U.S. patent application number 10/855594 was filed with the patent office on 2005-02-10 for composition for viral preservation.
Invention is credited to Setiwan, Kerrie.
Application Number | 20050032044 10/855594 |
Document ID | / |
Family ID | 25646857 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032044 |
Kind Code |
A1 |
Setiwan, Kerrie |
February 10, 2005 |
Composition for viral preservation
Abstract
This invention relates to a composition for the preservation of
a virus, the composition including a virus and a lipid.
Inventors: |
Setiwan, Kerrie; (South
Yarra, AU) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
25646857 |
Appl. No.: |
10/855594 |
Filed: |
May 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10855594 |
May 28, 2004 |
|
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PCT/AU02/01680 |
Dec 12, 2002 |
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Current U.S.
Class: |
435/5 ;
435/235.1 |
Current CPC
Class: |
Y10T 436/10 20150115;
C12N 15/86 20130101; C12N 2710/10332 20130101; C12N 7/00 20130101;
A61K 48/00 20130101; A61K 35/761 20130101; C12N 2710/10151
20130101; C12N 2710/10343 20130101; A61P 31/12 20180101 |
Class at
Publication: |
435/005 ;
435/235.1 |
International
Class: |
C12Q 001/70; C12Q
001/68; C12N 007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2001 |
AU |
PR9449 |
Feb 14, 2002 |
AU |
PS0545 |
Claims
1. A composition for the preservation of a virus, the composition
including a virus and a lipid.
2. A composition according to claim 1, wherein the lipid is a
cationic lipid, an anionic lipid, a zwitterionic lipid, a non-ionic
lipid or any combination of these lipids.
3. A composition according to claim 1, wherein the lipid is a
cationic lipid.
4. A composition according to claim 3, wherein the cationic lipid
has a hydrophilic moiety including one or more amino residues.
5. A composition according to claim 4, wherein the one or more
amino residues is derived from an amino acid.
6. A composition according to claim 5, wherein the one or more
amino acids is lysine, arginine or histidine.
7. A composition according to claim 3, wherein the cationic lipid
is a poly-cationic lipid.
8. A composition according to claim 7, wherein the poly-cationic
lipid has a hydrophilic moiety including three lysine amino
acids.
9. A composition according to claim 1, wherein the lipid has a
hydrophobic moiety including one or more hydrophobic groups.
10. A composition according to claim 9, wherein the one or more
hydrophobic groups includes an acyl, alkyl or alkoxy group.
11. A composition according to claim 10, wherein the acyl group has
a carbon chain length of 3 to 24 carbon atoms.
12. A composition according to claim 11, wherein the acyl group is
a laurate group.
13. A composition according to claim 9, wherein the lipid has a
hydrophobic moiety including three hydrophobic groups.
14. A composition according to claim 9, wherein the lipid further
includes a spacer group between the hydrophilic moiety and the
hydrophobic moiety.
15. A composition according to claim 14, wherein the spacer group
has a chain length equivalent to 1 to 30 carbon-carbon single
covalent bonds.
16. A composition according to claim 1, wherein the lipid is a
tris-conjugated cationic lipid.
17. A composition according to claim 16, wherein the
tris-conjugated cationic lipid has the following chemical formula:
3or a salt thereof, wherein: X is a positively charged hydrophilic
moiety; Y is spacer having a chain length equivalent to 1 to 30
carbon-carbon single covalent bonds or is absent; and R.sub.1,
R.sub.2, and R.sub.3 are the same or different and are acyl groups
derived from a fatty acid.
18. A composition according to claim 17, wherein the
tris-conjugated lipid has the following chemical formula: 4
19. A composition according claim 1, wherein the concentration of
lipid is in the range from 0.1 .mu.M to 1 mM.
20. A composition according to claim 1, wherein the concentration
of lipid is in the range from 1 .mu.M to 500 .mu.M.
21. A composition according to claim 1, wherein the concentration
of lipid is in the range from 5 .mu.M to 100 .mu.M.
22. A composition according to claim 18, wherein the concentration
of lipid is 5 .mu.M to 100 .mu.M.
23. A composition according to claim 1, wherein the virus is a
virus derived from one or more of the group consisting of
Adenoviridae, Herpesviridae, Poxviridae, Papovaviridae,
Orthohepadnavirus, Parvoviridae, Birnaviridae, Reoviridae,
Flaviviridae, Picornaviridae, Togaviridae, Filoviridae,
Paramyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae,
Orthomyxoviridae, and Retroviridae.
24. A composition according to claim 23, wherein the virus is
derived from the Adenoviridae family of viruses.
25. A composition according to claim 24, wherein the virus is an
ovine atadenovirus.
26. A composition according to claim 25, wherein the virus is
OAdV623 or a derivative of OAdV623.
27. A composition according to claim 1, wherein the concentration
of virus in the composition is in the range from 1.times.10.sup.6
to 1.times.10.sup.14 virus particles/ml.
28. A composition according to claim 1, wherein the concentration
of virus in the composition is in the range from 1.times.10.sup.8
to 5.times.10.sup.12 virus particles/ml.
29. A composition according to claim 1, wherein the pH of the
composition is in the range from 4 to 10.
30. A composition according to claim 1, wherein the pH of the
composition is in the range from 6 to 8.5.
31. A composition according to claim 1, wherein the virus is
storage stable.
32. A composition according to claim 31, wherein the virus is
storage stable when the composition is stored in liquid form.
33. A composition according to claim 32, wherein the composition is
stored at 0.degree. C. to 30.degree. C.
34. A composition according to claim 32, wherein the composition is
stored at 2.degree. C. to 25.degree. C.
35. A composition according to claim 32, wherein he composition is
stored at 2.degree. C. to 8.degree. C.
36. A composition according to claim 31, wherein the composition is
stored in the lyophilised, spray-dried or freeze-dried form.
37. A composition according to claim 36, wherein the composition is
stored at -20.degree. C. to 30.degree. C.
38. A composition according to claim 31, wherein the composition is
stored for 12 months or greater.
39. A composition according to claim 31, wherein the composition is
stored for 3 months or greater.
40. A composition according to claim 31, wherein the composition is
stored for 1 week or greater.
41. A composition according to claim 31, wherein the composition is
stored for 1 day or greater.
42. A composition according to claim 1, wherein the virus is stable
when the composition is subjected to one or more of agitation,
shearing forces or mechanical action.
