media" and
"medium" refers to any substance which can facilitate growth of
cells. According to one aspect of the present invention, the host
cells are grown in media that is serum-free media. In other
embodiments of the present invention, the host cells are grown in
media that is protein-free media. One example of a protein-free
media is CD293. Another example of media that can support host cell
growth in a particular embodiment of the invention is DMEM+2% FBS.
On of skill in the art would understand that various components and
agents can be added to the media to facilitate and control cell
growth. For example, the glucose concentration of the media can be
maintained at a certain level. In one embodiment of the present
methods for producing adenovirus, the glucose concentration is
maintained between about 0.5 and about 3.0 gm glucose/liter.
[0103] 1. Anchorage-Dependent Versus Non-Anchorage-Dependent
Cultures
[0104] In some embodiments of the present invention, the methods
for producing an adenovirus require growing host cells in
anchorage-dependent cultures, whereas other embodiments pertain to
methods for producing an adenovirus in non-anchorage-dependent
cultures. Animal and human cells can be propagated in vitro in two
modes: as non-anchorage dependent cells growing freely in
suspension throughout the bulk of the culture; or as
anchorage-dependent cells requiring attachment to a solid substrate
for their propagation (i.e., a monolayer type of cell growth).
[0105] Non-anchorage dependent or suspension cultures from
continuous established cell lines are the most widely used means of
large scale production of cells and cell products. Large scale
suspension culture based on microbial (bacterial and yeast)
fermentation technology has clear advantages for the manufacturing
of mammalian cell products. The processes are relatively simple to
operate and straightforward to scale up. Homogeneous conditions can
be provided in the reactor which allows for precise monitoring and
control of temperature, dissolved oxygen, and pH, and ensure that
representative samples of the culture can be taken.
[0106] However, suspension cultured cells cannot always be used in
the production of biologicals. Suspension cultures are still
considered to have tumorigenic potential and thus their use as
substrates for production put limits on the use of the resulting
products in human and veterinary applications (Petricciani, 1985;
Larsson, 1987). Viruses propagated in suspension cultures as
opposed to anchorage-dependent cultures can sometimes cause rapid
changes in viral markers, leading to reduced immunogenicity
(Bahnemann, 1980). Finally, sometimes even recombinant cell lines
can secrete considerably higher amounts of products when propagated
as anchorage-dependent cultures as compared with the same cell line
in suspension (Nilsson and Mosbach, 1987). For these reasons,
different types of anchorage-dependent cells are used extensively
in the production of different biological products.
[0107] 2. Reactors and Processes for Suspension
[0108] The bioreactors utilized in the context of selected
embodiments of the present invention may be stirred tank
bioreactors. Large scale suspension culture of mammalian cultures
in stirred tanks have been described. The instrumentation and
controls for bioreactors adapted, along with the design of the
fermentors, from related microbial applications. However,
acknowledging the increased demand for contamination control in the
slower growing mammalian cultures, improved aseptic designs were
quickly implemented, improving dependability of these reactors.
Instrumentation and controls are basically the same as found in
other fermentors and include agitation, temperature, dissolved
oxygen, and pH controls. More advanced probes and autoanalyzers for
on-line and off-line measurements of turbidity (a function of
particles present), capacitance (a function of viable cells
present), glucose/lactate, carbonate/bicarbonate and carbon dioxide
are available. In one embodiment of the present invention, the
autoanalyzer is a YSI-2700 SELECT.TM. analyzer.
[0109] Two suspension culture reactor designs are most widely used
in the industry due to their simplicity and robustness of
operation--the stirred reactor and the airlift reactor. The stirred
reactor design has successfully been used on a scale of 8000 liter
capacity for the production of interferon (Phillips et al., 1985;
Mizrahi, 1983). Cells are grown in a stainless steel tank with a
height-to-diameter ratio of 1:1 to 3:1. The culture is usually
mixed with one or more agitators, based on bladed disks or marine
propeller patterns. Agitator systems offering less shear forces
than blades have been described. Agitation may be driven either
directly or indirectly by magnetically coupled drives. Indirect
drives reduce the risk of microbial contamination through seals on
stirrer shafts.
[0110] The airlift reactor, also initially described for microbial
fermentation and later adapted for mammalian culture, relies on a
gas stream to both mix and oxygenate the culture. The gas stream
enters a riser section of the reactor and drives circulation. Gas
disengages at the culture surface, causing denser liquid free of
gas bubbles to travel downward in the downcomer section of the
reactor. The main advantage of this design is the simplicity and
lack of need for mechanical mixing. Typically, the
height-to-diameter ratio is 10:1. The airlift reactor scales up
relatively easily, has good mass transfer of gasses and generates
relatively low shear forces.
[0111] Most large-scale suspension cultures are operated as batch
or fed-batch processes because they are the most straightforward to
operate and scale up. However, continuous processes based on
chemostat or perfusion principles are available.
[0112] A batch process is a closed system in which a typical growth
profile is seen. A lag phase is followed by exponential, stationary
and decline phases. In such a system, the environment is
continuously changing as nutrients are depleted and metabolites
accumulate. This makes analysis of factors influencing cell growth
and productivity, and hence optimization of the process, a complex
task. Productivity of a batch process may be increased by
controlled feeding of key nutrients to prolong the growth cycle.
Such a fed-batch process is still a closed system because cells,
products and waste products are not removed.
[0113] In what is still a closed system, perfusion of fresh medium
through the culture can be achieved by retaining the cells with a
variety of devices (e.g. fine mesh spin filter, hollow fiber or
flat plate membrane filters, settling tubes). Spin filter cultures
can produce cell densities of approximately 5.times.10.sup.7
cells/ml. A true open system and the simplest perfusion process is
the chemostat in which there is an inflow of medium and an outflow
of cells and products. Culture medium is fed to the reactor at a
predetermined and constant rate which maintains the dilution rate
of the culture at a value less than the maximum specific growth
rate of the cells (to prevent washout of the cell mass from the
reactor). Culture fluid containing cells and cell products and
byproducts is removed at the same rate.
[0114] In certain embodiments of the present methods for producing
adenovirus, the bioreactor system is set up to include a system to
allow for media exchange. For example, filters may be incorporated
into the bioreactor system to allow for separation of cells from
spent media to facilitate media exchange. In some embodiments of
the present methods for producing adenovirus, media exchange and
perfusion is conducted beginning on a certain day of cell growth.
For example, media exchange and perfusion can begin on day 3 of
cell growth. The filter may be, external to the bioreactor, or
internal to the bioreactor.
[0115] In one embodiment of the present invention, the filter is a
floating flat filter that is internal to the bioreactor. The filter
provides for separation between the cells and spent medium. In
certain embodiments, the spent culture media is withdrawn through
the floating filer. Recirculation of the media may or may not be
required in the various embodiments of the present invention. In
one embodiment, wave action is used to minimize clogging of the
filter during media perfusion. The culture volume may be maintained
by a load cell used to trigger fresh medium addition. One of skill
in the art would be familiar with the various types of filters that
can be used for perfusion of media, and the various methods that
can be employed for attaching the filter to the bioreactor and
incorporating it into the cell growth process.
[0116] 3. Non-Perfused Attachment Systems
[0117] Traditionally, anchorage-dependent cell cultures are
propagated on the bottom of small glass or plastic vessels. The
restricted surface-to-volume ratio offered by classical and
traditional techniques, suitable for the laboratory scale, has
created a bottleneck in the production of cells and cell products
on a large scale. In an attempt to provide systems that offer large
accessible surfaces for cell growth in small culture volume, a
number of techniques have been proposed the roller bottle system,
the stack plate's propagator, the spiral film bottles, the hollow
fiber system, the packed bed, the plate exchanger system, and the
membrane tubing reel. Since these systems are non-homogeneous in
their nature, and are sometimes based on multiple processes, they
suffer from the following shortcomings--limited potential for
scale-up, difficulties in taking cell samples, limited potential
for measuring and controlling key process parameters and difficulty
in maintaining homogeneous environmental conditions throughout the
culture.
[0118] Despite these drawbacks, a commonly used process for large
scale anchorage-dependent cell production is the roller bottle.
Being little more than a large, differently shaped T-flask,
simplicity of the system makes it very dependable and, hence,
attractive. Fully automated robots are available that can handle
thousands of roller bottles per day, thus eliminating the risk of
contamination and inconsistency associated with the otherwise
required intense human handling. With frequent media changes,
roller bottle cultures can achieve cell densities of close to
0.5.times.10.sup.6 cells/cm.sup.2 (corresponding to approximately
10.sup.9 cells/bottle or almost 10.sup.7 cells/ml of culture
media).
[0119] 4. Cultures on Microcarriers
[0120] In an effort to overcome the shortcomings of the traditional
anchorage-dependent culture processes, van Wezel (1967) developed
the concept of the microcarrier culturing systems. In this system,
cells are propagated on the surface of small solid particles
suspended in the growth medium by slow agitation. Cells attach to
the microcarriers and grow gradually to confluency on the
microcarrier surface. In fact, this large scale culture system
upgrades the attachment dependent culture from a single disc
process to a unit process in which both monolayer and suspension
culture have been brought together. Thus, combining the necessary
surface for a cell to grow with the advantages of the homogeneous
suspension culture increases production.
[0121] The advantages of microcarrier cultures over most other
anchorage-dependent, large-scale cultivation methods are several
fold. First, microcarrier cultures offer a high surface-to-volume
ratio (variable by changing the carrier concentration) which leads
to high cell density yields and a potential for obtaining highly
concentrated cell products. Cell yields are up to
1-2.times.10.sup.7 cells/ml when cultures are propagated in a
perfused reactor mode. Second, cells can be propagated in one unit
process vessels instead of using many small low-productivity
vessels (i.e., flasks or dishes). This results in far better
nutrient utilization and a considerable saving of culture medium.
Moreover, propagation in a single reactor leads to reduction in
need for facility space and in the number of handling steps
required per cell, thus reducing labor cost and risk of
contamination. Third, the well-mixed and homogeneous microcarrier
suspension culture makes it possible to monitor and control
environmental conditions (e.g., pH, pO.sub.2, and concentration of
medium components), thus leading to more reproducible cell
propagation and product recovery. Fourth, it is possible to take a
representative sample for microscopic observation, chemical
testing, or enumeration. Fifth, since microcarriers settle out of
suspension quickly, use of a fed-batch process or harvesting of
cells can be done relatively easily. Sixth, the mode of the
anchorage-dependent culture propagation on the microcarriers makes
it possible to use this system for other cellular manipulations,
such as cell transfer without the use of proteolytic enzymes,
cocultivation of cells, transplantation into animals, and
perfusion, of the culture using decanters, columns, fluidized beds,
or hollow fibers for microcarrier retainment. Seventh, microcarrier
cultures are relatively easily scaled up using conventional
equipment used for cultivation of microbial and animal cells in
suspensions.
[0122] 5. Microencapsulation of Mammalian Cells
[0123] One method which has shown to be particularly useful for
culturing mammalian cells is microencapsulation. The mammalian
cells are retained inside a semipermeable hydrogel membrane. A
porous membrane is formed around the cells permitting the exchange
of nutrients, gases, and metabolic products with the bulk medium
surrounding the capsule. Several methods have been developed that
are gentle, rapid and non-toxic and where the resulting membrane is
sufficiently porous and strong to sustain the growing cell mass
throughout the term of the culture. These methods are all based on
soluble alginate gelled by droplet contacts with a
calcium-containing solution. Lim (1982, U.S. Pat. No. 4,352,883,
incorporated herein by reference) describes cells concentrated in
an approximately 1% solution of sodium alginate which are forced
through a small orifice, forming droplets, and breaking free into
an approximately 1% calcium chloride solution. The droplets are
then cast in a layer of polyamino acid that ionically bonds to the
surface alginate. Finally the alginate is reliquefied by treating
the droplet in a chelating agent to remove the calcium ions. Other
methods use cells in a calcium solution to be dropped into a
alginate solution, thus creating a hollow alginate sphere. A
similar approach involves cells in a chitosan solution dropped into
alginate, also creating hollow spheres.
[0124] Microencapsulated cells are easily propagated in stirred
tank reactors and, with beads sizes in the range of 150-1500 .mu.m
in diameter, are easily retained in a perfused reactor using a
fine-meshed screen. The ratio of capsule volume to total media
volume can be maintained from as dense as 1:2 to 1:10. With
intracapsular cell densities of up to 10.sup.8, the effective cell
density in the culture is 1-5.times.10.sup.7.
[0125] The advantages of microencapsulation over other processes
include the protection from the deleterious effects of shear
stresses which occur from sparging and agitation, the ability to
easily retain beads for the purpose of using perfused systems,
scale up is relatively straightforward and the ability to use the
beads for implantation.
[0126] The current invention includes cells which are
anchorage-dependent in nature. 293 cells, for example, are
anchorage-dependent, and when grown in suspension, the cells will
attach to each other and grow in clumps, eventually suffocating
cells in the inner core of each clump as they reach a size that
leaves the core cells unsustainable by the culture conditions.
Therefore, an efficient means of large-scale culture of
anchorage-dependent cells is needed in order to effectively employ
these cells to generate large quantities of adenovirus.
[0127] 6. Perfused Attachment Systems
[0128] Certain embodiments of the present invention involve methods
for producing an adenovirus that involve use of perfused attachment
systems. Perfusion refers to continuous flow at a steady rate,
through or over a population of cells (of a physiological nutrient
solution). It implies the retention of the cells within the culture
unit as opposed to continuous-flow culture which washes the cells
out with the withdrawn media (e.g., chemostat). The idea of
perfusion has been known since the beginning of the century, and
has been applied to keep small pieces of tissue viable for extended
microscopic observation. The technique was initiated to mimic the
cells milieu in vivo where cells are continuously supplied with
blood, lymph, or other body fluids. Without perfusion, cells in
culture go through alternating phases of being fed and starved,
thus limiting full expression of their growth and metabolic
potential.
