WO2003093486A1 - In situ generation of viral vectors - Google Patents

Abstract

Materials and methods useful in the in situ generation of viral vectors are described. Briefly, virus is liberated from cells by treatment with polysorbate and tri-butyl phosphate yielding non-viscous lysates with relatively little cell debris. Lysis is followed by step density ultracentrifugation to isolate crude virus. Finally, purification and helper removal is accomplished by dynamic banding ultracentrifugation.

Description

In situ Generation of Viral Vectors

BACKGROUND OF THE INVENTION

Field Of The Invention

The invention described herein relates to the fields of biology and medicine.

Specifically, the invention involves the in situ generation of viral vectors. The invention also involves improved materials and methods for the practice of in situ generation of viral vectors as well as materials and methods for production of biological compositions useful in the in situ generation of viral vectors.

Description Of The Related Art

Adenoviral biology, minimal adenoviral systems (e.g., including, but not limited to, mini-Ad, MAX-Ad, MAXIMUM-Ad^®, and the like) and complementary adenoviral systems {e.g., including, but not limited to, controlled Ad/supplemental Ad, Dual-Ad, and the like) are described in detail in related applications {see Cross-Reference to Related Applications, supra) as well as references therein, all of which are incorporated by reference to the extent that they are not contradictory.

The invention disclosed herein involves the in situ generation of viral vectors. The invention further comprises novel materials and methods useful for the production and use of biological compositions, in particular viral vectors and vector systems, specifically adenoviral vectors and vector systems. Such vectors and vector systems are useful to deliver genetic material for a variety of research and medical purposes including, but not limited to, the delivery of genes to express therapeutic proteins and the ability to generate and/or increase one or more immune responses to one or more antigens encoded by the vectors and/or vector systems.

It is known in the art that employing different antigenic compositions, formulations and/or vectors may stimulate a more robust immune response to a particular antigen {see generally Ertl, H.C.J. (2001) "Methods of augmenting mucosal immunity through systemic priming and mucosal boosting." U.S. Patent No. 6,210,663; Kong, Q. et al. (2001) "Oral immunization with hepatitis B surface antigen expressed in transgenic plants." Proc. Natl. Acad. Sci. USA, vol. 98, no. 20, pp. 11539-1 1544; Bruna-Romero, O. et al. (2001) "Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen." Proc. Natl. Acad. Sci. USA, vol. 98, no. 20, pp. 11491-11496; Tanghe, A. et al. (2001) "Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting." Infect. Immun., vol. 69, no. 5, pp. 3041-3047; Doolan, D.L. & Hoffman, S.L. (2001) "DNA-based vaccines against malaria: status and promise of the Multi-Stage Malaria DNA Vaccine Operation." Int. J. Parasitol, vol. 31, no. 8, pp. 753-762; Siegrist, CA. (2001) "Neonatal and early life vaccinology." Vaccine, vol. 19, nos. 25-26, pp. 3331-3346; Hill, AN. et al. (2000) "DΝA-based vaccines for malaria: a heterologous prime-boost immunization strategy." Dev. Biol, vol. 104, pp. 171-179; Gray, CM. & Puren, A.J. (2000) "Rethinking globally relevant vaccine strategies to human immunodeficiency virus type-1." Arch. Immunol. Ther. Exp., vol. 48, no. 4, pp. 235-241; Fedson, D.S. (1999) "The clinical effectiveness of pneumococcal vaccination: a brief review." Vaccine, vol. 17, suppl. 1 , pp. S85-90; Ada, G. (1999) "The coming of age of tumour immunotherapy." Immunol. Cell. Biol., vol. 77, no. 2, pp. 180- 185; Zheng, R. (1999) "Technology evaluation: HIVAC-le." Curr. Opin. Mol. Ther., vol. 1, no. 1, pp. 121-125; Richmond, J.F.L. et al. (1998) "Studies of the neutralizing activity and avidity of anti-human immunodeficiency virus type 1 env antibody elicited by DΝA priming and protein boosting." J. Virol, vol. 72, no. 1 1, pp. 9092-9100; Sedegah, M. et al. (1998) "Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DΝA vaccine." Proc. Natl. Acad. Sci. USA, vol. 95, pp. 7648-7653; Irvine, K.R. et al. (1997) "Enhancing efficacy of recombinant anticancer vaccines with prime boost regimens that use two different vectors." J. Natl. Cancer Inst, vol. 89, pp. 1595-1601 ; Cooney, E.L. et al. (1993) "Enhanced immunity to human immunodeficiency virus (HIV) envelope elicited by a combined vaccine regimen consisting of priming with vaccinia recombinant expressing HIV envelope and boosting with gpl60 protein." Proc. Natl. Acad. Sci. USA, vol. 90, pp. 1882-1886; references in any of the foregoing; and the like.).