43. A method of producing a composition for the preservation of a
virus, the method including the step of preparing a liquid
composition including a virus and a lipid.
44. A method according to claim 43, wherein the lipid is a cationic
lipid.
45. A method according to claim 44, wherein the cationic lipid is a
poly-cationic lipid.
46. A method according to claim 44, wherein the lipid is a
tris-conjugated cationic lipid.
47. A method according to claim 46, wherein the tris-conjugated
cationic lipid has the following chemical formula: 5or a salt
thereof, wherein: X is a positively charged hydrophilic moiety; Y
is spacer having a chain length equivalent to 1 to 30 carbon-carbon
single covalent bonds or is absent; and R.sub.1, R.sub.2, and
R.sub.3 are the same or different and are acyl groups derived from
a fatty acid.
48. A method according to claim 47, wherein the tris-conjugated
lipid has the following chemical formula: 6
49. A method according to claim 43, wherein the virus is a virus
derived from one or more of the group consisting of Adenoviridae,
Atadenoviridae, Herpesviridae, Poxviridae, Parvoviridae,
Papoviridae, Orthohepadnavirus, Parvoviridae, Birnaviridae,
Reoviridae, Flaviviridae, Picornaviridae, Togaviridae, Filoviridae,
Paramyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae,
Orthomyxoviridae, and Retroviridae.
50. A method according to claim 49, wherein the virus is derived
from the Adenoviridae family of viruses.
51. A method according to claim 43, wherein the virus is purified
by a method including chromatography or centrifugation.
52. A method according to claim 43, wherein the composition is
formed by combining a solution including the virus with a solution
including the lipid.
53. A method according to claim 43, wherein the virus is storage
stable.
54. A method according to claim 53, wherein the virus is storage
stable when the composition is stored in a liquid form.
55. A method according to claim 53, wherein the virus is storage
stable when the composition is stored in a lyophilised,
freeze-dried or spray dried form.
56. A method according to claim 43, wherein the virus is stable
when the composition is subjected to one or more of agitation,
shearing forces or mechanical action.
57. A composition for the preservation of a virus, the composition
including a virus and a lipid, wherein the virus is storage
stable.
58. A composition for the preservation of a virus, the composition
including a virus and a lipid, wherein the virus is stable when the
composition is subjected to one or more of agitation, shearing
forces or mechanical action.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions for the
preservation of viruses. The present invention also relates to
methods for preparing compositions for the preservation of
viruses.
[0002] It will become apparent from the following description that
the viral compositions according to the present invention are most
likely to be pharmaceutical compositions for the purposes of the
delivery of viral particles for gene therapy or vaccination.
However, it must be appreciated that the invention is not to be
limited in its application to only pharmaceutical compositions.
BACKGROUND OF THE INVENTION
[0003] Gene therapy broadly refers to the transfer of genetic
material into cells and the expression of that material in those
cells for a therapeutic purpose. The goal is to produce the desired
protein in the appropriate quantity and the proper location.
Although a variety of methods have been developed to deliver
therapeutic nucleic acids to cells, many of these methods are
limited by relatively inefficient transfer of the therapeutic
nucleic acid to the target cells. Because viruses are highly
efficient at infecting susceptible cells, viruses are now
recognized as being useful vehicles for the transfer of therapeutic
nucleic acids into cells for the purpose of gene therapy.
[0004] Viruses fall broadly into two distinct groups: those that
integrate into the genome of transduced cells and those that do
not. An integrating virus inserts its viral genome into host DNA to
facilitate long-term gene expression. For a non-integrating virus,
however, the viral genome exists extra-chromosomally as an episome
in the nucleus of transduced cells. Depending on the ability of the
virus to replicate, the viral genome is either passed on faithfully
to every daughter cell or is eventually lost during cell
division.
[0005] Retroviruses and adeno-associated viruses (AAVs) may
integrate into the host DNA to provide a steady level of expression
following transduction and incorporation into the host genome. As
the target DNA is replicated, so too is the inserted therapeutic
gene embedded in the transferred chromosomal DNA. Thus,
transduction via these vectors can produce durable gene expression.
This can be advantageous in tumour vaccine strategies in which a
steady level of gene expression may enhance efficacy.
[0006] In contrast, adenovirus and vaccinia virus vectors do not
integrate into the host DNA but exist as episomes. Thus, a
transferred gene is expressed without actual integration of the
gene into the target cell genome. Generally, non-integrating
viruses are used when transient gene expression is desired.
[0007] Examples of viruses that may be used to deliver nucleic
acids to cells for gene therapy purposes include adenovirus,
adeno-associated virus (AAV), retrovirus, herpes simplex virus,
vaccinia virus, poliovirus, sindbis virus, HIV-1, avian leukosis
virus, sarcoma virus, Epstein-Barr virus, papillomavirus, foamy
virus, influenza virus, Newcastle disease virus, sendai virus,
lymphocytic choriomeningitis virus, polyoma virus,
reticuloendotheliosis virus, Theiler's virus, and other types of
RNA and DNA viruses.
[0008] The use of attenuated and killed viruses for purposes of
vaccination is also well known. In addition, viruses are also
becoming increasingly important as tools for research and
diagnostics. The increasing importance of viruses as tools for gene
therapy, vaccination, and research and diagnosis has led to a need
to develop viral compositions that may be manufactured,
transported, stored and manipulated without compromising viral
efficacy. For example, viral compositions for vaccination must be
able to maintain the immunogenicity of a virus, or the
immunogenicity of a component of the virus. In the case of
compositions of viruses to be used for gene therapy, it is critical
that the efficacy of the live viral formulations carrying
therapeutic transgenes be maintained.
[0009] Because viruses are biological entities consisting of a
nucleic acid encapsulated by a protein coat, they are susceptible
to the same chemical and physical processes that may degrade or
inactivate proteins and nucleic acids. In particular, live viruses
may often be very susceptible to damage, as any change in the
conformation or integrity of one or more components of the virus
coat or the encapsulated nucleic acid may lead to a loss of
infectivity. As such, biopharmaceutical products containing
compositions of viruses for vaccination or gene therapy usually
require stringent conditions to avoid physicochemical degradation
and to maintain biological activity. Degradation of viruses in such
compositions may occur during isolation, production, purification,
formulation, storage, shipping or delivery of the virus.
