AU2002366654B2 - Composition for viral preservation - Google Patents

Description

WO 03/049764 PCT/AU02/01680 COMPOSITION FOR VIRAL PRESERVATION Field of the Invention 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.

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 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 recognised as being useful vehicles for the transfer of therapeutic nucleic acids into cells for the purpose of gene therapy.

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 WO 03/049764 PCT/AU02/01680 -2virus to replicate, the viral genome is either passed on faithfully to every daughter cell or is eventually lost during cell division.

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.

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, nonintegrating viruses are used when transient gene expression is desired.

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.

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 WO 03/049764 PCT/AU02/01680 -3compositions of viruses to be used for gene therapy, it is critical that the efficacy of the live viral formulations carrying therapeutic transgenes be maintained.

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).

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.

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.

WO 03/049764 PCT/AU02/01680 -4- 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.

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 0 C to -200C) for substantial periods of time represent a serious impediment to the widespread clinical use of many viruses.

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.

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.

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.

WO 03/049764 PCT/AU02/01680 It is therefore an aim of the present invention to provide a composition for the improved preservation of viruses.

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 The present invention provides a composition for the preservation of a virus, the composition including a virus and a lipid.

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.

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.

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 WO 03/049764 PCT/AU02/01680 -6provides improved preservation of the virus to the effects of agitation, shearing forces and mechanical action.

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.

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, nonviable and synthetic viral particles for non-medical applications, such as the preservation of viral preparations for research and diagnostic applications.

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.

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 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.

WO 03/049764 PCT/AU02/01680 -7- 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.

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.

The term "virus" as used throughout the specification is to be understood to mean any natural, recombinant, in vitro packaged or synthetic virus.

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.

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 WO 03/049764 PCT/AU02/01680 -8be 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.

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 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.

WO 03/049764 PCT/AU02/01680 -9- 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.

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 nonviable, 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.

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.

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.

A non-viable virus is to be understood to mean a viral particle that is not able to infect or transduce permissive host cells.

WO 03/049764 PCT/AU02/01680 A synthetic virus is to be understood to mean any nucleic acid packaged with a protein and/or lipid coat.

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.

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.

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.

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.

WO 03/049764 PCT/AU02/01680 -11- In the case of a solid composition, the composition may be a lyophilised composition, a freeze-dried composition or a spray dried composition.

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.

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.

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.

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 WO 03/049764 PCT/AU02/01680 -12particles or the size of the viral particles (as an indication of viral aggregation) in the product.

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.

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 1x106 to 1x1014 virus particles/ml. More preferably, the concentration of virus is in the range from 1x108 to 5x1012 virus particles/ml.

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.

The lipid present in the composition according to the various forms of the present invention may be a cationic lipid, anionic lipid, zwitterionic lipid, nonionic lipid or any combination of such lipids.

Examples of cationic lipids include 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate WO 03/049764 WO 03149764PCTIAU02/01680 -13- (DOS PA), dioctadecylaminoglycyl spermine (DOGS), dipalmitoyl phosphatidylethanolamyl spermine (OPPES), 1 ,3-dioleoyoxy-2-(6-carboxyspermyl)-propylamide (DOS PER), dioleyldimethylammonium chloride (DODAC), 1 -(2,3-dioleyloxy)propyl]-NNN-trimetylammonium chloride (DOTMA), 1 ,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP), 1 ,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMVRI E), 313-(N-((WV, Wdimethylamino)ethane)carbamoyl)-cholestero (DC-Chol), dimethyldioctadecyl ammonium bromide (DDAB), 1 -[2-(oleoyoxy)-ethyl]-2-oleyl- 3-(2-hydroxyethyl) imidazolinium chloride (DOTI bis(oleoyl)trimethylaminomethylphosphonate, 1 ,2-dimyristoylglycerolpentalysine salt, N,NV,N",N"Ltetramethyl-N,tiV,N' N"t-tetrapalmitylspermine (TMTPS), cetyltrimethylammoniumn bromide (CTAB) and the following proprietary cationic lipids: Lipofectamine (DOSPA:DOPE 3:1 Lipofectin (DOTMA:DOPE 1:1 Lipofectace (DDAB: DOPE 1:1.25 Transfectam, Cellfectin (TMTPS:DOPE 1:1.5 Superfect, LipoTaxi, DMRIE-C (DMRIE/cholesterol: 1:1) and trilysine-carpryloyl-tris-trilau rate (T-shape; CS087).