However, little is known about the usefulness or efficacy of such techniques when antigen delivery vectors derived from adenovirus are used for differential priming and/or boosting protocols (see Buge, S.L. et al. (1999) "Factors associated with slow disease progression in macaques immunized with an adenovirus-simian immunodeficiency virus (SIV) envelope priming-gpl20 boosting regimen and challenged vaginally with SIVmac251." J. Virol, vol. 73, no. 9, pp. 7430-7440; Buge, S.L. et al. (1999) "Factors associated with slow disease progression in macaques immunized with an adenovirus- simian immunodeficiency virus (SIV) envelope priming-gpl20 boosting regimen and challenged vaginally with SIVmac251 (Erratum)." J. Virol, vol. 73, no. 11, p. 9692; Robert-Guroff, M. et al. (1998) "Vaccine protection against a heterologous, non- syncytium-inducing, primary human immunodeficiency virus." J. Virol, vol. 72, no. 12, pp. 10275-10280).

Thus, it is apparent that improved materials and methods useful in the production of biological compositions, specifically vectors and/or vector systems, more specifically adenoviral vectors and vectors systems would be a distinct and unique advantage to persons skilled in the art.

It is also generally recognized that immune responses to vector system components can interfere with effective gene delivery and decrease the efficacy of these preparations. Disclosed and described herein are novel approaches to address this problem by utilizing liposomes to encapsulate vector systems to reduce these deleterious immune system effects.

This application is related to the following patent applications: U.S. App. No. 60/347,707, filed 10 Jan. 2002; U.S. App. No. 09/993,593, filed 05 Nov. 2001 ; U.S. App. No. 60/329798, filed 16 Oct. 2001; U.S. App. No. 09/974206, filed 10 Oct. 2001; Int. App. No. PCT/USOl/31842, filed 10 Oct. 2001; U.S. App. No. 60/323644, filed 20 Sep. 2001 ; Int. App. No. PCT US01/23005, filed 20 Jul. 2001 ; U.S. App. No. 09/837079, filed 18 Apr. 2001 ; U.S. App. No. 60/287850, filed 01 May 2001 ; U.S. App. No. 09/779570, filed 8 Feb. 2001 ; U.S. App. No. 60/241625, filed 19 Oct. 2000; U.S. App. No. 60/239224, filed 10 Oct. 2000; U.S. App. No. 09/619938, filed 20 Jul. 2000; U.S. App. No. 60/197734, filed 18 Apr. 2000; U.S. App. No. 60/198501, filed 18 Apr. 2000; U.S. App. No. 09/539698, filed 31 Mar. 2000; Int. App. No. PCT/US98/10330, filed 19 May 1998 and published 3 Dec. 1998 as WO 98/54345; Int. App. No. PCT/US98/03473, filed 23 Feb. 1998 and published 11 Sep. 1998 as WO 98/39411; Int. App. No. PCT/US98/01301, filed 23 January 1998 and published 13 Aug. 1998 as WO 98/35028; Int. App. No. PCT/US97/23685, filed 19 Dec. 1997 and published 30 Jul. 1998 as WO 98/32860; Int. App. No. PCT US97/10218, filed 30 May 1997 and published 4 Dec. 1997 as WO 97/45550; U.S. App. No. 08/866403, filed 30 May 1997; U.S. App. No. 08/810039, filed 4 Mar. 1997; U.S. App. No. 08/797160, filed 10 Feb. 1997; U.S. App. No. 08/791218, filed 31 Jan. 1997; U.S. App. No. 08/789886, filed 28 Jan. 1997; U.S. App. No. 08/791021, filed 27 Jan. 1997; and U.S. App. No. 08/658961 , filed 31 May 1996.