Accordingly, biopharmaceutical compositions of viruses must be
formulated to provide protection of the virus against factors such
as temperature, pH, pressure, oxidising agents, ionic content,
light, radiation, ultrasound, shear, agitation and changes in phase
(for example as occurs during lyophilization, spray-drying or
freeze-drying).
[0010] In addition to the factors already discussed, other factors
such as viral concentration, the size and structure of the
encapsulated nucleic acid, container composition, headspace gas,
and number of freeze-thaw cycles may all affect the activity of
viral compositions.
[0011] As a consequence, the utility of many viruses in
biopharmaceutical preparations is often limited by the instability
of compositions of the viruses, particularly the instability that
occurs upon manufacture, transportation, storage and manipulation
before use. For example, liquid compositions are often unstable
when stored, and show a significant loss of viral activity with
time. A further loss of activity may occur if the composition is
subjected to agitation, shearing forces or mechanical action.
[0012] With regard to the storage of viral compositions in the
lyophilised, spray-dried or freeze-dried states, there may also be
a loss of viral activity upon the change of phase from the liquid
to solid state. A loss of viral activity may also occur upon
reconstitution. The use of such viral compositions has the
additional disadvantage that upon reconstitution, the viral
composition must generally be left for an extended period of time
to reconstitute, usually at room temperature.
[0013] In addition, as low temperature storage conditions are not
always available, it would be advantageous to develop formulations
that can preserve viral formulations above freezing for extended
periods of time. Indeed, viral compositions that must be stored
below freezing and which cannot be stored at standard freezer
temperatures (for example -10.degree. C. to -20.degree. C.) for
substantial periods of time represent a serious impediment to the
widespread clinical use of many viruses.
[0014] There is also a need to develop viral compositions that can
maintain the desired pH of the composition for extended periods of
time despite being exposed to a variety of different
conditions.
[0015] Finally, increasingly high concentrations of virus are also
being required for therapeutic purposes. However, the concentration
of virus in a composition may present additional problems to the
ability to preserve a virus. In particular, a high concentration of
virus may contribute significantly to viral instability due to
aggregation and/or precipitation. In addition, a loss of viral
activity may also occur when concentrating the virus, in part due
to the mechanical shear forces that come to bear during the process
to concentrate the virus.
[0016] Therefore for many viruses a particular deficiency has been
the inability to formulate compositions that acceptably preserve
the virus during, for instance, manufacture, transportation,
storage and manipulation prior to use. Such deficiencies with the
ability to preserve the activity of viral compositions often
preclude their use for gene therapy, for vaccination, or for other
purposes.
[0017] It is therefore an aim of the present invention to provide a
composition for the improved preservation of viruses.
[0018] Throughout this specification reference may be made to
documents for the purpose of describing various aspects of the
invention. However, no admission is made that any reference cited
in this specification constitutes prior art. In particular, it will
be understood that the reference to any document herein does not
constitute an admission that any of these documents forms part of
the common general knowledge in the art in Australia or in any
other country. The discussion of the references states what their
authors assert, and the applicant reserves the right to challenge
the accuracy and pertinency of any of the documents cited
herein.
SUMMARY OF THE INVENTION
[0019] The present invention provides a composition for the
preservation of a virus, the composition including a virus and a
lipid.
[0020] The present invention further provides a method of producing
a composition for the preservation of a virus, the method including
the step of preparing a liquid composition including a virus and a
lipid.
[0021] In the context of the present invention, it has been
determined that the activity of a virus in a composition may be
preserved by including in the composition a lipid.
[0022] The composition according to the present invention provides
for improved preservation of a virus in the liquid or solid states.
The improved preservation of the virus in the composition is
evident over a broad range of storage temperatures, and over a
broad range of storage periods. The composition also provides
improved preservation of the virus to the effects of agitation,
shearing forces and mechanical action.
[0023] It is to be understood that while the composition according
to the present invention may be used for the preservation of viable
virus particles, the composition may also be used for the improved
preservation of attenuated virus particles, killed virus particles,
non-viable viral particles, synthetic viruses, or one or more
constituents of viable, killed, non-viable or synthetic
viruses.
[0024] It will also be appreciated that not only may the
composition be used for pharmaceutical compositions for medical
applications, such as the delivery of virus for the purposes of
gene therapy or the delivery of viruses or viral constituents for
vaccination, the compositions of the present invention may also be
used for compositions for the preservation of viable, attenuated,
killed, non-viable and synthetic viral particles for non-medical
applications, such as the preservation of viral preparations for
research and diagnostic applications.
[0025] Various terms that will be used throughout this
specification have meanings that will be well understood by a
skilled addressee. However, for ease of reference, some of these
terms will now be defined.
[0026] The term "preservation" as used throughout the specification
is to be understood to mean that a desired activity of a virus
(such as infectivity, transduction or immunogenicity) does not
decrease substantially over a given period of time, or that a
desired activity of a virus does not decrease substantially after a
particular treatment. For example (i) the activity of a virus in a
composition according to the present invention may not decrease
substantially when the composition is stored for a given period of
time; and/or (ii) the activity of a virus in a composition
according to the present invention may not decrease substantially
after the composition is agitated, subjected to shear forces, or
subjected to other types of mechanical action, phase change or
other condition that may reduce viral activity.
[0027] In the context of the present invention, the ability of a
composition to preserve a virus is to be understood to be improved
over similar compositions that do not contain a lipid. Accordingly,
the composition according to the present invention will show an
activity of the virus over a given period of time, or will show an
activity of the virus after a particular treatment (eg agitation,
shear or mechanical action), that is higher than a similar
composition that does not contain a lipid.
[0028] In this regard, the demonstration of the preservation of a
virus in a composition according to the various forms of the
present invention will be achieved by a suitable biological assay.
As will be appreciated, given the degree of variability in
biological systems, in determining the ability of a composition to
preserve a virus, sufficient repetitions of any biological assay
will need to be performed to statistically demonstrate that the
composition is able to preserve the virus.
[0029] The term "virus" as used throughout the specification is to
be understood to mean any natural, recombinant, in vitro packaged
or synthetic virus.
[0030] The term "viral composition" as used throughout the
specification is to be understood to mean any composition that may
be used for the preservation of a virus (or a part of a virus) for
therapeutic purposes, or for the preservation of virus (or part of
a virus) generally. The term not only encompasses the composition
according to the present invention, but also encompasses the
composition according to the present invention with other any other
additives, such as excipients.