[0129] The current use of perfused culture is in response to the
challenge of growing cells at high densities (i.e.,
0.1-5.times.10.sup.8 cells/ml). In order to increase densities
beyond 2-4.times.10.sup.6 cells/ml, the medium has to be constantly
replaced with a fresh supply in order to make up for nutritional
deficiencies and to remove toxic products. Perfusion allows for a
far better control of the culture environment (pH, pO.sub.2,
nutrient levels, etc.) and is a means of significantly increasing
the utilization of the surface area within a culture for cell
attachment.
[0130] The development of a perfused packed-bed reactor using a bed
matrix of a non-woven fabric has provided a means for maintaining a
perfusion culture at densities exceeding 10.sup.8 cells/ml of the
bed volume (CelliGen.TM., New Brunswick Scientific, Edison, N.J.;
Wang et al., 1992; Wang et al., 1993; Wang et al., 1994). Briefly
described, this reactor comprises an improved reactor for culturing
of both anchorage- and non-anchorage-dependent cells. The reactor
is designed as a packed bed with a means to provide internal
recirculation. Preferably, a fiber matrix carrier is placed in a
basket within the reactor vessel. A top and bottom portion of the
basket has holes, allowing the medium to flow through the basket. A
specially designed impeller provides recirculation of the medium
through the space occupied by the fiber matrix for assuring a
uniform supply of nutrient and the removal of wastes. This
simultaneously assures that a negligible amount of the total cell
mass is suspended in the medium. The combination of the basket and
the recirculation also provides a bubble-free flow of oxygenated
medium through the fiber matrix. The fiber matrix is a non-woven
fabric having a "pore" diameter of from 10 .mu.m to 100 .mu.m,
providing for a high internal volume with pore volumes
corresponding to 1 to 20 times the volumes of individual cells.
[0131] In comparison to other culturing systems, this approach
offers several significant advantages. With a fiber matrix carrier,
the cells are protected against mechanical stress from agitation
and foaming. The free medium flow through the basket provides the
cells with optimum regulated levels of oxygen, pH, and nutrients.
Products can be continuously removed from the culture and the
harvested products are free of cells and can be produced in
low-protein medium which facilitates subsequent purification steps.
Also, the unique design of this reactor system offers an easier way
to scale up the reactor. Currently, sizes up to 30 liter are
available. One hundred liter and 300 liter versions are in
development and theoretical calculations support up to a 1000 liter
reactor. This technology is explained in detail in WO 94/17178
(Aug. 4, 1994, Freedman et al.), which is hereby incorporated by
reference in its entirety.
[0132] The Cellcube.TM. (Corning-Costar) module provides a large
styrenic surface area for the immobilization and growth of
substrate attached cells. It is an integrally encapsulated sterile
single-use device that has a series of parallel culture plate
joined to create thin sealed laminar flow spaces between adjacent
plates.
[0133] The Cellcube.TM. module has inlet and outlet ports that are
diagonally opposite each other and help regulate the flow of media.
During the first few days of growth the culture is generally
satisfied by the media contained within the system after initial
seeding. The amount of time between the initial seeding and the
start of the media perfusion is dependent on the density of cells
in the seeding inoculum and the cell growth rate. The measurement
of nutrient concentration in the circulating media is a good
indicator of the status of the culture. When establishing a
procedure it may be necessary to monitor the nutrients composition
at a variety of different perfusion rates to determine the most
economical and productive operating parameters.
[0134] Cells within the system reach a higher density of solution
(cells/ml) than in traditional culture systems. Many typically used
basal media are designed to support 1-2.times.10.sup.6
cells/ml/day. A typical Cellcube.TM. run with an 85,000 cm.sup.2
surface, contains approximately 6 L media within the module. The
cell density often exceeds 10.sup.7 cells/mL in the culture vessel.
At confluence, 2-4 reactor volumes of media are required per
day.
[0135] The timing and parameters of the production phase of
cultures depends on the type and use of a particular cell line.
Many cultures require a different media for production than is
required for the growth phase of the culture. The transition from
one phase to the other will likely require multiple washing steps
in traditional cultures. However, the Cellcube.TM. system employs a
perfusion system. On of the benefits of such a system is the
ability to provide a gentle transition between various operating
phases. The perfusion system negates the need for traditional wash
steps that seek to remove serum components in a growth medium.
[0136] 7. Serum-Free Suspension Culture
[0137] In particular embodiments, adenoviral vectors for gene
therapy are produced from anchorage-dependent culture of 293 cells
(293A cells) as described above. Scale-up of adenoviral vector
production is constrained by the anchorage-dependency of 293A
cells. To facilitate scale-up and meet future demand for adenoviral
vectors, significant efforts have been devoted to the development
of alternative production processes that are amenable to scale-up.
Methods include growing 293A cells in microcarrier cultures and
adaptation of 293A producer cells into suspension cultures.
[0138] Microcarrier culture techniques have been described above.
This technique relies on the attachment of producer cells onto the
surfaces of microcarriers which are suspended in culture media by
mechanical agitation. The requirement of cell attachment may
present some limitations to the scaleability of microcarrier
cultures.
[0139] In certain embodiments of the present invention, the media
used in the methods for producing an adenovirus is a serum-free
media. In other embodiments of the present invention, the media is
a protein-free media. As previously discussed, certain embodiments
of the present invention involve use of bioreactors. The
bioreactors may be adapted for serum-free suspension culture of
cells. Filtration of media with media exchange may or may not be
included in the system.
[0140] D. Viral Infection
[0141] The present invention pertains to methods of producing an
adenovirus that include, infecting the host cells with an
adenovirus. Typically, the virus will simply be exposed to the
appropriate host cell under physiologic conditions, permitting
uptake of the virus. One of skill in the art would be familiar with
the wide range of techniques available for initiating virus
infection.
[0142] The present invention employs, in one example, adenoviral
infection of cells in order to generate therapeutically significant
vectors. Typically, the virus will simply be exposed to the
appropriate host cell under physiologic conditions, permitting
uptake of the virus. Though adenovirus is exemplified, the present
methods may be advantageously employed with other viral vectors, as
discussed below.
[0143] 1. Adenovirus
[0144] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid sized DNA genome, ease of
manipulation, high titer, wide target-cell range, and high
infectivity. The roughly 36 kB viral genome is bounded by 100-200
base pair (bp) inverted terminal repeats (ITR), in which are
contained cis-acting elements necessary for viral DNA replication
and packaging. The early (E) and late (L) regions of the genome
that contain different transcription units are divided by the onset
of viral DNA replication.
[0145] The E1 region (E1A and E1B) encodes proteins responsible for
the regulation of transcription of the viral genome and a few
cellular genes. The expression of the E2 region (E2A and E2B)
results in the synthesis of the proteins for viral DNA replication.
These proteins are involved in DNA replication, late gene
expression, and host cell shut off (Renan, 1990). The products of
the late genes (L1, L2, L3, L4 and L5), including the majority of
the viral capsid proteins, are expressed only after significant
processing of a single primary transcript issued by the major late
promoter (MLP). The MLP (located at 16.8 map units) is particularly
efficient during the late phase of infection, and all the mRNAs
issued from this promoter possess a 5' tripartite leader (TL)
sequence which makes them preferred mRNAs for translation.
[0146] In order for adenovirus to be optimized for gene therapy, it
is necessary to maximize the carrying capacity so that large
segments of DNA can be included. It also is very desirable to
reduce the toxicity and immunologic reaction associated with
certain adenoviral products. Elimination of large potions of the
adenoviral genome, and providing the delete gene products in trans,
by helper virus and/or helper cells, allows for the insertion of
large portions of heterologous DNA into the vector. This strategy,
also will result in reduced toxicity and immunogenicity of the
adenovirus gene products.
[0147] The large displacement of DNA is possible because the cis
elements required for viral DNA replication all are localized in
the inverted terminal repeats (ITR) (100-200 bp) at either end of
the linear viral genome. Plasmids containing ITR's can replicate in
the presence of a non-defective adenovirus (Hay et al., 1984).
Therefore, inclusion of these elements in an adenoviral vector
should permit replication.
[0148] In addition, the packaging signal for viral encapsidation is
localized between 194-385 bp (0.5-1.1 map units) at the left end of
the viral genome (Hearing et al., 1987). This signal mimics the
protein recognition site in bacteriophage lambda. DNA where a
specific sequence close to the left end, but outside the cohesive
end sequence, mediates the binding to proteins that are required
for insertion of the DNA into the head structure. E1 substitution
vectors of Ad have demonstrated that a 450 bp (0-1.25 map units)
fragment at the left end of the viral genome could direct packaging
in 293 cells (Levrero et al., 1991).
[0149] Previously, it has been shown that certain regions of the
adenoviral genome can be incorporated into the genome of mammalian
cells and the genes encoded thereby expressed. These cell lines are
capable of supporting the replication of an adenoviral vector that
is deficient in the adenoviral function encoded by the cell line.
There also have been reports of complementation of replication
deficient adenoviral vectors by "helping" vectors, e.g., wild-type
virus or conditionally defective mutants.
[0150] Replication-deficient adenoviral vectors can be
complemented, in trans, by helper virus. This observation alone
does not permit isolation of the replication-deficient vectors,
however, since the presence of helper virus, needed to provide
replicative functions, would contaminate any preparation. Thus, an
additional element was needed that would add specificity to the
replication and/or packaging of the replication-deficient vector.
That element, as provided for in the present invention, derives
from the packaging function of adenovirus.
[0151] It has been shown that a packaging signal for adenovirus
exists in the left end of the conventional adenovirus map
(Tibbetts, 1977). Later studies showed that a mutant with a
deletion in the E1A (194-358 bp) region of the genome grew poorly
even in a cell line that complemented the early (E1A) function
(Hearing and Shenk, 1983). When a compensating adenoviral DNA
(0-353 bp) was recombined into the right end of the mutant, the
virus was packaged normally. Further mutational analysis identified
a short, repeated, position-dependent element in the left end of,
the Ad5 genome. One copy of the repeat was found to be sufficient
for efficient packaging if present at either end of the genome, but
not when moved towards the interior of the Ad5 DNA molecule
(Hearing et al., 1987).
[0152] By using mutated versions of the packaging signal, it is
possible to create helper viruses that are packaged with varying
efficiencies. Typically, the mutations are point mutations or
deletions. When helper viruses with low efficiency packaging are
grown in helper cells, the virus is packaged, albeit at reduced
rates compared to wild-type virus, thereby permitting propagation
of the helper. When these helper viruses are grown in cells along
with virus that contains wild-type packaging signals, however, the
wild-type packaging signals are recognized preferentially over the
mutated versions. Given a limiting amount of packaging factor, the
virus containing the wild-type signals are packaged selectively
when compared to the helpers. If the preference is great enough,
stocks approaching homogeneity should be achieved.
[0153] 2. Retrovirus
[0154] Although adenoviral infection of cells for the generation of
therapeutically significant vectors is a preferred embodiments of
the present invention, it is contemplated that the present
invention may employ retroviral infection of cells for the purposes
of generating such vectors. The retroviruses are a group of
single-stranded RNA viruses characterized by an ability to convert
their RNA to double-stranded DNA in infected cells by a process of
reverse-transcription (Coffin, 1990). The resulting DNA then stably
integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins. The integration results in the
retention of the viral gene sequences in the recipient cell and its
descendants. The retroviral genome contains three genes--gag, pol
and env--that code for capsid proteins, polymerase enzyme, and
envelope components, respectively. A sequence found upstream from
the gag gene, termed Y, functions as a signal for packaging of the
genome into virions. Two long terminal repeat (LTR) sequences are
present at the 5' and 3' ends of the viral genome. These contain
strong promoter and enhancer sequences and are also required for
integration in the host cell genome (Coffin, 1990).
[0155] In order to construct a retroviral vector, a nucleic acid
encoding a promoter is inserted into the viral genome in the place
of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol and env genes but without the LTR
and Y components is constructed (Mann et al., 1983). When a
recombinant plasmid containing a human cDNA, together with the
retroviral LTR and Y sequences is introduced into this cell line
(by calcium phosphate precipitation for example), the Y sequence
allows the RNA transcript of the recombinant plasmid to be packaged
into viral particles, which are then secreted into the culture
media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al.,
1983). The media containing the recombinant retroviruses is then
collected, optionally concentrated, and used for gene transfer.
Retroviral vectors are able to infect a broad variety of cell
types. However, integration and stable expression require the
division of host cells (Paskind et al., 1975).
[0156] A novel approach designed to allow specific targeting of
retrovirus vectors was recently developed based on the chemical
modification of a retrovirus by the chemical addition of galactose
residues to the viral envelope. This modification could permit the
specific infection of cells such as hepatocytes via
asialoglycoprotein receptors, should this be desired.
[0157] A different approach to targeting of recombinant
retroviruses was designed in which biotinylated antibodies against
a retroviral envelope protein and against a specific cell receptor
were used. The antibodies were coupled via the biotin components by
using streptavidin (Roux et al., 1989). Using antibodies against
major histocompatibility complex class I and class II antigens, the
infection of a variety, of human cells that bore those surface
antigens was demonstrated with an ecotropic virus in vitro (Roux et
al., 1989).
[0158] 3. Other Viral Vectors
[0159] Other viral vectors may be employed as expression constructs
in the present invention. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988), adeno-associated virus (AAV) (Ridgeway, 1988;
Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), herpes
viruses and lentivirus may be employed. These viruses offer several
features for use in gene transfer into various mammalian cells.