Examples of anionic lipids include 1 ,2-dioleoyl-sn-glycero-3-[phospho-L-serineI (DOPS), 1 ,2-dimyristoyl-sn-glycero-3-phosphoglycero (DM PG), and PEG-PE lipids such as 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol) 2000] (PEG2000 DM PE), 1 ,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-[poly(ethyleneglycol) 2000] (PEG2000 DPPE), 1,2distearoyl-sn-glycero-3- phos ph oeth ano lam ine- N-[poly(ethyleneg lycol) 2000] (PEG2000 DSPE).

Examples of zwitterionic/neutral lipids include 1 ,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3-phosphcholine (DOPO), 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DM PC) and 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine (DM PE).

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 WO 03/049764 PCT/AU02/01680 -14preferably, 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.

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 polycationic 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.

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.

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.

WO 03/049764 PCT/AU02/01680 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.

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:

CH

2 0-R 1 X-Y-NH-C-C H 2 0-R 2

CH

2 0-R 3 In this general formula, X represents the hydrophilic moiety, Y represents a spacer group (which may or may not be present), and R 1 Ra and R 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 carbon-carbon single covalent bonds.

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: WO 03/049764 PCT/AU02/01680 -16-

NH,*

HN 0 0 0

HN

NH"

To preserve virus, the concentration of the lipid in the composition is preferably in the range from 0.1 tLM to 1 mM. More preferably the concentration of the lipid is 1 [M to 500 gM. In the most preferred embodiment, the concentration of the lipid is 5 pM to 100 pM.

In the case where the lipid in the composition is trilysine-carpryloyl-tris-trilaurate (CS087), the concentration of the lipid is preferably 10 gM to 50 CIM. Most preferably, the concentration of trilysine-carpryloyl-tris-trilaure is 10 pM.

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.

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.

Preferably, the surfactant is present in the composition at a concentration in the range from 0.0001% to 50% volumelvolume. More preferably, the surfactant is present in the composition at a concentration in the range from 0.001% to volume/volume.

WO 03/049764 PCT/AU02/01680 -17- 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.

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.

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 volume/volume.

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 to 9. In the most preferred form of the invention, the pH of the composition is in the range of 6 to 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 WO 03/049764 PCT/AU02/01680 -18acids. 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).

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.

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.

Preferably, the temperature of storage of the composition in liquid form is in the range from OOC to 30 0 C. More preferably, the temperature of storage is in the range from 20C to 250C. Most preferably, the temperature of storage is in the range from 20C to 8C.

In the case where the virus in the composition is stored in the solid state, the composition is preferably stored from -20°C to 300C.

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.

WO 03/049764 PCT/AU02/01680 -19- 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.

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.

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.

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.

WO 03/049764 PCT/AU02/01680 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.

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.

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.

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 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.

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 WO 03/049764 PCT/AU02/01680 -21 is filtered to remove unwanted micro-organisms. Most preferably, the solution is filtered through a 0.2 micron membrane filter.

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.

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 Preferred Embodiments 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 CsCI purified OAdV623 virus was suspended in a pH 8.0 buffer containing 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 10mM Tris, 8.5% sucrose, 2% WO 03/049764 PCT/AU02/01680 -22polyethylene glycol 400, in a polystyrene tube, at two-times the final concentration.

The suspensions of OAdV623 and CS087 were filtered separately through a 0.2utm 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 180C-200C, 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.

The stability of various OAdV623 compositions (approximately 6x10 8 VP/ml) stored at refrigeration temperature (approximately 40C) 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.

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.