[0031] The term "lipid" as used throughout the specification is to
be understood to mean any fatty acid and derivatives of fatty
acids, glycerol-derived lipids including phospholipids,
sphingosine-derived lipid (including ceramides, cerebrosides,
gangliosides and sphingomyelins) and glycolipids, terpenes and
their derivatives, long chain alcohols and waxes. In referring to
such lipids, it will be appreciated that these molecules are
amphiphilic and will contain a substantially hydrophilic moiety
coupled to a substantially hydrophobic moiety. The hydrophilic
moiety will contain one or more substantially hydrophilic groups,
and the hydrophobic moiety will contain one or more substantially
hydrophobic groups.
[0032] The term "surfactant" as used throughout the specification
is to be understood to mean any compound that can reduce the
interfacial tension between two immiscible phases. In this regard,
it will be understood that a molecule with surfactant function may
also perform one or more additional functions in any particular
composition. Accordingly, the demonstration that a molecule has a
surfactant capacity will be achieved by a suitable method known in
the art to test whether the molecule has the ability to reduce the
interfacial tension between two immiscible phases.
GENERAL DESCRIPTION OF THE INVENTION
[0033] As mentioned above, the composition according to the present
invention provides a composition for the improved preservation of
virus particles. Preferably, the virus particles are selected from
one or more of the group consisting of Adenoviridae including
Mastadenovirus such as Human Adenovirus and Atadenovirus such as
Ovine Adenovirus; Herpesviridae; Poxviridae including vaccinia,
fowlpox, swinepox and sheeppox; Papovaviridae; Orthohepadnavirus;
Parvoviridae including adeno-associated virus; Birnaviridae;
Reoviridae; Flaviviridae; Picornaviridae including poliovirus;
Togaviridae including Sindbis virus and Semliki Forest virus;
Filoviridae; Paramyxoviridae; Rhabdoviridae; Arenaviridae;
Bunyaviridae; Orthomyxoviridae; Retroviridae including Lentivirus.
More preferably, the virus particle is derived from the
Adenoviridae family of viruses. More preferably, the virus is an
Atadenovirus. Most preferably, the virus is an ovine
atadenovirus.
[0034] For the purposes of the various forms of the present
invention, the virus is preferably a recombinant virus. More
preferably, the virus is a recombinant virus that has utility for
the purposes of gene therapy. In a particularly preferred
embodiment, the virus is a recombinant ovine adenovirus, such as
the adenoviral vector OAdV623 or derivatives of this vector.
OAdV623 encodes the purine nucleoside phosphorylase (PNP) gene
which catalyses the conversion of the immunosuppressive prodrug
Fludarabine to the toxic 2-fluoro-adenine product. Adenoviral
vector OAdV623 is as described in Lockett L. J. and Both G. W.
(2002) Virology 294:333-341.
[0035] The composition according to the present invention may be
used for the preservation of viral particles that retain the
ability to infect or transduce cells, or for the preservation of
viral particles that have been attenuated, killed, are non-viable,
have been produced by in vitro packaging or are of synthetic
origin. The composition may also be used for the preservation of
parts of a virus, such as the preservation of one or more
constituents of the virus coat. Preferably, the viral particles are
viable viral particles.
[0036] In this regard, an attenuated virus is to be understood to
mean a virus whose virulence has been lowered by a biological,
physical or chemical process. For example, the virulence of a virus
may be attenuated by passaging through a semi-permissive host.
[0037] A killed virus is to be understood to mean a viral particle
that has been inactivated by a treatment so that the viral particle
no longer retains the ability to infect a permissive host. Examples
of treatments that may kill a viral particle are heat or chemical
modification.
[0038] A non-viable virus is to be understood to mean a viral
particle that is not able to infect or transduce permissive host
cells.
[0039] A synthetic virus is to be understood to mean any nucleic
acid packaged with a protein and/or lipid coat.
[0040] The composition according to the present invention may be
used for the preservation of viruses that are to be used for
medical applications. Preferably, the composition according to the
present invention is for the preservation of viruses that are to be
used for the purposes of gene therapy. More preferably, the
composition according to the present invention is for the
preservation of viruses that are to be used for the delivery of
therapeutic nucleic acids to prostatic cells for gene therapy.
[0041] The composition according to the present invention may also
be used for the preservation of viruses that are to be used for the
purposes of eliciting an immunogenic response, such as for
vaccination. It will be understood in this regard that the
composition may be used for the preservation of whole viruses, or
for the preservation of one or more immunogenic constituents of a
virus, such as the preservation of one or more polypeptides that
make up part of the virus coat.
[0042] When the composition according to the present invention is
used for the preservation of a virus to be used for medical
applications, the composition may also include one or more
pharmaceutically acceptable additives, such as pharmaceutically
acceptable salts, amino acids, polypeptides, polymers, solvents,
buffers and bulking agents.
[0043] The composition according to the present invention may be a
liquid or solid composition. In the case of a liquid composition,
the composition is preferably a substantially aqueous composition
or a composition composed of one or more other solvents. Most
preferably, the composition is a substantially aqueous
composition.
[0044] In the case of a solid composition, the composition may be a
lyophilised composition, a freeze-dried composition or a spray
dried composition.
[0045] The composition may be stored in a container suitable for
the preservation of the virus, such as borosilicate glass. The
composition according to the present invention may also be stored
under a gaseous atmosphere that is suitable for the preservation of
the virus including air, argon or nitrogen.
[0046] The composition according to the present invention may also
be used for the preservation of viable, attenuated, killed,
non-viable or synthetic viruses for research applications. For
example, the composition may be used for the preservation of viral
particles that have use in research applications, such as the use
of viral preparations for immunological research. The composition
may also be used for the preservation of viral preparations for use
in molecular biological research, such as the use of viral
preparations for the infection or transduction of cells in
culture.
[0047] In a similar fashion, the compositions according to the
present invention may also be used for the preservation of viral
particles that have use in diagnostic applications, such as the use
of viral preparations as positive and negative test standards for
diagnostic applications.
[0048] With regard to viral activity, the activity of the virus may
be measured by any suitable assay that is known in the art. Such
assays include both direct and indirect biological and
physicochemical assays of viral activity. Examples of direct assays
include the measurement of the number of infectious viral particles
in the product, the expression of a reporter gene or other
transgene carried by the virus, the cell killing or cell viability
following viral infection or transduction of a suitable cell line,
or the quantity of components produced following administration of
the viral particles or constituents to a suitable model (eg. immune
response in case of vaccination). Examples of indirect assays
include the measurement of the number of intact and non-aggregated
viral particles or the size of the viral particles (as an
indication of viral aggregation) in the product.