[0160] 4. Methods of Gene Transfer
[0161] In order to create the helper cell lines of the present
invention, and to create recombinant adenovirus vectors for use
therewith, various genetic (i.e. DNA) constructs must be delivered
to a cell. One way to achieve this is via viral transductions using
infectious viral particles, for example, by transformation with an
adenovirus vector of the present invention. Alternatively,
retroviral or bovine papilloma virus may be employed, both of which
permit permanent transformation of a host cell with a gene(s) of
interest. In other situations, the nucleic acid to be transferred
is not infectious, i.e., contained in an infectious virus particle.
This genetic material must rely on non-viral methods for
transfer.
[0162] Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells also are contemplated by
the present invention. These include calcium phosphate
precipitation (Graham, and Van Der Eb, 1973; Chen and Okayama,
1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985),
electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),
direct microinjection (Harland and Weintraub, 1985), DNA-loaded
liposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell
sonication (Fechheimer, et al., 1987), gene bombardment using high
velocity microprojectiles (Yang et al., 1990), and
receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,
1988)
[0163] Once the construct has been delivered into the cell the
nucleic acid encoding the therapeutic gene may be positioned and
expressed at different sites. In certain embodiments, the nucleic
acid encoding the therapeutic gene may be stably integrated into
the genome of the cell. This integration may be in the cognate
location and orientation via homologous recombination (gene
replacement) or it may be integrated in a random, non-specific
location (gene augmentation). In yet further embodiments, the
nucleic acid may be stably maintained in the cell as a separate,
episomal segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication
independent of or in synchronization with the host cell cycle. How
the expression construct is delivered to a cell and where in the
cell the nucleic acid remains is dependent on the type of
expression construct employed.
[0164] In one embodiment of the invention, the expression construct
may simply consist of naked recombinant DNA or plasmids. Transfer
of the construct may be performed by any of the methods mentioned
above which physically or chemically permeabilize the cell
membrane. This is particularity applicable for transfer in vitro,
however, it may be applied for in vivo use as well. Dubensky et al.
(1984) successfully injected polyomavirus DNA in the form of
CaPO.sub.4 precipitates into liver and spleen of adult and newborn
mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif (1986) also demonstrated that direct
intraperitoneal injection of CaPO.sub.4 precipitated plasmids
results in expression of the transfected genes. It is envisioned
that DNA encoding a CAM may also be transferred in a similar manner
in vivo and express CAM.
[0165] Another embodiment of the invention for transferring a naked
DNA expression construct into cells may involve particle
bombardment. This method depends on the ability to accelerate DNA
coated microprojectiles to a high velocity allowing them to pierce
cell membranes and enter cells without killing them (Klein et al.,
1987). Several devices; for accelerating small particles have been
developed. One such device relies on a high voltage discharge to
generate an electrical current, which in turn provides the motive
force (Yang et al., 1990). The microprojectiles used have consisted
of biologically inert substances such as tungsten or gold
beads.
[0166] In a further embodiment of the invention, the expression
construct may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and an
inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before, the formation
of closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991).
[0167] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful. Using the
.beta.-lactamase gene, Wong et al. (1980) demonstrated the
feasibility of liposome-mediated delivery and expression of foreign
DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et
al. (1987) accomplished successful liposome-mediated gene transfer
in rats after intravenous injection. Also included are various
commercial approaches involving "lipofection" technology.
[0168] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear nonhistone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression constructs have been successfully employed in
transfer and expression of nucleic acid in, vitro and in vivo, then
they are applicable for the present invention.
[0169] Other expression constructs which can be employed to deliver
a nucleic acid encoding a therapeutic gene into cells are
receptor-mediated delivery vehicles. These take advantage of the
selective uptake of macromolecules by receptor-mediated endocytosis
in almost all eukaryotic cells. Because of the cell type-specific
distribution of various receptors, the delivery can be highly
specific (Wu and Wu, 1993).
[0170] Receptor-mediated gene targeting vehicles generally consist
of two components: a cell receptor-specific ligand and a
DNA-binding agent. Several ligands have been used for
receptor-mediated gene transfer. The most extensively characterized
ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and
transferring (Wagner et al., 1990). Recently, a synthetic
neoglycoprotein, which recognizes the same receptor as ASOR, has
been used as a gene delivery vehicle (Ferkol et al., 1993; Perales
et al., 1994) and epidermal growth factor (EGF) has also been used
to deliver genes to squamous carcinoma cells (Myers, EPO
0273085).
[0171] In other embodiments, the delivery vehicle may comprises a
ligand and a liposome. For example, Nicolau et al. (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated into liposomes and observed an increase in the uptake
of the insulin gene by hepatocytes. Thus, it is feasible that a
nucleic acid encoding a therapeutic gene also may be specifically
delivered into a cell type such as prostate, epithelial or tumor
cells, by any number of receptor-ligand systems with or without
liposomes. For example, the human prostate-specific antigen (Watt
et al., 1986) may be used as the receptor for mediated delivery of
a nucleic acid in prostate tissue.
[0172] In certain embodiments of the present invention, the
temperature at which infection of the host cells is performed is
37.degree. C. However, in other embodiments, the infection
temperature is at temperature that is less than 37.degree. C. This
is based on the inventors' discovery that infection temperatures
less than 37.degree. C. provide for optimal production of
adenovirus. Thus, for example, the temperature may be 32.1.degree.
C., 32.2.degree. C., 32.3.degree. C., 32.4.degree. C., 32.5.degree.
C., 32.6.degree. C., 32.7.degree. C., 32.8.degree. C., 32.9.degree.
C., 33.0.degree. C., 33.1.degree. C., 33.2.degree. C., 33.3.degree.
C., 33.4.degree. C., 33.5.degree. C., 33.6.degree. C., 33.7.degree.
C., 33.8.degree. C., 33.9.degree. C., 34.0.degree. C., 34.1.degree.
C., 34.2.degree. C., 34.3.degree. C., 34.4.degree. C., 34.5.degree.
C., 34.6.degree. C., 34.7.degree. C., 34.8.degree. C., 34.9.degree.
C., 35.0.degree. C., 35.1.degree. C., 35.2.degree. C., 35.3.degree.
C., 35.4.degree. C., 35.5.degree. C., 35.6.degree. C., 35.7.degree.
C., 35.8.degree. C., 35.9.degree. C., 36.0.degree. C., 36.1.degree.
C., 36.2.degree. C., 36.3.degree. C., 36.4.degree. C., 36.5.degree.
C., 36.6.degree. C., 36.7.degree. C., 36.8.degree. C., and
36.9.degree. C. and any range of temperature or increments of
temperature derivable therein. Any method known to those of skill
in the art may be used to measure the temperature of the cell
culture media. One of skill in the art would be familiar with the
wide range of methods available for measuring the temperature of
culture media.
[0173] For example, one convenient way to measure temperature would
be to use a real time digital device to measure the temperature
inside an incubator. Prior to the procedure, the digital device can
be calibrated using traceable temperature calibrations equipment to
verify accuracy of the digital device.
[0174] In certain embodiments of the present invention, the methods
for producing an adenovirus may involve initiating virus infection
by diluting the host cells with fresh media and adenovirus. This
avoids the need for a separate medium exchange step prior to
infection. The invention contemplates that any amount of dilution
of the host cells is contemplated by the present invention. In a
certain embodiment, the host cells are diluted 10-fold with fresh
media. The invention also contemplates any amount of virus added to
initiate infection. However, in a certain embodiment of the present
invention, virus infection will be initiated by adding 50 vp/host
cell.
[0175] The embodiments of the present invention contemplate that
virus infection can be allowed to proceed for any length of time.
However, in a certain embodiment, virus infection is allowed to
proceed for 4 days. In another embodiment of the present invention,
host cell growth is allowed to occur in one bioreactor, and
infection of host cells is conducted in a second bioreactor.
[0176] The term "adenovirus preparation" will be used herein to
describe the reaction mixture following initiation of infection
with adenovirus. The adenovirus preparation may include host cells
that have undergone lysis, cell fragments, adenovirus, media, and
any other components present in the reaction mixture during
infection. The adenovirus preparation may include intact host
cells, depending on how long infection was allowed to proceed. Some
or all of the host cells may have undergone cell lysis, with
release of viral particles into the surrounding media. The present
invention contemplates that in the embodiments of the methods for
producing an adenovirus, adenovirus isolation will occur at any
time and by any means known to those of skill in the art following
infection. For example, in one embodiment of the present invention,
isolating the adenovirus from the adenovirus preparation occurs 4
days after viral infection is completed.
[0177] E. Engineering of Viral Vectors
[0178] 1. Viral Vectors
[0179] In particular embodiments, a recombinant adenovirus is
contemplated for the delivery of expression constructs.
"Recombinant adenovirus," "adenovirus vector" or "adenoviral
expression vector" is meant to include those constructs containing
adenovirus sequences sufficient to (a) support packaging of the
construct and (b) to ultimately express a tissue or cell-specific
construct that has been cloned therein. The recombinant adenovirus
may encode a recombinant gene. Thus, a recombinant adenovirus may
include any of the engineered vectors that comprise adenoviral
sequences.
[0180] An adenovirus expression vector according to the present
invention comprises a genetically engineered form of the
adenovirus. The nature of the adenovirus vector is not believed to
be crucial to the successful practice of the invention. The
adenovirus may be of any of the known serotypes and/or subgroups
A-F. Adenovirus type 5 of subgroup C is the preferred starting
material in order to obtain one adenovirus vector for use in the
present invention. This is because adenovirus type 5 is a human
adenovirus about which a great deal of biochemical and genetic
information is known, and it has historically been used for most
constructions employing adenovirus as a vector.
[0181] Advantages of adenoviral gene transfer include the ability
to infect a wide variety of cell types, including non-dividing
cells, a mid-sized genome, ease of manipulation, high infectivity
and they can be grown to high titers (Wilson, 1996). Further,
adenoviral infection of host cells does not result in chromosomal
integration because adenoviral DNA can replicate in an episomal
manner, without potential genotoxicity associated with other viral
vectors. Adenoviruses also are structurally stable (Marienfeld et
al., 1999) and no genome rearrangement has been detected after
extensive amplification (Parks et al., 1997; Bett et al.,
1993).
[0182] Adenovirus growth and manipulation is known to those of
skill in the art, and exhibits broad host range in vitro and in
vivo (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat.
No. 5,824,544). This group of viruses can be obtained in high
titers, e.g., 10.sup.9 to 10.sup.11 plaque-forming units per ml,
and they are highly infective. The life cycle of adenovirus does
not require integration into the host cell genome. The foreign
genes delivered by adenovirus vectors are episomal and, therefore,
have low genotoxicity to host cells.
[0183] Although adenovirus based vectors offer several unique
advantages over other vector systems, they often are limited by
vector immunogenicity, size constraints for insertion of
recombinant genes, low levels of replication, and low levels of
transgene expression. A major concern in using adenoviral vectors
is the generation of a replication-competent virus during vector
production in a packaging cell line or during gene therapy
treatment of an individual. The generation of a
replication-competent virus could pose serious threat of an
unintended viral infection and pathological consequences for the
patient.
[0184] Certain embodiments of the present invention pertain to
methods of producing an adenovirus that involve
replication-deficient adenovirus. Armentano et al., describe the
preparation of a replication-deficient adenovirus vector, claimed
to eliminate the potential for the inadvertent generation of a
replication-competent adenovirus (U.S. Pat. No. 5,824,544). The
replication-deficient adenovirus method comprises a deleted E1
region and a relocated protein IX gene, wherein the vector
expresses a heterologous, mammalian gene.
[0185] A common approach for generating a denoviruses for use as a
gene transfer vector is the deletion of the E1 gene (E1.sup.-),
which is involved in the induction of the E2, E3 and E4 promoters
(Graham and Prevec, 1995). Subsequently, a therapeutic gene or
genes can be inserted recombinantly in place of the E1 gene,
wherein expression of the therapeutic gene(s) is driven by the E1
promoter or a heterologous promoter. The E1.sup.-,
replication-deficient virus is then proliferated in a "helper" cell
line that provides the E1 polypeptides in trans (e.g., the human
embryonic kidney cell line 293). Alternatively, the E3 region,
portions of the E4 region or both may be deleted, wherein a
heterologous nucleic acid sequence under the control of a promoter
operable in eukaryotic cells is inserted into the adenovirus genome
for use in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No.
5,932,210).
[0186] 2. Viral Vectors Encoding Therapeutic Genes
[0187] In certain embodiments, the invention may include methods of
producing an adenovirus where the adenovirus is a recombinant
adenovirus encoding a recombinant gene. The recombinant gene may be
operatively linked to a promoter. In certain other embodiments, the
recombinant gene is a therapeutic gene. The invention contemplates
use of any gene that has therapeutic or potential therapeutic value
in the treatment of a disease or genetic disorder. One of skill in
the art would be familiar with the wide range of such genes that
have been identified.
[0188] Gene therapy generally involves the introduction into cells
of therapeutic genes, also known as transgenes, whose expression
results in amelioration or treatment of disease or genetic
disorders. The therapeutic genes involved may be those that encode
proteins, structural or enzymatic RNAs, inhibitory products such as
antisense RNA or DNA, or any other gene product. Expression is the
generation of such a gene product or the resultant effects of the
generation of such a gene product. Thus, enhanced expression
includes the greater production of any therapeutic gene or the
augmentation of that product's role in determining the condition of
the cell, tissue, organ or organism. The delivery of therapeutic
genes by adenoviral vectors involves what may be termed
transduction of cells. As used here, transduction is defined as the
introduction into a cell a therapeutic gene, transgene, or
transgene construct by an adenoviral or related vector.