To determine the extent of cell killing, an aliquot of virus in the relevant composition containing approximately 6x10 6 virus particles were used to transduce 1x104 PC3 cells in culture. The ability of the virus to kill PC3 cells by converting the prodrug fludarabine, supplied to the cells as fludarabine WO 03/049764 PCT/AU02/01680 -23phosphate, 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.

The concentration of the various components used was as follows: mM Tris mM sodium hydrogen maleate sucrose 50 gM CS087 2% polyethylene glycol 400 (PEG400) 0.005% polysorbate 80 The whole composition was buffered to the desired pH with Tris or maleate buffer.

Stability at pH 8.

pH 8 (4C) 100 0 Day 0 a Tris/sucrose O Tris/sucrose/CS087 m Tris/sucrose/CS087/PEG400 m Tris/sucrose/CS087/PS80 Trs/sucrose/PEG400 a 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 40C for 7 days. The addition of a non-ionic surfactant to the tris/sucrose/lipid composition further WO 03/049764 PCT/AU02/01680 -24enhanced 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 nonionic surfactant.

Stability at pH 6.

pH 6 (4C) 100 0' Day 0 m Maleate/sucrose Maleate/sucrose/CS087 m Maleate/sucrose/CS087/PEG400 [1 Maleate/sucrose/CS087/PS80 M Maleate/sucrose/PEG400 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 40C 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 Preservation of various viral compositions (pH 8) upon storage (-800C) and when subjected to gentle agitation, transportation and multiple freeze-thaw cycles (-800C to wet ice temperature of approximately 0°C and -800C to 200C) WO 03/049764 PCT/AU02/01680 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: a storage period of 11 weeks, packed in dry ice and transported by road for approximately 1 hour, a freeze-thaw cycle, mixing by gentle agitation (eg. pipetting up and down), frozen, packed in dry ice and again transported by road for approximately 1 hour, an additional storage period of 2 weeks and a second freeze-thaw cycle.

The storage temperature was -80 0 C. For the first freeze-thaw cycle, virus was thawed on wet ice (approximately 0°C) for a total of 6 hours. For the second freeze-thaw cycle, virus was thawed at 20 0 C for 30 minutes.

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: Formulation A: OAdV623 (1x1012 VP/ml) was stored in tris/sucrose at -800C 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°C) for 6 hours before being re-frozen at -800C, packed in dry ice and again transported and then stored for a further 2 weeks. This formulation containing OAdV623 at 1x10 12 VP/ml was then thawed again at 200C for 30 minutes immediately prior to the cell killing assay.

Formulation B: OAdV623 (1xl 012 VP/ml) was stored in tris/sucrose at -800C for 11 weeks. This formulation was subsequently packed in dry ice and transported, thawed on wet ice (approximately 0°C) for 2 hours, mixed with lipid WO 03/049764 PCT/AU02/01680 -26with gentle agitation, held at room temperature (approximately 200C) for minutes and then left to stand on wet ice for 3.5 hours before being re-frozen at -800C, packed in dry ice and again transported and then stored for a further 2 weeks. This formulation containing OAdV623 at 5x1011 VP/ml was then thawed again at 200C for 30 minutes immediately prior to the cell killing assay.

Formulation C: OAdV623 (1xl 012 VP/ml) was stored in tris/sucrose at -800C for 11 weeks. This formulation was subsequently packed in dry ice and transported, thawed on wet ice (approximately 0°C) for 2 hours, mixed with lipid/PEG400 with gentle agitation, held at room temperature (approximately 200C) for 30 minutes and then left to stand on wet ice for 3.5 hours before being re-frozen at -800C, packed in dry ice and again transported and then stored for a further 2 weeks. This formulation containing OAdV623 at 5x10 1 1 VP/ml was then thawed again at 200C for 30 minutes immediately prior to the cell killing assay.

Formulation D: OAdV623 (1x10 1 2 VP/ml) in tris/sucrose was mixed with lipid/PEG400 at room temperature (approximately 20°C) for 30 minutes. This OAdV623 formulation in tris/sucrose/lipid/PEG400 (5x10 1 1 VP/ml) was stored at -80 0 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°C) for 6 hours before being re-frozen at -800C, packed in dry ice and again transported and then stored for a further 2 weeks. This formulation containing OAdV623 at 5x10 11 VP/ml was then thawed again at 200C for 30 minutes immediately prior to the cell killing assay.