[0049] For example, for determining the activity of viable viral
particles, the number of permissive cells killed following
infection or transduction with a defined amount of virus may be
determined by any suitable assay. Alternatively, as an indirect
measure of viral activity, the number of intact and non-aggregated
viral particles in the product may be determined by anion-exchange
HPLC and the particle size determined by light scattering
analysis.
[0050] The concentration of virus in the composition may also
affect the ability of the composition to preserve the virus.
Preferably, the concentration of virus in the composition is in the
range from 1.times.10.sup.6 to 1.times.10.sup.14 virus
particles/ml. More preferably, the concentration of virus is in the
range from 1.times.10.sup.8 to 5.times.10.sup.12 virus
particles/ml.
[0051] The lipid in the composition of the various forms of the
present invention is any fatty acid or derivative of a fatty acid,
glycerol-derived lipid including a phospholipid,
sphingosine-derived lipid (including ceramides, cerebrosides,
gangliosides and sphingomyelins) and glycolipid, terpene and their
derivatives, long chain alcohol and wax. The lipid is an
amphiphilic molecule that contains a substantially hydrophilic
moiety coupled (directly or by way of a spacer) to a substantially
hydrophobic moiety. The hydrophilic moiety will contain one or more
substantially hydrophilic groups and the hydrophobic moiety will
contain one or more substantially hydrophobic groups.
[0052] The lipid present in the composition according to the
various forms of the present invention may be a cationic lipid,
anionic lipid, zwitterionic lipid, non-ionic lipid or any
combination of such lipids.
[0053] Examples of cationic lipids include
2,3-dioleyloxy-N-[2(sperminecar-
boxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate
(DOSPA), dioctadecylaminoglycyl spermine (DOGS), dipalmitoyl
phosphatidylethanolamyl spermine (DPPES),
1,3-dioleoyoxy-2-(6-carboxy-spe- rmyl)-propylamide (DOSPER),
dioleyidimethylammonium chloride (DODAC),
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimetylammonium chloride
(DOTMA), 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane
(DOTAP), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium
bromide (DMRIE),
3.beta.-(N-((N',N'-dimethylamino)ethane)carbamoyl)-cholesterol
(DC-Chol), dimethyldioctadecyl ammonium bromide (DDAB),
1-[2-(oleoyoxy)-ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium
chloride (DOTIM), bis(oleoyl)-trimethylaminomethylphosphonate,
1,2-dimyristoylglycerolpentalysine salt,
N,N',N",N'"-tetramethyl-N,N', N",N'"-tetrapalmitylspermine (TMTPS),
cetyltrimethylammonium bromide (CTAB) and the following proprietary
cationic lipids: Lipofectamine (DOSPA:DOPE 3:1 w/w), Lipofectin
(DOTMA:DOPE 1:1 w/w), Lipofectace (DDAB:DOPE 1:1.25 w/w),
Transfectam, Cellfectin (TMTPS:DOPE 1:1.5 M/M), Superfect,
LipoTaxi, DMRIE-C (DMRIE/cholesterol: 1:1) and
trilysine-carpryloyl-tris-trilaurate (T-shape; CS087).
[0054] Examples of anionic lipids include
1,2-dioleoyl-sn-glycero-3-[phosp- ho-L-serine] (DOPS),
1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), and PEG-PE
lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamin-
e-N-[poly(ethyleneglycol) 2000] (PEG2000 DMPE),
1,2-dipalmitoyl-sn-glycero-
-3-phosphoethanolamine-N-[poly(ethyleneglycol) 2000] (PEG2000
DPPE),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)
2000] (PEG2000 DSPE).
[0055] Examples of zwitterionic/neutral lipids include
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dioleoyl-sn-glycero-3-phosphcholine (DOPC),
1,2-dimyristoyl-sn-glycer- o-3-phosphocholine (DMPC) and
1,2-dimyristoyl-sn-glycero-3-phosphoethanola- mine (DMPE).
[0056] Preferably the lipid is a cationic lipid. More preferably,
the lipid is a cationic lipid that has a hydrophilic moiety that
includes one or more amino residues. More preferably, the lipid is
a cationic lipid that has a hydrophilic moiety that includes one or
more groups derived from amino acids. More preferably, the lipid is
a cationic lipid that has a hydrophilic moiety that includes one or
more groups derived from a positively charged amino acid, such as
lysine, arginine or histidine. Most preferably, the lipid is a
cationic lipid that has a hydrophilic moiety including one or more
lysine groups.
[0057] In a particularly preferred embodiment, the lipid is a
poly-cationic lipid. Preferably, the lipid is a poly-cationic lipid
that has a hydrophilic moiety that includes two or more amino
residues. More preferably, the lipid is a poly-cationic lipid that
has a hydrophilic moiety that includes two or more groups derived
from amino acids. More preferably, the lipid is a poly-cationic
lipid that has a hydrophilic moiety that includes two or more
groups derived from positively charged amino acids, such as lysine,
arginine or histidine. Most preferably, the lipid is a
poly-cationic lipid that has a hydrophilic moiety that includes
three lysine groups.
[0058] The hydrophobic moiety of the lipid in the composition
according to the present invention includes one or more hydrophobic
groups. Hydrophobic groups include, but are not restricted to,
acyl, alkyl, or alkoxy chains. Preferably, the one or more
hydrophobic groups are derived from an acyl group of a fatty acid.
More preferably, the one or more acyl groups have a carbon chain
length of 3 to 24 carbon atoms. Most preferably, the one or more
acyl groups is a laurate group.
[0059] Preferably, the lipid in the composition according to the
present invention has a hydrophobic moiety that includes two or
more hydrophobic groups. More preferably the lipid has a
hydrophobic moiety that includes three hydrophobic groups. Most
preferably, the lipid has a hydrophobic moiety that includes three
laurate groups.
[0060] The lipid in the composition according to the present
invention may also include a spacer group between the hydrophilic
moiety and the hydrophobic moiety. The spacer group may include any
combination or series of atoms that covalently join the hydrophilic
and hydrophobic moieties. Preferably, the spacer region has a chain
length equivalent to 1 to 30 carbon-carbon single covalent
bonds.