[0189] Many experiments, innovations, preclinical studies and
clinical trials are currently under investigation for the use of
adenoviruses as gene delivery vectors. For example, adenoviral gene
delivery-based gene therapies are being developed for liver
diseases (Han et al., 1999), psychiatric diseases (Lesch, 1999),
neurological diseases (Hermens and Verhaagen, 1998), coronary
diseases (Feldman et al., 1996), muscular diseases (Petrof, 1998),
and various cancers such as colorectal (Dorai et al., 1999),
bladder (Irie et al., 1999), prostate (Mincheff et al., 2000), head
and neck (Blackwell et al., 1999), breast (Stewart et al., 1999),
lung (Batra et al., 1999) and ovarian (Vanderkwaak et al.,
1999).
[0190] The particular therapeutic gene encoded by the adenoviral
vector is not limiting and includes those useful for various
therapeutic and research purposes, as well as reporter genes and
reporter gene systems and constructs useful in tracking the
expression of transgenes and the effectiveness of adenoviral and
adenoviral vector transduction. Thus, by way of example, the
following are classes of possible genes whose expression may be
enhanced by using the compositions and methods of the present
invention: developmental genes (e.g. adhesion molecules, cyclin
kinase inhibitors, Wnt family members, Pax family members, Winged
helix family members, Hox family members, cytokines/lymphokines and
their receptors, growth or differentiation factors and their
receptors, neurotransmitters and their receptors), oncogenes (e.g.
ABLI, BLC1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1,
ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL,
MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and
YES), tumor suppresser genes (e.g. APC, BRCA1, BRCA2, MADH4, MCC,
NF1, NF2, RB1, TP53 and WT1), enzymes (e.g. ACP desaturases and
hycroxylases, ADP-glucose pyrophorylases, ATPases, alcohol
dehycrogenases, amylases, amyloglucosidases, catalases, cellulases,
cyclooxygenases, decarboxylases, dextrinases, esterases, DNA and,
RNA polymerases, hyaluron synthases, galactosidases, glucanases,
glucose oxidases, GTPases, helicases, hemicellulases,
hyaluronidases, integrases, invertases, isomersases, kinases,
lactases, lipases, lipoxygenases, lyases, lysozymes,
pectinesterases, peroxidases, phosphatases, phospholipases,
phophorylases, polygalacturonases, proteinases and peptideases,
pullanases, recombinases, reverse transcriptases, topoisomerases,
xylanases), reporter genes (e.g. Green fluorescent protein and its
many color variants, luciferase, CAT reporter systems,
Beta-galactosidase, etc.), blood derivatives, hormones, lymphokines
(including interleukins), interferons, TNF, growth factors,
neurotransmitters or their precursors or synthetic enzymes, trophic
factors (such as BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5,
and the like), apolipoproteins (such as ApoAI, ApoAIV, ApoE, and
the like), dystrophin or a minidystrophic, tumor suppressor genes
(such as p53, Rb, Rap1A, DCC, k-rev, and the like), genes coding
for factors involved in coagulation (such as factors V II, VIII,
IX, and the like), suicide genes (such as thymidine kinase),
cytosine deaminase, or all or part of a natural or artificial
immunoglobulin (Fab, ScFv, and the like). Other examples of
therapeutic genes include fus, interferon .alpha., interferon
.beta., interferon .gamma., ADP (adenoviral death protein).
[0191] The therapeutic gene can also be an antisense gene or
sequence whose expression in the target cell enables the expression
of cellular genes or the transcription of cellular mRNA to be
controlled, or instance ribozymes. Such sequence can, for example,
be transcribed in the target cell into RNAs complementary to
cellular mRNAs. The therapeutic gene can also be a gene coding for
an antigenic peptide capable of generating an immune response in
man. In this particular embodiment, the invention hence makes it
possible to produce vaccines enabling humans to be immunized, in
particular against microorganisms and viruses.
[0192] The tumor suppressor oncogenes function to inhibit excessive
cellular proliferation. The inactivation of these genes destroys
their inhibitory activity, resulting in unregulated proliferation.
The tumor suppressors p53, p16 and C-CAM are described below.
[0193] p53 currently is recognized as a tumor suppressor gene. High
levels of mutant p53 have been found in many cells transformed by
chemical carcinogenesis, ultraviolet radiation, and several
viruses, including SV40. The p53 gene is a frequent target of
mutational inactivation in a wide variety of human tumors and is
already documented to be the most frequently-mutated gene in common
human cancers. It is mutated in over 50% of human NSCLC (Hollstein
et al., 1991) and in a wide spectrum of other tumors.
[0194] The p53 gene encodes a 393-amino-acid phophoprotein that can
form complexes with host proteins, such as large-T antigen and E1B.
The protein is found in normal tissues and cells, but at
concentrations which are minute by comparison with transformed
cells or tumor tissue. Interestingly, wild-type p53 appears to be
important in regulating cell growth and division. Overexpression of
wild-type p53 has been shown in some cases to be anti-proliferative
in human tumor cell lines. Thus, p53 can act as a negative
regulator of cell growth (Weinberg, 1991) and may directly suppress
uncontrolled cell growth or indirectly activate genes that suppress
this growth. Thus, absence or inactivation of wild-type p53 may
contribute to transformation. However, some studies indicate that
the presence of mutant p53 may be necessary for full expression of
the transforming potential of the gene.
[0195] Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are essential for the transforming ability of the oncogene. A
single genetic change prompted by point mutations can create
carcinogenic p53. Unlike other oncogenes, however, p53 point
mutations are know to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism
and passed on in the germ line. Various mutant alleles appear to
range from minimally dysfunctional to strongly penetrant, dominant
negative alleles (Weinberg, 1991).
[0196] Casey and colleagues have reported that transfection of DNA
encoding wild-type p53 into two human breast cancer cell lines
restores growth suppression control in such cells (Casey et al.,
1991). A similar effect has also been demonstrated on transfection
of wild-type, but not mutant, p53 into human lung cancer cell lines
(Takahasi et al., 1992). p53 appears dominant over the mutant gene
and will select against proliferation when transfected into cells
with the mutant gene. Normal expression of the transfected p53 is
not detrimental to normal cells with endogenous wild-type p53.
Thus, such constructs might be taken up by normal cells without
adverse effects. It is thus proposed that the treatment of
p53-associated cancers with wild-type p53 expression constructs
will reduce the number of malignant cells or their growth rate.
Furthermore, recent studies suggest that some p53 wild-type tumors
are also sensitive to the effects of exogenous p53 expression.
[0197] The major transitions of the eukaryotic cell cycle are
triggered by cyclin-dependent kinases, or CDK's. One CDK,
cyclin-dependent kinase 4 (CDK4), regulates progression through the
G.sub.1 phase. The activity of this enzyme may be to phosphorylate
Rb at late G.sub.1. The activity of CDK4 is controlled by an
activating subunit, D-type cyclin, and by an inhibitory subunit,
e.g. p16.sup.INK4, which has been biochemically characterized as a
protein that specifically binds to and inhibits CDK4, and thus may
regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,
1995). Since the p16.sup.INK4 protein is a CDK4 inhibitor (Serrano,
1993), deletion of this gene may increase the activity of CDK4,
resulting in hyperphosphorylation of the Rb protein p16 also is
known to regulate the function of CDK6.
[0198] p16.sup.INK4 belongs to a newly described class of
CDK-inhibitory proteins that also includes p16.sup.B, p21.sup.WAF1,
CIP1, SDI1, and p27.sup.KIP1. The p16.sup.INK4 gene maps to 9p21, a
chromosome region frequently deleted in many tumor types.
Homozygous deletions and mutations of the p16.sup.INK4 gene are
frequent in human tumor cell lines. This evidence suggests that the
p16.sup.INK4 gene is a tumor suppressor gene. This interpretation
has been challenged, however, by the observation that the frequency
of the p16.sup.INK4 gene alterations is much lower in primary
uncultured tumors than in cultured cell lines (Caldas et al., 1994;
Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994a;
Kamb et al., 1994b; Mori et al., 1994; Okamoto et al., 1994; Nobori
et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration
of wild-type p16.sup.INK4 function by transfection with a plasmid
expression vector reduced colony formation by some human cancer
cell lines (Okamoto, 1994; Arap, 1995).
[0199] C-CAM is expressed in virtually all epithelial cells (Odin
and Obrink, 1987). C-CAM, with an apparent molecular weight of 105
kD, was originally isolated from the plasma membrane of the rat
hepatocyte by its reaction with specific antibodies that neutralize
cell aggregation (Obrink, 1991). Recent studies indicate that,
structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily
and its sequence is highly homologous to carcinoembryonic antigen
(CEA) (Lin and Guidotti, 1989). Using a baculovirus expression
system, Cheung et al. (1993a; 1993b and 1993c) demonstrated that
the first Ig domain of C-CAM is critical for cell adhesion
activity.
[0200] Cell adhesion molecules, or CAMs are known to be involved in
a complex network of molecular interactions that regulate organ
development and cell differentiation (Edelman, 1985). Recent data
indicate that aberrant expression of CAMs may be involved in the
tumorigenesis of several neoplasms; for example, decreased
expression of E-cadherin, which is predominantly expressed in
epithelial cells, is associated with the progression of several
kinds of neoplasms (Edelman and Crossin, 1991; Frixen et al., 1991;
Bussemakers et al., 1992; Matsura et al., 1992; Umbas et al.,
1992). Also, Giancotti and Ruoslahti (1990) demonstrated that
increasing expression of .alpha..sub.5.beta..sub.1. integrin by
gene transfer can reduce tumorigenicity of Chinese hamster ovary
cells in vivo. C-CAM now has been shown to suppress tumor growth in
vitro and in vivo.
[0201] Other tumor suppressors that may be employed according to
the present invention include BRCA1, BRCA2, zac1, p73, MMAC-1, ATM,
HIC-1, DPC-4, FHIT, NF2, APC, DCC, PTEN, ING1, NOEY1, NOEY2, PML,
OVCA1, MADR2, WT1, 53BP2, and IRF-1. Other genes that may be
employed according to the present invention include Rb, APC, DCC,
NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1/PTEN,
DBCCR-1, FCC, rsk-3, p27, p57 p27/p16 fusions, p21/p27 fusions,
anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc,
neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes
involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1,
GDAIF, or their receptors) and MCC. Inducers of apoptosis, such as
Bax, Bak, Bcl-X..sub.s, Bik, Bid, Harakiri, Ad E1B, Bad and
ICE-CED3 proteases, similarly could find use according to the
present invention.
[0202] In certain embodiments the adenovirus comprises an exogenous
gene construct that is an mda-7 gene. MDA-7 is another putative
tumor suppressor that has been shown to suppress the growth of
cancer cells that are p53-wild-type, p53-null and p53-mutant. Also,
the observed upregulation of the apoptosis-related Bax gene in p53
null cells indicates that MDA-7 is capable of using p53-independent
mechanisms to induce the destruction of cancer cells.
[0203] Studies have shown that elevated expression of MDA-7
suppressed cancer cell growth in vitro and selectively induced
apoptosis in human breast cancer cells as well as inhibiting tumor
growth in nude mice (Jiang et al., 1996 and Su et al., 1998). Jiang
et al. (1996) report findings that MDA-7 is a potent growth
suppressing gene in cancer cells of diverse origins including
breast, central nervous system, cervix, colon, prostate, and
connective tissue. A colony inhibition assay was used to
demonstrate that elevated expression of MDA-7 enhanced growth
inhibition in human cervical carcinoma (HeLa), human breast
carcinoma (MCF-7 and T47D), colon carcinoma (LS174T and SW480),
nasopharyngeal carcinoma (HONE-1), prostate carcinoma (DU-145),
melanoma (HO-1 and C8161), gliobiastome multiforme (GBM-18 and
T98G), and osteosarcoma (Saos-2). MDA-7 overexpression in normal
cells (HMECs, HBL-100, and CREF-Trans6) showed limited growth
inhibition indicating that MDA-7 transgene effects are not manifest
in normal cells. Taken together, the data indicates that growth
inhibition by elevated expression of MDA-7 is more effective in
vitro in cancer cells than in normal cells. Su et al. (1998)
reported investigations into the mechanism by which MDA-7
suppressed cancer cell growth. The studies reported that ectopic
expression of MDA-7 in breast cancer cell lines MCF-7 and T47D
induced apoptosis as detected by cell cycle analysis and TUNEL
assay without an effect on the normal HBL-100 cells. Western blot
analysis of cell lysates from cells infected with adenovirus MDA-7
("Ad-MDA-7") showed an upregulation of the apoptosis stimulating
protein BAX. Ad-MDA-7 infection elevated levels of BAX protein only
in MCF-7 and T47D cells and not normal HBL-100 or HMEC cells. These
data lead the investigators to evaluate the effect of ex vivo
Ad-MDA-7 transduction on xenograft tumor formation of MCF-7 tumor
cells. Ex vivo transduction resulted in the inhibition of tumor
formation and progression in the tumor xenograft model. These
characteristics indicate that MDA-7 has broad therapeutic,
prognostic and diagnostic potential as an inducer of PKR and,
consequently, an enhancer of an induced immune response.
[0204] Various enzyme genes are also considered therapeutic genes.