To determine the extent of cell killing, virus particles in the range of 2x10 6 to 2x10 8 were used to transduce 5x10 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 WO 03/049764 PCT/AU02/01680 -27measure the number of viable cells in treated wells compared to a standard curve of cells not treated with the virus.

The concentration of the various components in the final formulations were as follows: mM Tris sucrose IM CS087 0.5% polyethylene glycol 400 (PEG400) The whole composition was buffered to the desired pH with Tris or maleate buffer.

The results are as shown in the following figure: (j a c 0 ca 0 o 6° 0 Formulation A Formulation B Formulation C Formulation D Note: Cell kill at virus particle input of 2x10- 7 upon harvest and prior to preparation of formulations was As can be seen, the composition containing tris/sucrose/lipid/PEG400 (Formulation D) provided the most protection to the virus against the effects of storage, agitation, transportation and freeze-thawing. In contrast, the WO 03/049764 PCT/AU02/01680 -28composition 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.

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.

Claims (14)

1. A composition for the preservation of a virus, the composition including a virus and a lipid. A composition according to claim 1, wherein the lipid is a cationic lipid. IN IN 3. A composition according to claim 2, wherein the cationic lipid has a hydrophilic moiety including one or more amino residues. 0

4. A composition according to claim 3, wherein the one or more amino residues is derived from an amino acid. A composition according to any one of claims 1 to 4, wherein the lipid has a hydrophobic moiety including one or more hydrophobic groups.

6. A composition according to claim 5, wherein the one or more hydrophobic groups includes an acyl, alkyl or alkoxy group.

7. A composition according to claim 6, wherein the acyl group has a carbon chain length of 3 to 24 carbon atoms.

8. A composition according to any one of claims 1 to 7, wherein the lipid has the following chemical formula: CH 2 O-R 1 I X-Y-N H-C-C H 2 0-R 2 I CH 2 0-R 3 or a salt thereof, wherein: 00 0 (O O O C X is a positively charged hydrophilic moiety; Y is spacer having a chain length equivalent to 1 to carbon-carbon single covalent bonds or is absent; and R 1 R 2 and R 3 are the same or different and are acyl groups derived from a fatty acid.

9. A composition according to claim 8, wherein the tris-conjugated lipid has the following chemical formula: HN 0 NH,' A composition according to any one of claims 1 to 9, wherein the composition further includes a surfactant.

11. A composition according to claim 10, wherein the surfactant is a non- ionic surfactant.

12. A composition according to any one of claims 1 to 11, wherein the virus is selected from the group consisting of Adenoviridae, Herpesviridae, Poxviridae, Papovaviridae, Orthohepadnavirus, Parvoviridae, Birnaviridae, Reoviridae, Flaviviridae, Picornaviridae, Togaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, and Retroviridae. -31 00 O

13. A composition according to any one of claims 1 to 12, wherein the virus C is storage stable.

14. A composition according to any one of claims 1 to 13, wherein the virus \0 is stable when the composition is subjected to one or more of agitation, IND shearing forces or mechanical action. S 10 15. 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.

16. A method according to claim 15, wherein the lipid is a cationic lipid.

17. A method according to claims 15 or 16, wherein the lipid has the following chemical formula: CH 2 0-R 1 X-Y-NH-C-CH 2 0-R 2 I CH 2 0-R 3 or a salt thereof, wherein: X is a positively charged hydrophilic moiety; Y is spacer having a chain length equivalent to 1 to carbon-carbon single covalent bonds or is absent; and R 1 R 2 and R 3 are the same or different and are acyl groups derived from a fatty acid.

18. A method according to any one of claims 15 to 17, 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, -32- 00 O Picornaviridae, Togaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, and Retroviridae.

19. A method according to claim any one of claims 15 to 18, wherein the virus is storage stable. In 11 20. A composition produced according to the method of any one of claims IN 15 to 19. .i