[0061] In a preferred embodiment, the lipid in the composition
according to the present invention is derived from a
tris-conjugated cationic lipid (or a salt thereof) according to the
following general formula: 1
[0062] In this general formula, X represents the hydrophilic
moiety, Y represents a spacer group (which may or may not be
present), and R.sub.1, R.sub.2 and R.sub.3 are acyl groups of fatty
acids. Preferably, a spacer group Y is present in the molecule.
Most preferably the spacer group has a chain length equivalent to 1
to 30 carbon-carbon single covalent bonds.
[0063] Most preferably, the lipid in the composition according to
the various forms of the present invention is the molecule
trilysine-carpryloyl-tris-trilaurate (T-shape; CS087), or a salt
thereof, the structure of which is as follows: 2
[0064] To preserve virus, the concentration of the lipid in the
composition is preferably in the range from 0.1 .mu.M to 1 mM. More
preferably the concentration of the lipid is 1 .mu.M to 500 .mu.M.
In the most preferred embodiment, the concentration of the lipid is
5 .mu.M to 100 .mu.M.
[0065] In the case where the lipid in the composition is
trilysine-carpryloyl-tris-trilaurate (CS087), the concentration of
the lipid is preferably 10 .mu.M to 50 .mu.M. Most preferably, the
concentration of trilysine-carpryloyl-tris-trilaure is 10
.mu.M.
[0066] It has also been found that the presence of a surfactant in
the composition may further improve the ability of the composition
to preserve a virus. The surfactant is any molecule that can reduce
the interfacial tension between two immiscible phases. Preferably
the surfactant is a non-ionic surfactant.
[0067] The determination of whether a molecule may function as a
surfactant may be by a suitable method known in the art in which
the function of a molecule to reduce the interfacial tension
between two immiscible phases may be tested.
[0068] Preferably, the surfactant is present in the composition at
a concentration in the range from 0.0001% to 50% volume/volume.
More preferably, the surfactant is present in the composition at a
concentration in the range from 0.001% to 10% volume/volume.
[0069] In a preferred embodiment, the non-ionic surfactant is a
molecule that includes an oxyethylene group and a hydroxy group.
Most preferably, the non-ionic surfactant is polysorbate 80 or
polyethylene glycol 400, or any combination of these non-ionic
surfactants.
[0070] When polysorbate 80 is used in the composition, the
concentration of the polysorbate 80 is preferably 0.0001 to 1%
volume/volume. More preferably, the concentration of polysorbate 80
in the composition is 0.001 to 0.1% volume/volume. Most preferably,
the concentration of polysorbate 80 in the composition is 0.005%
volume/volume.
[0071] When polyethylene glycol 400 is used in the composition, the
concentration is preferably 0.001 to 50% volume/volume. More
preferably, the concentration of polyethylene glycol 400 is 0.01 to
10% volume/volume. More preferably, the concentration of
polyethylene glycol 400 is 0.01 to 5% volume/volume. Most
preferably, the concentration of polyethylene glycol 400 in the
composition is 0.5% volume/volume.
[0072] The pH of the composition may also be selected to improve
viral preservation. The pH may also be selected to be compatible
with the administration of the composition to a subject for
therapeutic purposes. Preferably, the pH of the composition is in
the range of 4 to 10. More preferably the pH is in the range of 5
to 9. In the most preferred form of the invention, the pH of the
composition is in the range of 6 to 8.5.
[0073] The pH of the composition according to the present invention
may be obtained by buffering with a pharmaceutically acceptable
buffer. Preferably, the buffer is selected from one or more buffers
selected from the group consisting of monobasic acids including
acetic, benzoic, gluconic, glyceric and lactic acids, dibasic acids
including aconitic, adipic, ascorbic, carbonic, glutamic, maleic,
malic, succinic, tartaric acids, polybasic acids including citric
and phosphoric acids. The buffer may also be selected from one or
more buffers selected from the group consisting of bases including
ammonia or ammonium chloride, diethanolamine, glycine, tromethamine
(also known as Tris and Tham).
[0074] Preferably, the buffer is selected from one or more buffers
selected from the group consisting of a tris-based buffer, a sodium
hydrogen maleate buffer, succinate buffer, or phosphate buffer.
Tris-based buffers and sodium hydrogen maleate buffers are
particularly preferred.
[0075] As stated previously, the composition according to the
present invention provides a composition for the preservation of a
virus. In one embodiment, the present invention provides a
composition for the preservation of a virus, the composition
including a virus and a lipid, wherein the virus is storage stable.
The composition in this form of the invention may be in liquid form
or in lyophilised, spray-dried or freeze-dried forms.
[0076] Preferably, the temperature of storage of the composition in
liquid form is in the range from 0.degree. C. to 30.degree. C. More
preferably, the temperature of storage is in the range from
2.degree. C. to 25.degree. C. Most preferably, the temperature of
storage is in the range from 2.degree. C. to 8C.
[0077] In the case where the virus in the composition is stored in
the solid state, the composition is preferably stored from
-20.degree. C. to 30.degree. C.
[0078] With respect to the period of time over which the
composition according to the various forms of the present invention
shows improved preservation, the composition according to the
present invention may be stored for a period of greater than 24
months. Preferably the period of storage is 12 months or greater.
More preferably, the period of storage is 6 months or greater. More
preferably, the period of storage is 3 months or greater. More
preferably, the period of storage is 1 week or greater. Most
preferably, the period of storage is 1 day or greater.
[0079] In a further preferred embodiment, the present invention
provides a composition for the preservation of a virus, the
composition including a virus and a lipid, wherein the virus is
stable when the composition is subjected to one or more of
agitation, shearing forces or mechanical action.
[0080] In this regard, as has been discussed previously, the
improved preservation of the composition in the various forms of
the present invention will be as compared to a composition that
does not contain lipid. That is, the activity of the virus will not
decrease substantially with time when the composition is stored at
the abovementioned temperatures and/or for the abovementioned
periods of time, or when subjected to the abovementioned
conditions, as compared to a composition not containing lipid. The
activity of the virus may be a desired activity of the virus in the
composition, such as infectivity, ability to transduce or
immunogenicity.
[0081] The composition according to the present invention may also
be in a dosage form suitable for administration to a human or an
animal subject. The dosage form includes the composition according
to the present invention and may further include other
pharmaceutically acceptable additives.