Particularly appropriate genes for expression include those genes
that are thought to be expressed at less than normal level in the
target cells of the subject mammal. Examples of particularly useful
gene products include carbamoyl synthetase I, ornithine
transcarbamylase, arginosuccinate synthetase, arginosuccinate
lyase, and arginase. Other desirable gene products include
fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1
antitrypsin, glucose-6-phosphatase, low-density-lipoprotein
receptor, porphobilinogen deaminase, factor VIII, factor IX,
cystathione .beta.-synthase, branched chain ketoacid decarboxylase,
albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase,
methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,
.beta.-glucosidase, pyruvate carboxylase, hepatic phosphorylase,
phosphorylase kinase, glycine decarboxylase (also referred to as
P-protein), H-protein, T-protein, Menkes disease
copper-transporting ATPase, and Wilson's disease
copper-transporting ATPase. Other examples of gene products
include, cytosine deaminase, hypoxanthine-guanine
phosphoribosyltransferase, galactose-1-phosphate uridyltransferase,
phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,
.alpha.-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV
thymidine kinase and human thymidine kinase. Hormones are another
group of genes that may be used in the vectors described herein.
Included are growth hormone, prolactin, placental lactogen,
luteinizing hormone, follicle-stimulating hormone, chorionic
gonadotropin, thyroid-stimulating hormone, leptin,
adrenocorticotropin (ACTH), angiotensin I and II, .beta.-endorphin,
.beta.-melanocyte stimulating hormone (.beta.-MSH),
cholecystokinin, endothelin I, galanin, gastric inhibitory peptide
(GIP), glucagon, insulin, lipotropins, neurophysins, somatostatin,
calcitonin, calcitonin gene related peptide (CGRP),
.beta.-calcitonin gene related peptide, hypercalcemia of malignancy
factor (1-40), parathyroid hormone-related protein (107-139)
(PTH-rP), parathyroid hormone-related protein (107-111) (PTH-rP),
glucagon-like peptide (GLP-1), pancreastatin, pancreatic peptide,
peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP),
oxytocin, vasopressin (AVP), vasotocin, enkephalinamide,
metorphinamide, alpha melanocyte stimulating hormone (alpha-MSH),
atrial natriuretic factor (5-28) (ANF), amylin, amyloid P component
(SAP-1), corticotropin releasing hormone (CRH), growth hormone
releasing factor (GHRH), luteinizing hormone-releasing hormone
(LHRH), neuropeptide Y, substance K (neurokinin A), substance P and
thyrotropin releasing hormone (TRH). Other classes of genes that
are contemplated to be inserted into the vectors of the present
invention include interleukins and cytokines. Interleukin 1 (IL-1),
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12,
GM-CSF and G-CSF.
[0205] Examples of diseases for which the present viral vector
would be useful include, but are not limited to, adenosine
deaminase deficiency, human blood clotting factor IX deficiency in
hemophilia B, and cystic fibrosis, which would involve the
replacement of the cystic fibrosis conductance regulator gene. The
vectors embodied in the present invention could also be used for
treatment of hyperproliferative disorders such as rheumatoid
arthritis or restenosis by transfer of genes encoding angiogenesis
inhibitors or cell cycle inhibitors. Transfer of prodrug activators
such as the HSV-TK gene can be also be used in the treatment of
hyperploiferative disorders, including cancer.
[0206] 3. Antisense Constructs
[0207] Oncogenes such as ras, myc, neu, raf erb, src, fms, jun,
trk, ret, gsp, hst, bcl and abl also are suitable targets. However,
for therapeutic benefit, these oncogenes would be expressed as an
antisense nucleic acid, so as to inhibit the expression of the
oncogene. The term "antisense nucleic acid" is intended to refer to
the oligonucleotides complementary to the base sequences of
oncogene-encoding DNA and RNA. Antisense oligonucleotides, when
introduced into a target cell, specifically bind to their target
nucleic acid and interfere with transcription, RNA processing,
transport and/of translation. Targeting double-stranded (ds) DNA
with oligonucleotide leads to triple-helix formation; targeting RNA
will lead to double-helix formation.
[0208] Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. Antisense RNA constructs, or DNA encoding
such antisense RNAs, may be employed to inhibit gene transcription
or translation or both within a host cell, either in vitro or in
vivo, such as within a host animal, including a human subject.
Nucleic acid sequences comprising "complementary nucleotides" are
those which are capable of base-pairing according to the standard
Watson-Crick complementarity rules. That is, that the larger
purines will base pair with the smaller pyrimidines to form only
combinations of guanine paired with cytosine (G:C) and adenine
paired with either thymine (A:T), in the case of DNA, or adenine
paired with uracil (A:U) in the case of RNA.
[0209] As used herein, the terms "complementary" or "antisense
sequences" mean nucleic acid sequences that are substantially
complementary over their entire length and have very few base
mismatches. For example, nucleic acid sequences of fifteen bases in
length may be termed complementary when they have a complementary
nucleotide at thirteen or fourteen positions with only single or
double mismatches. Naturally, nucleic acid sequences which are
"completely complementary" will be nucleic acid sequences which are
entirely complementary throughout their entire length and have no
base mismatches.
[0210] While all or part of the gene sequence may be employed in
the context of antisense construction, statistically, any sequence
17 bases long should occur only once in the human genome and,
therefore, suffice to specify a unique target sequence. Although
shorter oligomers are easier to make and increase in vivo
accessibility, numerous other factors are involved in determining
the specificity of hybridization. Both binding affinity and
sequence specificity of an oligonucleotide to its complementary
target increases with increasing length. It is contemplated that
oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 or more base pairs will be used. One can readily determine
whether a given antisense nucleic acid is effective at targeting of
the corresponding host cell gene simply by testing the constructs
in vitro to determine whether the endogenous gene's function is
affected or whether the expression of related genes having
complementary sequences is affected.
[0211] In certain embodiments, one may wish to employ antisense
constructs which include other elements, for example, those which
include C-5 propyne pyrimidines. Oligonucleotides which contain C-5
propyne analogues of uridine and cytidine have been shown to bind
RNA with high affinity and to be potent antisense inhibitors of
gene expression (Wagner et al., 1993).
[0212] As an alternative to targeted antisense delivery, targeted
ribozymes may be used. The term "ribozyme" refers to an RNA-based
enzyme capable of targeting and cleaving particular base sequences
in oncogene DNA and RNA. Ribozymes can either be targeted directly
to cells, in the form of RNA oligo-nucleotides incorporating
ribozyme sequences, or introduced into the cell as an expression
construct encoding the desired ribozymal RNA. Ribozymes may be used
and applied in much the same way as described for antisense nucleic
acids.
[0213] 4. Antigens for Vaccines
[0214] Other therapeutics genes might include genes encoding
antigens such as viral antigens, bacterial antigens, fungal
antigens or parasitic antigens. Viruses include picornavirus,
coronavirus, togavirus, flavirvirus, rhabdovirus, paramyxovirus,
orthomyxovirus, bunyavirus, arenvirus, reovirus, retrovirus,
papovavirus, parvovirus, herpesvirus, poxvirus, hepadnavirus, and
spongiform virus. Preferred viral targets include influenza, herpes
simplex virus 1 and 2, measles, small pox, polio or HIV. Pathogens
include trypanosomes, tapeworms, roundworms, helminths. Also, tumor
markers, such as fetal antigen or prostate specific antigen, may be
targeted in this manner. Preferred examples include HIV env
proteins and hepatitis B surface antigen. Administration of a
vector according to the present invention for vaccination purposes
would require that the vector-associated antigens be sufficiently
non-immunogenic to enable long term expression of the transgene,
for which a strong immune response would be desired. Preferably,
vaccination of an individual would only be required infrequently,
such as yearly or biennially, and provide long term immunologic
protection against the infectious agent.
[0215] 5. Control Regions
[0216] In order for the viral vector to effect expression of a
transcript encoding a therapeutic gene, the polynucleotide encoding
the therapeutic gene will be under the transcriptional control of a
promoter and a polyadenylation signal. Therefore, certain
embodiments of the present invention involve methods for producing
an adenovirus wherein the adenovirus comprises an adenoviral vector
encoding an exogenous gene construct that is operatively linked to
a promoter. A "promoter" refers to a DNA sequence recognized by the
synthetic machinery of the host cell, or introduced synthetic
machinery, that is required to initiate the specific transcription
of a gene. A polyadenylation signal refers to a DNA sequence
recognized by the synthetic machinery of the host cell, or
introduced synthetic machinery, that is required to direct the
addition of a series of nucleotides on the end of the mRNA
transcript for proper processing and trafficking of the transcript
out of the nucleus into the cytoplasm for translation. The phrases
"operatively linked," "under control," and "under transcriptional
control" mean that the promoter is in the correct location in
relation to the polynucleotide to control RNA polymerase initiation
and expression of the polynucleotide.
[0217] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0218] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0219] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either cooperatively or independently to activate
transcription.
[0220] The particular promoter that is employed to control the
expression of a therapeutic genesis not believed to be critical, so
long as it is capable of expressing the polynucleotide in the
targeted cell. The promoter may be a tissue-specific promoter or an
inducible promoter. Examples of promoters that may be employed
include SV40 EI, RSV LTR, .beta.-actin, CMV-IE, adenovirus major
late, polyoma F9-1, .alpha.-fetal protein promoter, egr-1, or
tyrosinase promoter. One of skill in the art would be familiar with
the range of options available for promoters that can be used to
control the expression of a therapeutic gene. Thus, where a human
cell is targeted, it is preferable to position the polynucleotide
coding region adjacent to and under the control of a promoter that
is capable of being expressed in a human cell. Generally speaking,
such a promoter might include either a human or viral promoter. A
list of promoters is provided in the Table 2. TABLE-US-00002 TABLE
2 PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light Chain
T-Cell Receptor HLA DQ .alpha. and DQ .beta. .beta.-Interferon
Interleukin-2 Interleukin-2 Receptor MHC Class II 5 MHC Class II
HLA-DR.alpha. .beta.-Actin Muscle Creatine Kinase Prealbumin
(Transthyretin) Elastase I Metallothionein Collagenase Albumin Gene
.alpha.-Fetoprotein .tau.-Globin .beta.-Globin c-fos c-HA-ras
Insulin Neural Cell Adhesion Molecule (NCAM) .alpha.1-Antitrypsin
H2B (TH2B) Histone Mouse or Type I Collagen Glucose-Regulated
Proteins (GRP94 and GRP78) Rat Growth Hormone Human Serum Amyloid A
(SAA) Troponin I (TN I) Platelet-Derived Growth Factor Duchenne
Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma Virus
Hepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus
Gibbon Ape Leukemia Virus
[0221] The promoter may be a constitutive promoter, an inducible
promoter, or a tissue-specific promoter. An inducible promoter is a
promoter which is inactive or exhibits low activity except in the
presence of an inducer substance. Some examples of promoters that
may be included as a part of the present invention include, but are
not limited to, MT II, MMTV, Collagenase, Stromelysin, SV40, Murine
MX gene, .alpha.-2-Macroglobulin, MHC class I gene h-2kb, HSP70,
Proliferin, Tumor Necrosis Factor, or Thyroid Stimulating Hormone
.alpha. gene. The associated inducers are shown in Table 3. It is
understood that any inducible promoter may be used in the practice
of the present invention and that all such promoters would fall
within the spirit and scope of the claimed invention. A promoter
that is "endogenous" or "constitutive" is a promoter that is one
naturally associated with a gene or sequence, as may be obtained by
isolating the 5' non-coding sequences located upstream of the
coding segment and/or exon. TABLE-US-00003 TABLE 3 Element Inducer
MT II Phorbol Ester (TPA) Heavy metals MMTV (mouse mammary tumor
Glucocorticoids virus) .beta.-Interferon poly(rI)X poly(rc)
Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H.sub.2O.sub.2
Collagenase Phorbol Ester (TPA) Stromelysin Phorbol Ester (TPA),
IL-1 SV40 Phorbol Ester (TPA) Murine MX Gene Interferon, Newcastle
Disease Virus GRP78 Gene A23187 .alpha.-2-Macroglobulin IL-6
Vimentin Serum MHC Class I Gene H-2kB Interferon HSP70 Ela, SV40
Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis Factor
FMA Thyroid Stimulating Hormone .alpha. Thyroid Hormone Gene
[0222] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter and the Rous
sarcoma virus long terminal repeat can be used to obtain high-level
expression of the polynucleotide of interest. The use of other
viral or mammalian cellular or bacterial phage promoters which are
well-known in the art to achieve expression of polynucleotides is
contemplated as well, provided that the levels of expression are
sufficient to produce a growth inhibitory effect.
[0223] By employing a promoter with well-known properties, the
level and pattern of expression of a polynucleotide following
transfection can be optimized. For example, selection of a promoter
which is active in specific cells, such as tyrosinase (melanoma),
alpha-fetoprotein and albumin (liver tumors), CC10 (lung tumor) and
prostate-specific antigen (prostate tumor) will permit
tissue-specific expression of the therapeutic gene.
[0224] Enhancers were originally detected as genetic elements that
increased transcription from a promoter located at a distant
position on the same molecule of DNA. This ability to act over a
large distance had little precedent in classic studies of
prokaryotic transcriptional regulation. Subsequent work showed that
regions of DNA with enhancer activity are organized much like
promoters. That is, they are composed of many individual elements,
each of which binds to one or more transcriptional proteins.
[0225] The basic distinction, between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization.
[0226] Additionally any promoter/enhancer combination (as per the
Eukaryotic Promoter Data Base (EPDB)) could also be used to drive
expression of a particular construct. Use of a T3, T7 or SP6
cytoplasmic expression system is another possible embodiment.
Eukaryotic cells can support cytoplasmic transcription from certain
bacteriophage promoters if the appropriate bacteriophage polymerase
is provided, either as part of the delivery complex or as an
additional genetic expression vector.