[0082] The addition of such pharmaceutically acceptable additives
to the dosage form may be to improve the ability of the virus to
infect or transduce target cells, or to improve the activity
elicited by the administration of virus. For example, local
bystander killing can be enhanced by co-administration of a
pharmaceutical or genetic agent which enhances cell-cell
communications. Another example is the co-administration of a DNA
encoding a cytokine to increase the immunogenicity of tumour cells.
Another example is the inclusion of an adjuvant compound in a
vaccine to enhance immune response.
[0083] The present invention also provides a method of producing a
composition for the preservation of a virus, the method including
the step of preparing a liquid composition including a virus and a
lipid.
[0084] As will be appreciated, the method according to the various
forms of the present invention will also embody the same preferred
features as those for the composition as discussed in detail
above.
[0085] With regard to the preparation of virus, the virus may be
purified by any suitable means. Preferably, the virus is purified
by a chromatographic method including ion-exchange chromatography
or HPLC, or centrifugation including CsCI centrifugation, after the
virus has been recovered from infected permissive cells and/or the
supernatant thereof. Preferably, the virus is purified by a
chromatographic method. When purified by CsCI centrifugation, the
virus is prepared after recovery from infected permissive cells by
centrifugation through a CsCI step gradient and centrifugation to
equilibrium on a CsCI gradient. When virus is purified in this
manner, the CsCI is preferably removed by column
chromatography.
[0086] Preferably, the concentrated virus so formed is diluted in a
solution that includes a suitable buffer. More preferably, the
solution further includes a non-ionic surfactant. In a preferred
embodiment, the concentrated virus is diluted in a solution
including a Tris buffer, and polyethylene glycol 400 and/or
polysorbate 80. In a particularly preferred embodiment, the
concentrated virus is diluted in a solution (at pH 8.0) including
10 mM Tris buffer, and 2% polyethylene glycol 400. Preferably, the
solution (which may exist as a suspension) containing virus is then
filtered to remove unwanted micro-organisms. Most preferably, the
solution is filtered through a 0.2 micron membrane filter.
[0087] For the preparation of a composition according to the
present invention, the lipid is preferably first dispersed in a
solution identical to that used for the dilution of virus.
Preferably, the solution (which may exist as a suspension)
containing lipid is filtered to remove unwanted micro-organisms.
Most preferably, the solution is filtered through a 0.2 micron
membrane filter.
[0088] To prepare a composition for the preservation of virus, the
diluted solution of virus (which may exist as a suspension) may
then be combined with a solution containing lipid (which may also
exist as a suspension), the relative proportions of each selected
so as to achieve the desired final concentrations of virus and
lipid. Accordingly, the method according to the present invention
provides a method for producing a composition for the preservation
of a virus, wherein the composition is formed by combining a
solution including a virus with a solution including lipid.
[0089] The composition so formed may be stored in a suitable closed
container. Preferably the composition is stored in borosilicate
glass vials. In addition, the composition may be stored under a
suitable gas or mixture of gases.
DESCRIPTION OF THE PRERERRED EMBODIMENTS
[0090] Reference will now be made to examples that embody the above
general principles of the present invention. However, it is to be
understood that the following description is not to limit the
generality of the above description.
EXAMPLE 1
Preparation of a Composition for the Preservation of Virus
[0091] CsCI purified OAdV623 virus was suspended in a pH 8.0 buffer
containing 10 mM Tris, 8.5% sucrose, 2% PEG buffer, in a
polypropylene tube, at two-times the final concentration. CS087 was
supplied as a freeze-dried solid that was first dissolved in
ethanol and the ethanol then removed to produce a film. The film
was dispersed in a pH 8.0 buffer containing 10 mM Tris, 8.5%
sucrose, 2% polyethylene glycol 400, in a polystyrene tube, at
two-times the final concentration.
[0092] The suspensions of OAdV623 and CS087 were filtered
separately through a 0.2 .mu.m membrane filter. An equal volume of
OAdV623 and CS087 were combined aseptically. The suspension was
then gently agitated continuously at approximately 40 rpm for 60 to
90 minutes at 18.degree. C.-20.degree. C., to ensure viral mixing.
The final product was then aseptically dispensed into washed and
autoclaved Type I borosilicate glass vials and stored at the
appropriate temperature.
EXAMPLE 2
Storage Stability of Various Viral Compositions at Refrigeration
Temperature
[0093] The stability of various OAdV623 compositions (approximately
6.times.10.sup.8 VP/ml) stored at refrigeration temperature
(approximately 4.degree. C.) was assessed by determining the extent
of cell killing at Day 0 and after storage for 7 days. Cell killing
was determined for compositions stored at pH 8 and pH 6.
[0094] OAdV623 encodes the PNP gene which catalyses the conversion
of the immunosuppressive prodrug Fludarabine to the toxic
2-fluoro-adenine product. This results in the death of cells
producing PNP and to a limited extent, cells in the vicinity with a
near neighbour bystander effect. The death of susceptible cells,
such as the PC3 cell line, following transduction with OAdV623 and
treatment with Fludarabine phosphate, is a direct indicator of the
potency of the OAdV623 preparation.
[0095] To determine the extent of cell killing, an aliquot of virus
in the relevant composition containing approximately
6.times.10.sup.6 virus particles were used to transduce
1.times.10.sup.4 PC3 cells in culture. The ability of the virus to
kill PC3 cells by converting the prodrug fludarabine, supplied to
the cells as fludarabine phosphate, to active 2-fluoroadenine, was
then determined quantitatively. Cell killing was determined by an
MTS assay (Promega) to measure the number of viable cells in
treated wells compared to a standard curve of cells not treated
with the virus.
[0096] The concentration of the various components used was as
follows:
[0097] 10 mM Tris
[0098] 10 mM sodium hydrogen maleate
[0099] 8.5% sucrose
[0100] 50 .mu.M CS087
[0101] 2% (v/v) polyethylene glycol 400 (PEG400)
[0102] 0.005% (v/v) polysorbate 80 (PS80)
[0103] The whole composition was buffered to the desired pH with
Tris or maleate buffer.
[0104] (a) Stability at pH 8.
[0105] As can be seen, the addition of lipid to the tris/sucrose
composition enhanced the preservation of the virus, when the virus
was stored at 4.degree. C. for 7 days. The addition of a non-ionic
surfactant to the tris/sucrose/lipid composition further enhanced
the preservation of virus. Thus the preservation of the virus after
storage was improved by the addition of lipid and, in particular,
lipid plus a non-ionic surfactant.
[0106] (b) Stability at pH 6.
[0107] As can be seen, the addition of lipid to the maleate/sucrose
composition enhanced the preservation of the virus, when the virus
was stored at 4.degree. C. for 7 days. The addition of a non-ionic
surfactant to the maleate/sucrose/lipid composition also enhanced
the preservation of virus compared to a composition without any
lipid or surfactant. Thus the preservation of the virus after
storage was improved by the addition of lipid with or without a
non-ionic surfactant.
EXAMPLE 3
Preservation of Various Viral Compositions Upon Storage and when
Subjected to Gentle Agitation, Transportation and Multiple
Freeze-thaw Cycles
[0108] Preservation of various viral compositions (pH 8) upon
storage (-80.degree. C.) and when subjected to gentle agitation,
transportation and multiple freeze-thaw cycles (-80.degree. C. to
wet ice temperature of approximately 0.degree. C. and -80.degree.
C. to 20.degree. C.)
[0109] The ability of various OAdV623 compositions to be preserved
upon storage and when subjected to gentle agitation, transportation
and multiple freeze-thaw cycles was assessed by determining the
extent of cell killing after the formulations had been subjected to
the following:
[0110] a storage period of 11 weeks,
[0111] packed in dry ice and transported by road for approximately
1 hour,
[0112] a freeze-thaw cycle,
[0113] mixing by gentle agitation (eg. pipetting up and down),
[0114] frozen, packed in dry ice and again transported by road for
approximately 1 hour,
[0115] an additional storage period of 2 weeks and
[0116] a second freeze-thaw cycle.
[0117] The storage temperature was -80.degree. C. For the first
freeze-thaw cycle, virus was thawed on wet ice (approximately
0.degree. C.) for a total of 6 hours. For the second freeze-thaw
cycle, virus was thawed at 20.degree. C. for 30 minutes.
[0118] A batch of virus was first harvested in tris/sucrose and the
potency determined using a cell-killing assay. Four formulations
were then prepared from this batch of virus as follows:
[0119] Formulation A: OAdV623 (1.times.10.sup.12 VP/ml) was stored
in tris/sucrose at -80.degree. C. for 11 weeks. This formulation
was subsequently packed in dry ice and transported, thawed, mixed
by gentle agitation and held on wet ice (approximately 0.degree.
C.) for 6 hours before being re-frozen at -80.degree. C., packed in
dry ice and again transported and then stored for a further 2
weeks. This formulation containing OAdV623 at 1.times.10.sup.12
VP/ml was then thawed again at 20.degree. C. for 30 minutes
immediately prior to the cell killing assay.
[0120] Formulation B: OAdV623 (1.times.10.sup.12 VP/ml) was stored
in tris/sucrose at -80.degree. C. for 11 weeks. This formulation
was subsequently packed in dry ice and transported, thawed on wet
ice (approximately 0.degree. C.) for 2 hours, mixed with lipid with
gentle agitation, held at room temperature (approximately
20.degree. C.) for 30 minutes and then left to stand on wet ice for
3.5 hours before being re-frozen at -80.degree. C., packed in dry
ice and again transported and then stored for a further 2 weeks.
This formulation containing OAdV623 at 5.times.10.sup.11 VP/ml was
then thawed again at 20.degree. C. for 30 minutes immediately prior
to the cell killing assay.
[0121] Formulation C: OAdV623 (1.times.10.sup.12 VP/ml) was stored
in tris/sucrose at -80.degree. C. for 11 weeks. This formulation
was subsequently packed in dry ice and transported, thawed on wet
ice (approximately 0.degree. C.) for 2 hours, mixed with
lipid/PEG400 with gentle agitation, held at room temperature
(approximately 20.degree. C.) for 30 minutes and then left to stand
on wet ice for 3.5 hours before being re-frozen at -80.degree. C.,
packed in dry ice and again transported and then stored for a
further 2 weeks. This formulation containing OAdV623 at
5.times.10.sup.11 VP/ml was then thawed again at 20.degree. C. for
30 minutes immediately prior to the cell killing assay.
[0122] Formulation D: OAdV623 (1.times.10.sup.12 VP/ml) in
tris/sucrose was mixed with lipid/PEG400 at room temperature
(approximately 20.degree. C.) for 30 minutes. This OAdV623
formulation in tris/sucrose/lipid/PEG400 (5.times.10.sup.11 VP/ml)
was stored at -80.degree. C. for 11 weeks. This formulation was
subsequently packed in dry ice and transported, thawed, mixed by
gentle agitation and held on wet ice (approximately 0.degree. C.)
for 6 hours before being re-frozen at -80.degree. C., packed in dry
ice and again transported and then stored for a further 2 weeks.
This formulation containing OAdV623 at 5.times.10.sup.11 VP/ml was
then thawed again at 20.degree. C. for 30 minutes immediately prior
to the cell killing assay.
[0123] To determine the extent of cell killing, virus particles in
the range of 2.times.10.sup.6 to 2.times.10.sup.8 were used to
transduce 5.times.10.sup.3 PC3 cells in culture. The ability of the
virus to kill PC3 cells by converting the prodrug fludarabine,
supplied to the cells as fludarabine phosphate, to active
2-fluoroadenine, was then determined quantitatively. Cell killing
was determined by an MTS assay (Promega) to measure the number of
viable cells in treated wells compared to a standard curve of cells
not treated with the virus.
[0124] The concentration of the various components in the final
formulations were as follows:
[0125] 10 mM Tris
[0126] 8.5% sucrose
[0127] 10 .mu.M CS087
[0128] 0.5% (v/v) polyethylene glycol 400 (PEG400)
[0129] The whole composition was buffered to the desired pH with
Tris or maleate buffer.
[0130] The results are as shown in the following figure:
[0131] As can be seen, the composition containing
tris/sucrose/lipid/PEG40- 0 (Formulation D) provided the most
protection to the virus against the effects of storage, agitation,
transportation and freeze-thawing. In contrast, the composition
containing tris/sucrose without any lipid or PEG400 (Formulation A)
provided the least protection to the virus against the effects of
storage, agitation, transportation and freeze-thawing.
[0132] Finally, it will be appreciated that various modifications
and variations of the described compositions and methods of the
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention, which are apparent to those skilled in the field of
virology, molecular biology, or related fields are intended to be
within the scope of the present invention.
* * * * *