[0227] Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed. Such polyadenylation signals as that from SV40, bovine
growth hormone, and the herpes simplex virus thymidine kinase gene
have been found to function well in a number of target cells.
[0228] F. Methods of Isolating Adenovirus
[0229] Adenoviral infection results in the lysis of the cells being
infected. The lytic characteristics of adenovirus infection permit
two different modes of virus isolation and production. One is
harvesting infected cells prior to cell lysis. The other mode is
harvesting virus supernatant after complete cell lysis by the
produced virus. For the latter mode, longer incubation times are
required in order to achieve complete cell lysis. This prolonged
incubation time after virus infection creates a serious concern
about increased possibility of generation of replication competent
adenovirus (RCA), particularly for the current first generation
adenoviral vectors (E1-deleted vector). Therefore, in certain
embodiments of the present invention, the methods for producing an
adenovirus involve harvesting the host cells and then lysing the
host cells. Table 4 lists the most common methods that have been
used for lysing cells after cell harvest. TABLE-US-00004 TABLE 4
Methods used for cell lysis Methods Procedures Comments Freeze-thaw
Cycling between dry ice Easy to carry out at lab and 37.degree. C.
water bath scale. High cell lysis efficiency Not scaleable Not
recommended for large scale manufacturing Solid Shear French Press
Capital equipment Hughes Press investment Virus containment
concerns Lack of experience Detergent Non-ionic detergent Easy to
carry out at both lab lysis solutions such as Tween .RTM., and
manufacturing Triton .RTM., NP-40, etc. scale Wide variety of
detergent choices Concerns of residual detergent in finished
product Hypotonic water, citric buffer Low lysis efficiency
solution lysis Liquid Shear Homogenizer Capital equipment Impinging
Jet investment Microfluidizer Virus containment concerns
Scaleability concerns Sonication ultrasound Capital equipment
investment Virus containment concerns Noise pollution Scaleability
concern
[0230] 1. Detergents
[0231] In certain embodiments of the present invention, the methods
for producing an adenovirus involve isolating the adenovirus by
lysing the host cells with a detergent. Cells are bounded by
membranes. In order to release components of the cell, it is
necessary to break open the cells. The most advantageous way in
which this can be accomplished, according to the present invention,
is to solubilize the membranes with the use of detergents.
Detergents are amphipathic molecules with an apolar end of
aliphatic or aromatic nature and a polar end which may be charged
or uncharged. Detergents are more hydrophilic than lipids and thus
have greater water solubility than lipids. They allow for the
dispersion of water insoluble compounds into aqueous media and are
used to isolate and purify proteins in a native form.
[0232] Any detergent capable of lysing the host cells is
contemplated by the claimed invention. One of skill in the art
would be familiar with the wide range of detergents available for
lysing cells. Detergents can be denaturing or non-denaturing. The
former can be anionic such as sodium dodecyl sulfate or cationic
such as ethyl trimethyl ammonium bromide. These detergents totally
disrupt membranes and denature the protein by breaking
protein-protein interactions. Non denaturing detergents can be
divided into non-anionic detergents such as Triton.RTM. X-100, bile
salts such as cholates and zwitterionic detergents such as CHAPS.
Zwitterionics contain both cationic and anion groups in the same
molecule, the positive electric charge is neutralized by the
negative charge on the same or adjacent molecule.
[0233] Denaturing agents such as SDS bind to proteins as monomers
and the reaction is equilibrium driven until saturated. Thus, the
free concentration of monomers determines the necessary detergent
concentration. SDS binding is cooperative i.e. the binding of one
molecule of SDS increase the probability of another molecule
binding to that protein, and alters proteins into rods whose length
is proportional to their molecular weight.
[0234] Non-denaturing agents such as Triton.RTM. X-100 do not bind
to native conformations nor do they have a cooperative binding
mechanism. These detergents have rigid and bulky apolar moieties
that do not penetrate into water soluble proteins. They bind to the
hydrophobic parts of proteins. Triton.RTM. X100 and other
polyoxyethylene nonanionic detergents are inefficient in breaking
protein-protein interaction and can cause artifactual aggregations
of protein. These detergents will, however, disrupt protein-lipid
interactions but are much gentler and capable of maintaining the
native form and functional capabilities of the proteins.
[0235] Detergent removal can be attempted in a number of ways.
Dialysis works well with detergents that exist as monomers.
Dialysis is somewhat ineffective with detergents that readily
aggregate to form micelles because they micelles are too large to
pass through dialysis. Ion exchange chromatography can be utilized
to circumvent this problem. The disrupted protein solution is
applied to an ion exchange chromatography column and the column is
then washed with buffer minus detergent. The detergent will be
removed as a result of the equilibration of the buffer with the
detergent solution. Alternatively the protein solution may be
passed through a density gradient. As the protein sediments through
the gradients the detergent will come off due to the chemical
potential.
[0236] Often a single detergent is not versatile enough for the
solubilization and analysis of the milieu of proteins found in a
cell. The proteins can be solubilized in one detergent and then
placed in another suitable detergent for protein analysis. The
protein detergent micelles formed in the first step should separate
from pure detergent micelles. When these are added to an excess of
the detergent for analysis, the protein is found in micelles with
both detergents. Separation of the detergent-protein micelles can
be accomplished with ion exchange or gel filtration chromatography,
dialysis or buoyant density type separations.
[0237] a. Triton.RTM.X-Detergents
[0238] This family of detergents (Triton.RTM.-100, X114 and NP-40)
have the same basic characteristics but are different in their
specific hydrophobic-hydrophilic nature. All of these heterogeneous
detergents have a branched 8-carbon chain attached to an aromatic,
ring. This portion of the molecule contributes most of the
hydrophobic nature of the detergent. Triton.RTM.X detergents are
used to solublize membrane proteins under non-denaturing
conditions. The choice of detergent to solubilize proteins will
depend on the hydrophobic nature of the protein to be solubilized.
Hydrophobic proteins require hydrophobic detergents to effectively
solubilize them.
[0239] Triton.RTM.X-100 and NP-40 are very similar in structure and
hydrophobicity and are interchangeable in most applications
including cell lysis, delipidation protein dissociation and
membrane protein and lipid solubilization. Generally 2 mg of
detergent is used to solubilize 1 mg membrane protein or 10 mg
detergent/1 mg of lipid membrane. Triton.RTM.X-114 is useful for
separating hydrophobic from hydrophilic proteins.
[0240] b. Brij.RTM. Detergents
[0241] These are similar in structure to Triton.RTM.X detergents in
that they have varying lengths of polyoxyethylene chains attached
to a hydrophobic chain. However, unlike Triton.RTM.X detergents,
the Brij.RTM. detergents do not have an aromatic ring and the
length of the carbon chains can vary. The Brij.RTM. detergents are
difficult to remove from solution using dialysis but may be removed
by detergent removing gels. Brij.RTM.58 is most similar to
Triton.RTM.X100 in its hydrophobic/hydrophilic characteristics.
Brij.RTM.-35 is a commonly used detergent in HPLC applications.
[0242] c. Dializable Nonionic Detergents
[0243] .eta.-Octyl-.beta.-D-glucoside (octylglucopyranoside) and
.eta.-Octyl-.beta.-D-thioglucoside (octylthioglucopyranoside, OTG)
are nondenaturing nonionic detergents which are easily dialyzed
from solution. These detergents are useful for solubilizing
membrane proteins and have low UV absorbances at 280 nm.
Octylglucoside has a high CMC of 23-25 mM and has been used at
concentrations of 1.1-1.2% to solubilize membrane proteins.
[0244] Octylthioglucoside was first synthesized to offer an
alternative to octylglucoside. Octylglucoside is expensive to
manufacture and there are some inherent problems in biological
systems because it can be hydrolyzed by .beta.-glucosidase.
[0245] d. Tween.RTM. Detergents
[0246] The Tween.RTM. detergents are nondenaturing, nonionic
detergents. They are polyoxyethylene sorbitan esters of fatty
acids. Tween.RTM. 20 and Tween.RTM. 80 detergents are used as
blocking agents in biochemical applications and are usually added
to protein solutions to prevent nonspecific binding to hydrophobic
materials such as plastics or nitrocellulose. They have been used
as blocking agents in ELISA and blotting applications. Generally,
these detergents are use at concentrations of 0.01-1.0% to prevent
nonspecific binding to hydrophobic materials.
[0247] Tween.RTM. 20 and other nonionic detergents have been shown
to remove some proteins from the surface of nitrocellulose.
Tween.RTM. 80 has been used to solubilize membrane proteins,
present nonspecific binding of protein to multiwell plastic tissue
culture plates and to reduce nonspecific binding by serum proteins
and biotinylated protein A to polystyrene plates in ELISA.
[0248] The difference between these detergents is the length of the
fatty acid chain. Tween.RTM. 80 is derived from oleic acid with a
C.sub.18 chain while Tween.RTM. 20 is derived from lauric acid with
a C.sub.12 chain. The longer fatty acid chain makes the Tween.RTM.
80 detergent less hydrophilic than Tween.RTM. 20 detergent. Both
detergents are very soluble in water.
[0249] The Tween.RTM. detergents are difficult to remove from
solution by dialysis, but Tween.RTM. 20 can be removed by detergent
removing gels. The polyoxyethylene chain found in these detergents
makes them subject to oxidation (peroxide formation) as is true
with the Triton.RTM. X and Brij.RTM. series detergents.
[0250] e. Zwitterionic Detergents
[0251] The zwitterionic detergent, CHAPS, is a sulfobetaine
derivative of cholic acid. This zwitterionic detergent is useful
for membrane protein solubilization when protein activity is
important. This detergent is useful over a wide range of pH (pH
2-12) and is easily removed from solution by dialysis due to high
CMCs (8-10 mM). This detergent has low absorbances at 280 nm making
it useful when protein monitoring at this wavelength is necessary.
CHAPS is compatible with the BCA Protein Assay and can be removed
from solution by detergent removing gel. Proteins can be iodinated
in the presence of CHAPS
[0252] CHAPS has been successfully, used to solubilize intrinsic
membrane proteins and receptors and maintain the functional
capability of the protein. When cytochrome P-450 is solubilized in
either Triton.RTM. X-100 or sodium cholate aggregates are
formed.
[0253] 2. Non-Detergent Methods
[0254] Various non-detergent methods, though not preferred, may be
employed in conjunction with other advantageous aspects of the
present invention:
[0255] a. Freeze-Thaw
[0256] This has been a widely used technique for lysis cells in a
gentle and effective manner. Cells are generally frozen rapidly in,
for example, a dry ice/ethanol bath until completely frozen, then
transferred to a 37.degree. C. bath until completely thawed. This
cycle is repeated a number of times to achieve complete cell
lysis.
[0257] b. Sonication
[0258] High frequency ultrasonic oscillations have been found to be
useful for cell disruption. The method by which ultrasonic waves
break cells is not fully understood but it is known that high
transient pressures are produced when suspensions are subjected to
ultrasonic vibration. The main disadvantage with this technique is
that considerable amounts of heat are generated. In order to
minimize heat effects specifically designed glass vessels are used
to hold the cell suspension. Such designs allow the suspension to
circulate away from the ultrasonic probe to the outside of the
vessel where it is cooled as the flask is suspended in ice.
[0259] c. High Pressure Extrusion
[0260] This is a frequently used method to disrupt microbial cell.
The French pressure cell employs pressures of 10.4.times.10.sup.7
Pa (16,000 p.s.i.) to break cells open. These apparatus consists of
a stainless steel chamber which opens to the outside by means of a
needle valve. The cell suspension is placed in the chamber with the
needle valve in the closed position. After inverting the chamber,
the valve is opened and the piston pushed in to force out any air
in the chamber. With the valve in the closed position, the chamber
is restored to its original position, placed on a solid based and
the required pressure is exerted on the piston by a hydraulic
press. When the pressure has been attained the needle valve is
opened fractionally to slightly release the pressure and as the
cells expand they burst. The valve is kept open while the pressure
is maintained so that there is a trickle of ruptured cell which may
be collected.
[0261] d. Solid Shear Methods
[0262] Mechanical shearing with abrasives may be achieved in Mickle
shakers which oscillate suspension vigorously (300-3000 time/min)
in the presence of glass beads of 500 nm diameter. This method may
result in organelle damage. A more controlled method is to use a
Hughes press where a piston forces most cells together with
abrasives or deep frozen paste of cells through a 0.25 mm, diameter
slot in the pressure, chamber. Pressures of up to
5.5.times.10.sup.7 Pa (8000 p.s.i.) may be used to lyse bacterial
preparations.
[0263] e. Liquid Shear Methods
[0264] These methods employ blenders, which use high speed
reciprocating or rotating blades, homogenizers which use an
upward/downward motion of a plunger and ball and microfluidizers or
impinging jets which use high velocity passage through small
diameter tubes or high velocity impingement of two fluid streams.
The blades of blenders are inclined at different angles to permit
efficient mixing. Homogenizers are usually operated in short high
speed bursts of a few seconds to minimize local heat. These
techniques are not generally suitable for microbial cells but even
very gentle liquid shear is usually adequate to disrupt animal
cells.
[0265] f. Hypotonic/Hypertonic Methods
[0266] Cells are exposed to a solution with a much lower
(hypotonic) or higher (hypertonic) solute concentration. The
difference in solute concentration creates an osmotic pressure
gradient. The resulting flow of water into the cell in a hypotonic
environment causes the cells to swell and burst. The flow of water
out of the cell in a hypertonic environment causes the cells to
shrink and subsequently burst.
[0267] G. Methods of Concentration and Filtration
[0268] The present invention involve methods of producing an
adenovirus that involve isolating the adenovirus. Methods of
isolating the adenovirus from host cells include any methods known
to those of skill in the art. For example, these methods may
include clarification, concentration and diafiltration. One step in
the purification process can include clarification of the cell
lysate to remove large particulate matter, particularly cellular
components, from the cell lysate. Clarification of the lysate can
be achieved using a depth filter or by tangential flow filtration.
In one embodiment of the present invention, the cell lysate is
concentrated. Concentrating the crude cell lysate, may include any
step known to those of skill in the art. For example, the crude
cell lysate may be passed through a depth filter, which consists of
a packed column of relatively non-adsorbent material (e.g.
polyester resins, sand, diatomeceous earth, colloids, gels, and the
like). In tangential flow filtration (TFF), the lysate solution
flows across a membrane surface which facilitates back diffusion of
solute from the membrane surface into the bulk solution. Membranes
are generally arranged within various types of filter apparatus
including open channel plate and frame, hollow fibers, and
tubules.
[0269] After clarification and prefiltration of the cell lysate,
the resultant virus supernatant may be concentrated and buffer may
be exchanged by diafiltration. The virus supernatant can be
concentrated by tangential flow filtration across an
ultrafiltration membrane of 100-300K nominal molecular weight
cutoff. Ultrafiltration is a pressure-modified convective process
that uses semi-permeable membranes to separate species by molecular
size, shape and/or charge. It separates solvents from solutes of
various sizes, independent of solute molecular size.
Ultrafiltration is gentle, efficient and can be used to
simultaneously concentrate and desalt solutions. Ultrafiltration
membranes generally have two distinct layers: a thin (0.1-1.5
.mu.m), dense skin with a pore diameter of 10-400 angstroms and an
open substructure of progressively larger voids which are largely
open to the permeate side of the ultrafilter. Any species capable
of passing through the pores of the skin can therefore freely pass
through the membrane. For maximum retention of solute, a membrane
is selected that has a nominal molecular weight cut-off well below
that of the species being retained. In macromolecular
concentration, the membrane enriches the content of the desired
biological species and provides filtrate cleared of retained
substances. Microsolutes are removed convectively with the solvent.
As concentration of the retained solute increases, the
ultrafiltration rate diminishes.
[0270] Some embodiments of the present invention involve use of
exchanging buffer of the crude cell lysate. Buffer exchange, or
diafiltration, involves using ultrafilters is an ideal way for
removal and exchange of salts, sugars, non-aqueous solvents
separation of free from bound species, removal of material of low
molecular weight, or rapid change of ionic and pH environments.
Microsolutes are removed most efficiently by adding solvent to the
solution being ultrafiltered at a rate equal to the ultrafiltration
rate. This washes microspecies from the solution at constant
volume, purifying the retained species.
[0271] H. Removing Nucleic Acid Contaminants
[0272] Certain embodiments of the methods for producing an
adenovirus involve reducing the concentration, of contaminating
nucleic acids in a crude cell lysate. The present invention employs
nucleases to remove contaminating nucleic acids. Exemplary
nucleases include Benzonase.RTM., Pulmozyme.RTM.; or any other
DNase or RNase commonly used within the art.
[0273] Enzymes such as Benzonaze.RTM. degrade nucleic acid and have
no proteolytic activity. The ability of Benzonase.RTM. to rapidly
hydrolyze nucleic acids makes the enzyme ideal for reducing cell
lysate viscosity. It is well known that nucleic acids may adhere to
cell derived particles such as viruses. The adhesion may interfere
with separation due to agglomeration, change in size, of the
particle or change in particle charge, resulting in little if any
product being recovered with a given purification scheme.
Benzonase.RTM. is well suited for reducing the nucleic acid load
during purification, thus eliminating the interference and
improving yield.
[0274] As with all endonucleases, Benzonase.RTM. hydrolyzes
internal phosphodiester bonds between specific nucleotides. Upon
complete digestion, all free nucleic acids present in solution are
reduced to oligonucleotides 2 to 4 bases in length.
[0275] I. Size Partitioning Purification
[0276] According to one aspect of the invention it has been found
that size partitioning purification techniques may be used to
provide adenoviral preparations of sufficient purity that they may
be therapeutically administered without additional purification
steps such as chromatographic and other methods previously
considered necessary. Without intending to be bound by any
particular theory of the invention it is believed that the steps of
processing viral host cells in a cell suspension culture in a serum
free media results in a viral particle product with a reduced load
of contaminants. Moreover, the contaminants are of a size and
nature that they may be readily separated from viral particles by a
simple size partitioning purification step.
[0277] Membrane filtration is a well known technique in the art of
bioprocessing. A membrane is defined as a structure having lateral
dimensions much greater than its thickness, through which mass
transfer may occur under a variety of driving forces. While many
filters are may be considered membranes, filters also include
materials whose lateral dimensions are not usually 100 times
greater than their depth and whose separation function is primarily
by capture of species or particles through their depth. The most
common parameters used to characterize membranes fall in three
general categories. These are transport properties, pore
(geometric) characteristics, and surface (or predominantely
chemical) properties. Nevertheless, the transport properties depend
significantly upon the pore and surface characteristics. While
membrane separation can be slower and a lower volume process than
other separton processes, its effectiveness makes it a preferred
method for retrieving small amounts of valuable products.
[0278] Membrane filter systems may be designed in a variety of
manners to have different filtration properties. Design criteria
include: operation in dead-end (with or without stirring) or cross
flow mode; full or partial recovery of the feed mixture;
application of an external transmembrane pressure via pumping,
inert gas blanket, or evacuation of the permeate side of the
device; and the use of flat sheets (either singly or multiply),
hollow fiber bundle, or tubular membranes. Preferred size
partitioning separation methods utilize a size partitioning
membrane which may be a dialysis or other similar membrane as would
be known to those of ordinary skill in the art. Suitable dialysis
membrane materials useful in the size partitioning membrane
filtration fo the invention include those commercially available
such as those produced from polyethersulphone, polycarbonate,
nylon, polypropylene and the like. Suppliers of these dialysis
membrane materials include Akzo-Nobel, Millipore, Inc., Poretics,
Inc., and Pall Corp., by way of example. Size partitioning
membranes having pore sizes of less than 0.08 microns are useful in
practice of the invention with those having pore sizes less than
0.05 microns and less than 0.02 microns and greater than 0.001
microns being particularly preferred. Such membranes are capable of
allowing the passage of desired viral particles while retaining
undesired contaminants.
[0279] According to one aspect of the invention, tangential flow
filtration (TFF) units, also known as "cross-flow filtration", have
been found to be particularly advantageous for practice of the
invention. Tangential flow filtration is a pressure driven
separation process wherein fluid is pumped tangentially long the
surface of a membrane. An applied pressure serves to force a
portion of the fluid including contaminants through the membrane to
the filtrate size. Particulates and macromolecules that are too
large to pass through the membrane pores are retained on the
upstream side. In contrast to normal flow filtration (NFF)
techniques in which the retained components build up on the surface
of the membrane, tangential flow filtration sweeps the retained
components along by the flow of the fluid.
[0280] TFF is classified based on the size of components being
separated. A membrane pore size rating is typically given as a
micron value and indicates that particles larger than the rating
will be retained by the membrane. A nominal molecular weight limit
(NMWL), on the other hand, is an indication that most dissolved
macromolecules with molecular weights higher than the NMWL and some
with molecular weights lower than the NMWL will be retained by the
membrane. A component's shape, its ability to deform, and its
interaction with other components in the solution all affect
retention. Different membrane manufacturers use different criteria
to assign the NMWL ratings to a family of membranes but those of
ordinary skill would be able to determine the appropriate rating
empirically.
[0281] Ultrafiltration is one of the most widely used forms of TFF
and is used to separate proteins from buffer components for buffer
exchange, desalting or concentration but may also be used for Virus
Filtration. Typical NMWL ratings for Virus Filtration range from
100 kD to 500 kD, or up to 0.05 to 0.08 microns.
[0282] Diafiltration is a TFF process than can be performed in
combination with any of the other categories of separation to
enhance either produce yield or purity. During diafiltration,
buffer is introduced into the recycle tank while filtrate is
removed from the unit operation. In processes where the product is
in the retentate, diafiltration washes componenets out of the
product pool into the filtrate, thereby exchanging buffers and
reducing the concentration of undesirable species. When the product
is in the filtrate, diafiltration washes it through the membrane
into a collection vessel.
[0283] In TFF unit operation, a pump is used to generate flow of
the feed stream through the channel between two membrane surfaces.
During each pass of fluid over the surface of the membrane, the
applied pressure forces a portion of the fluid through the membrane
and into the filtrate stream. The result is a gradient in the
feedstock concentration from the bulk conditions at the center of
the channel to the more concentrated wall conditions at the
membrane surface. There is also a concentration gradient along the
length of the feed channel from the inlet to the outlet (retentate)
at progressively more fluid passes to the filtrate side. The flow
of feedstock along the length of the membrane causes a pressure
drop from the feed to the retentate end of the channel. The flow on
the filtrate side of the membrane is typically low and there is
little restriction, so the pressure along the length of the
membrane on the filtrate side is approximately constant.
[0284] Membranes may be fabricated from various materials offering
alternatives in flushing characteristics and chemical
compatibility. Suitable materials include cellulose,
polyethersulfone and other materials known to those of skill in the
art with polyethersulfone being particularly preferred. Typical
polyethersulfone membranes tend to adsorb protein as well as other
biological components, leading to membrane fouling and lowered
flux. Some membranes are hydrophilically modified to be more
resistant to fouling such as Biomax.RTM. (Millipore)
[0285] Those of skill in the art would recognize that various types
of TFF modules would be useful in practice of the invention. Useful
TFF modules include but are not limited to flat plate modules (also
known as cassettes), spiral wound modules, and hollow fiber
modules. In flat plate modules, layers of membrane either with or
without alternating layers of separator screen are stacked together
and then sealed into a package. Feed fluid is pumped into
alternating channels at one end of the stack and the filtrate
passes through the membrane into the filtrate channels. Flat plat
modules generally have high packing densities (area of membrane
surface per area of floor space), allow linear scaling, and some
offer the choice of open or turbulence promoted channels.
[0286] Spiral wound modules comprise alternating layers of membrane
and separator screen wound around a hollow central core. the feed
stream is pumped into one end and flows down the axis of the
cartridge. Filtrate passes through the membrane and spirals to the
core, where it is removed. The separator screens increase
turbulence in the flowpath, leading to a higher efficiency module
than hollow fibers. One drawback to spiral wound modules is that
they are not linearly scaleable because either the feed flowpath
length (cartridge length) or the filtrate flowpath length
(cartridge width) must be changed within scales.
[0287] Hollow fiber modules are comprises of a bundle of membrane
tubes with narrow diameters (typically in the range of 0.1 to 2.0
mm). In a hollow fiber module, the feed stream is pumped into the
lumen (inside) of the tube and the filtrate passes through the
membrane to the shell side, where it is removed. Because of the
very open feed flowpath, low shear is generated even with moderate
cross flow rates.
[0288] For any given module, key process parameters may then be
readily optimized by those of ordinary skill. Such parameters
include cross flow rate, transmembrane pressure (TMP), filtrate
control, membrane area and diafiltration design. Cross flow rate
depends upon which module is selected. In general, a higher cross
flow rate gives higher flux at equal TMP and increases the sweeping
action across the membrane and reduces the concentration gradient
towards the membrane surface. Many TFF applications apply a cross
flow and pressure set point and the filtrate flows uncontrolled and
unrestricted out of the module. This is the simplest type of
operation but in some circumstances it might be desired to use some
type of filtrate control beyond that achieved by simply adjusting
the pressure with the retentate valve. Membrane area is selected
after determining the process flow and the total volume to be
processed and is also dependent upon process time.
[0289] According to one aspect of the invention a plate and frame
TFF system was used with each of a 300 KD, a 500 KD or a 1000 KD
polysulfone membrane having a surface area of 1.1 ft.sup.2. The
cross flow rate was 900 mL/ft.sup.2/min. and the transmembrane
pressure was about 7 psi. The filtrate rate was not actively
controlled and the diafiltration was performed using the consistent
volume method.
[0290] The invention provides methods of producing purified
adenovirus compositions which avoid the necessity of multiple
purification steps including chromatographic purification steps.
Nevertheless, additional purification steps including those known
to the art may be practiced if desired. Such methods include those
taught in U.S. Pat. No. 6,194,191, the disclosure of which is
incorporated by reference, including density gradient
centrifugation; chromatography including size exclusion
chromatography, ion exchange chromatography, high performance
liquid chromatography (HPLC), and the like.
[0291] J. Pharmaceutical Formulations
[0292] The present invention includes, in certain embodiments,
methods for producing an adenovirus that involve placing the
adenovirus into a pharmaceutically acceptable composition. The
present invention also includes compositions of adenovirus prepared
by one of the processes disclosed herein, wherein the composition
is a pharmaceutically acceptable composition.
[0293] When purified according to the methods set forth above, the
viral particles of the present invention will be administered in
various manners with in vitro, ex vivo or in vivo being
contemplated. Thus, it will be desirable to prepare the complex as
a pharmaceutical composition appropriate for the intended
application. Generally this will entail preparing a pharmaceutical
composition that is essentially free of pyrogens, as well as any
other impurities that could be harmful to humans or animals. It may
also be desired to employ appropriate salts and buffers to render
the complex stable and allow for complex uptake by target
cells.
[0294] The phrase "pharmaceutically acceptable composition" refers
to molecular entities and compositions that do not produce an
adverse, allergic or other untoward reaction when administered to
an animal, or a human, as appropriate. As used herein,
"pharmaceutically acceptable composition" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the composition. In
addition, the composition can include supplementary inactive
ingredients. For instance, the composition for use as a mouthwash
may include a flavorant or the composition may contain
supplementary ingredients to make the formulation
timed-release.
[0295] Aqueous compositions of the present invention comprise an
effective amount of the expression cassette, dissolved or dispersed
in a pharmaceutically acceptable carrier or aqueous medium. Such
compositions also are referred to as inocula. Examples of aqueous
compositions include a formulation for intravenous administration
or a formulation for topical application.
[0296] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene
glycols, mixtures thereof and in oils. Under ordinary conditions of
storage and use, these preparations contain a preservative to
prevent the growth of microorganisms.
[0297] The viral particles of the present invention may include
classic pharmaceutical preparations for use in therapeutic
regimens, including their administration to humans. Administration
of therapeutic compositions according to the present invention will
be via any common route so long as the target tissue is available
via that route. This includes oral, nasal, buccal, rectal, vaginal
or topical. Alternatively, administration may be by orthotopic,
intradermal subcutaneous, intramuscular, intraperitoneal, or
intravenous injection. Such compositions would normally be
administered as pharmaceutically acceptable compositions that
include physiologically acceptable carriers, buffers or other
excipients. For application against tumors, direct intratumoral
injection, inject of a resected tumor bed, regional (i.e.,
lymphatic) or general administration is contemplated. It also may
be desired to perform continuous perfusion over hours or days via a
catheter to a disease site, e.g., a tumor or tumor site.
[0298] The therapeutic and preventive compositions of the present
invention are advantageously administered in the form of liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to topical use may also be prepared. A
typical composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per
ml of phosphate buffered saline. Other pharmaceutically acceptable
carriers include aqueous solutions, non-toxic excipients, including
salts, preservatives, buffers and the like. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oil
and injectable organic esters such as ethyloleate. Aqueous carriers
include water, alcoholic/aqueous solutions, saline solutions,
parenteral vehicles such as sodium chloride, Ringer's dextrose,
etc. Preservatives include antimicrobial agents, anti-oxidants,
chelating agents and inert gases. The pH and exact concentration of
the various components of the pharmaceutical composition are
adjusted according to well-known parameters.
[0299] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and/or the like. These compositions take the form of
solutions such as mouthwashes and mouthrinses, suspensions,
tablets, pills, capsules, sustained release formulations and/or
powders. In certain defined embodiments, oral pharmaceutical
compositions will comprise an inert diluent and/or assimilable
edible carrier, and/or they may be enclosed in hard and/or soft
shell gelatin capsule, and/or they may be compressed into tablets,
and/or they may be incorporated directly with the food of the diet.
For oral therapeutic administration, the active compounds may be
incorporated with excipients and/or used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and/or the like. Such compositions and/or
preparations should contain at least 0.1% of active compound. The
percentage of the compositions and/or preparations may, of course,
be varied and/or may conveniently be between about 2 to about 75%
of the weight of the unit, and/or preferably between 25-60%. The
amount of active compounds in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
[0300] The tablets, troches, pills, capsules and/or the like may
also contain the following: a binder, as gum tragacanth, acacia,
cornstarch, and/or gelatin; excipients, such as dicalcium
phosphate; a disintegrating agent, such as corn starch, potato
starch, alginic acid and/or the like; a lubricant, such as
magnesium stearate; and/or a sweetening agent, such as sucrose,
lactose and/or saccharin may be added and/or a flavoring agent,
such as peppermint, oil of wintergreen, and/or cherry flavoring.
When the dosage unit form is a capsule, it may contain, in addition
to materials of the above type, a liquid carrier. Various other
materials may be present as coatings and/or to otherwise modify the
physical form of the dosage unit. For instance, tablets, pills,
and/or capsules may be coated with shellac, sugar and/or both. A
syrup of elixir may contain the active compounds sucrose as a
sweetening agent methyl and/or propylparabens as preservatives, a
dye and/or flavoring, such as cherry and/or orange flavor.
[0301] For oral administration the expression cassette of the
present invention may be incorporated with excipients and used in
the form of non-ingestible mouthwashes and dentifrices. A mouthwash
may be prepared incorporating the active ingredient in the required
amount in an appropriate solvent, such as a sodium borate solution
(Dobell's Solution). Alternatively, the active ingredient may be
incorporated into an antiseptic wash containing sodium borate,
glycerin and potassium bicarbonate. The active ingredient also may
be dispersed in dentifrices, including: gels, pastes, powders and
slurries. The active ingredient may be added in a therapeutically
effective amount to a paste dentifrice that may include water,
binders, abrasives, flavoring agents, foaming agents, and
humectants.
[0302] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or, ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0303] One may also use solutions and/or sprays, hyposprays,
aerosols and/or inhalants in the present invention for
administration. One example is a spray for administration to the
aerodigestive tract. The sprays are isotonic and/or slightly
buffered to maintain a pH of 5.5 to 6.5. In addition, antimicrobial
preservatives, similar to those used in ophthalmic preparations,
and/or appropriate drug stabilizers, if required, may be included
in the formulation. Additional formulations which are suitable for
other modes of administration include vaginal or rectal
suppositories and/or pessaries. Formulations for other types of
administration that is topical include, for example, a cream,
suppository, ointment or salve.
[0304] An effective amount of the therapeutic agent is determined
based on the intended goal, for example (i) inhibition of tumor
cell proliferation, (ii) elimination or killing, of tumor cells,
(iii) vaccination, or (iv) gene transfer for long term expression
of a therapeutic gene. The term "unit dose" refers to physically
discrete units suitable for use in a subject, each unit containing
a predetermined-quantity of the therapeutic composition calculated
to produce the desired responses, discussed above, in association
with its administration, i.e., the appropriate route and treatment
regimen. The quantity to be administered, both according to number
of treatments and unit dose, depends on the subject to be treated,
the state of the subject and the result desired. Multiple gene
therapeutic regimens are expected, especially for adenovirus.
[0305] In certain embodiments of the present invention, an
adenoviral vector encoding a tumor suppressor gene will be used to
treat cancer patients. Typical amounts of an adenovirus vector used
in gene therapy of cancer is 10.sup.3-10.sup.15 PFU/dose,
(10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11 10.sup.12, 10.sup.13, 10.sup.14,
10.sup.15) wherein the dose may be divided into several injections,
at different sites within a solid tumor. The treatment regimen also
may involve several cycles of administration of the gene transfer
vector over a period of 3-10 weeks. Administration of the vector
for longer periods of time from months to years may be necessary
for continual therapeutic benefit.
[0306] In another embodiment of the present invention, an
adenoviral, vector encoding a therapeutic gene may be used to
vaccinate, humans or other mammals. Typically, an amount of virus
effective to produce the desired effect, in this case vaccination,
would be administered to a human or mammal so that long term
expression of the transgene is achieved and a strong host immune
response develops. It is contemplated that a series of injections,
for example, a primary injection followed by two booster
injections, would be sufficient to induce an long term immune
response. A typical dose would be from 10.sup.6 to 10.sup.15
PFU/injection depending on the desired result. Low doses of antigen
generally induce a strong cell-mediated response, whereas high
doses of antigen generally induce an antibody-mediated immune
response. Precise amounts of the therapeutic composition also
depend on the judgment of the practitioner and are peculiar to each
individual.
EXAMPLES
[0307] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0308] According to this example, cells were cultured and
adenoviral vectors produced with medium perfusion using a 10 L (5 L
working volume) Wave Bioreactor.RTM. (20/50EH (Wave Biotech, LLC)
equipped with a YSI-2700 SELECT.TM. biochemistry analyzer according
to the production and purification process depicted in FIG. 1. FIG.
3 depicts a perfusion Wave bioreactor (10) comprising an inflated
plastic bag (12) containing cell culture media (14) and an internal
flat perfusion filter (16) to provide separation between the cells
and spent medium. Media is fed to the bioreactor from a feed bag
(18) by feed pump (22) Spent culture medium is withdrawn through
the floating filter (16) to a harvest bag (20) by harvest pump
(24). Controller (26) controls the functions of the pumps and
bioreactor (10). No medium recirculation is required, and
consequently this mode of medium perfusion is very gentle to the
cells in culture. The wave action minimizes filter clogging during
perfusion. The culture volume during perfusion is maintained by a
load cell used to a trigger fresh medium addition. HEK293 (human
epithelial embryonic kidney cells) adapted to serum-free suspension
culture according to the method of U.S. Pat. No. 6,194,191 were
seeded at 4.8.times.10.sup.5 cells/ml and were allowed to grow to
1.2.times.10.sup.6 cells/ml in protein-free CD293 medium
(Invitrogen.TM.). On day 3 of culture, medium perfusion was started
at a cell concentration of 1.7.times.10.sup.6 cells/ml. Cell
concentration increased approximately exponentially to
1.times.10.sup.7 cells/ml on day 6, and cell viability was
maintained above 90%. The cell growth, the viability and
nutrient/metabolite concentrations during culture are shown in FIG.
4 and FIG. 5
[0309] The rocking speed was set at 10 and the rocking angle was
set at 11. The culture pH was maintained by adjusting CO.sub.2 gas
percentage delivered by the gas mixer. The dissolved oxygen tension
(DOT) in the culture medium was monitored using a disposable DOT
probe supplied by Wave Biotech.TM..
[0310] When the cell concentration reached 1.times.10.sup.7
cells/ml, the cell culture was diluted 10-fold fold with fresh
CD293 medium to supplement nutrients and dilute potentially toxic
metabolites into a Wave Biotech.TM. 200 Bioreactor without a
perfusion filter. The cells were then infected with an adenoviral
vector (AdCMVp53) at a MOI of 50 vp/cell. AdCMVp53 is a genetically
engineered, replication-incompetent human type 5 adenovirus
expressing the human wild type p53 protein under control of the
cytomegalovirus (CMV) immediate early promoter. Infection was
allowed to proceed for 2 days. The culture was harvested on day 2
post-infection. The virus harvest was then subjected to TFF
concentration using a Pellicon 2 mini system fitted with a 500 KD
Biomax membrane cassette and subjected to enzyme treatment with
Benzonase.RTM..
[0311] Adenoviral vector productions was measured using an anion
exchange HPLC method. The adenoviral vector concentration in the
bioreactor was found to be 1.1.times.10.sup.11 vp/ml, the virus
yield was 1.1.times.10.sup.16 vp, and the cell-specific vector
productivity was 126,000 vp/cell.
Example 2
[0312] According to this example, the product of Example 1 was
subjected to diafiltration using a tangential flow filtration (TFF)
membrane using a Pellicon 2 mini system fitted with a 500 KD Biomax
membrane cassette The clarified harvest was concentrated 20-fold
using the Pellicon 2 mini system prior to diafiltration using a 500
mM Tris buffer at pH 8.0. Diafiltration was performed by the
consistent volume method. Fresh diafiltration buffer was
continuously added to the system as filtrate was permeated out of
the membrane. Studies carried out using the 100 L production scale
are set out in Table 5 below. The lack of fetal bovine serum in the
culture medium makes is feasible to use TFF membrane partitioning
diafiltration as a method of virus purification with high recovery.
TABLE-US-00005 TABLE 5 Titer HPLC Purity Recovery Total Yield
(vp/mL) (%) (%) (vp) Clarified Harvest 1.2 .times. 10.sup.11 5.3 NA
1.20 .times. 10.sup.16 10-fold DF 2.3 .times. 10.sup.12 78.6 90
1.08 .times. 10.sup.16 20-fold DF 2.2 .times. 10.sup.12 89.5 89
1.07 .times. 10.sup.16 30-Fold DF 2.3 .times. 10.sup.12 93.5 89
1.06 .times. 10.sup.16 40-Fold DF 1.8 .times. 10.sup.12 97.1 90
1.08 .times. 10.sup.16 60-Fold DF 1.5 .times. 10.sup.12 98.5 79
9.50 .times. 10.sup.15
[0313] Table 6 below depicts the infectivity (PFU/vp ratio) of 2
viral vector products produced by the protein free suspension
process. Viral particle concentration was determined by OD.sub.260
analysis and Infections unit (IU) concentration was determined by
TCID.sub.50 assay. This demonstrates that viruses produced by the
protein free suspension process are as infectious as those from
serum containing production processes. TABLE-US-00006 TABLE 6
Infections Viral Viral particle conc. unit conc. Vectors (vp/mL)
(IU/mL) VP/IU 1 1.2 .times. 10.sup.12 8 .times. 10.sup.10 15 2 1.0
.times. 10.sup.12 6 .times. 10.sup.10 17
[0314] Each of the resulting diafiltration products described in
Table 6 above along with a viral preparation purified by
traditional column chromatography were subjected to SDS-PAGE
analysis to determine the presence of contaminants. The results
depicted gin FIG. 2 show that impurities were still present in the
diafiltration purified virus preparation even though initial HPLC
analysis demonstrated good purity.
[0315] The resulting purified viral product was compared to viral
preparations prepared by conventional methods utilizing
chromatographic purification. SDS-PAGE analysis reveals that the
column purified virus is still significantly more pure. While
significant purification is realized by the size partitioning as
supported by HPLC analysis SDS-PAGE analysis reveals that
impurities remain.
[0316] Further tests were conducted comparing the gene expression
of products produced by the Wave bioreactor process with those
produced by using CellCube bioreactors and, are shown in FIG. 6.
The virus produced by practice of the Wave suspension process is
comparable to that produced by the CellCube process in terms of
infectiousness and activity.
[0317] The use of the wave bioreactor with a suspension culture in
a serum-free medium combined with use of tangential flow filtration
provides improved scalability and virus yields in the production of
purified virus preparations.
[0318] All of the methods and compositions disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the methods and compositions and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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