All patents, published and unpublished patent applications as well as any other scientific, technical and general writings referred to anywhere herein are hereby incorporated by reference to the extent that they are not contradictory.

BRIEF SUMMARY OF THE INVENTION

The invention disclosed herein comprises novel materials and methods useful in the in situ generation of viral vectors.

The invention disclosed herein further comprises novel materials and methods useful in the production of biological compositions, specifically vectors and/or vector systems, more specifically adenoviral vectors and/or adenoviral vector systems, which are in turn useful in the in situ generation of viral vectos.

Large scale production techniques for the commercial scale manufacturing of minimal adenoviral vectors {e.g. , so-called "gutted" adenoviral vectors) are disclosed and described herein. The requirement for scaled down testing has resulted in new and novel techniques applicable to bench scale vector purification and removal of helper virus. These bench scale techniques can be performed in a fraction of the time needed with conventional techniques, and require only standard laboratory equipment. Briefly, virus is liberated from cells using a ten minute treatment with polysorbate and tri-butyl phosphate. Virus recovery is excellent and relative volumes are low with virus concentrations exceeding 10 vp/ml (virus particles/milliliter). Also, in contrast to traditional freeze/thaw methods, as well as other chemical lysis techniques, treatment with polysorbate and tri-butyl phosphate yields non-viscous lysates that are relatively low in cell debris. Lysis is followed by step density gradient ultracentrifugation to isolate crude virus. Finally, purification and helper removal is accomplished by twice using a novel ultracentrifugation technique dubbed "dynamic banding".

Dynamic banding resembles a traditional isopycnic separation in that it employs a self-forming CsCl gradient and the species of interest is isolated according to its buoyant density. However, a traditional isopycnic separation is limited by the rate of formation of an equilibrium density gradient. Dynamic banding occurs prior to reaching stable equilibrium and therefore requires substantially less time. The technique requires that conditions for a conventional self forming CsCl density gradient are known. Standard centrifugation equations are applied to the Cs ion rather than to the species of interest that can be used to predict effective separations at unexpectedly high relative centrifugal forces.

The power of this technique is illustrated herein with the separation of a "gutted" adenovirus from its helper virus. Using conventional CsCl techniques, the two viruses resolve into distinct bands in approximately 20 hours. Identical resolution can be accomplished using dynamic banding in 135 minutes, and calculations suggest that substantially faster separations can be accomplished. The novel use of polysorbate and tri-butyl phosphate for cell lysis in combination with dynamic banding was compared to freeze/thaw lysis in combination with conventional CsCl banding and shown to be equivalent. Two Cell Factories (Nunclon^® Δ; Nalge/Nunc) were seeded with A549E1 cells and subsequently coinfected with a gutted adenovirus containing the human factor VIII gene, and its helper virus. Once the cultures exhibited a cytopathic effect, virus was purified according to one or the other of the above protocols. Yields and purities were equivalent, namely 3.7 x 10¹² gutted adenovirus particles per factory with a vector purity of greater than 99%. The different methods also yielded vectors with indistinguishable biological activity. Finally, day to day and operator to operator variability is low, making these new techniques useful in research as well as in large scale GMP settings.

DETAILED DESCRIPTION OF THE INVENTION The following examples illustrate the nature of the invention disclosed herein and are not to be construed as limiting the invention in any way. They are provided merely as illustrative examples of the disclosed invention.

EXAMPLE 1

In situ Generation of Viral Vectors

Two or more genomes (DNA or RNA) are delivered to cells in such a manner as to assure that transfected/transduced cells receive at least one copy of each genome. Gene delivery can be accomplished using viral or non viral vectors. The genome set is comprised of at least one "payload" genome and at least one "dissemination" genome. The payload genome, carries gene(s) of interest, and contains necessary cz^'s-acting elements for its own replication and packaging into viral particles. The genome(s) can present as plasmid constructs, viral constructs, or a combination of the two.

The "dissemination" genome(s) contains necessary trα s-acting elements for viral replication and packaging, but can lack cώ-acting elements necessary for packaging. Dissemination genomes can also be deficient or lacking in cts-acting elements necessary for their own replication. Dissemination genomes can also be of an inappropriate length for packaging.

In other embodiments, the payload and the dissemination genomes can be complementary for vector propagation similar to the Dual-Ad systems described herein and in related applications. The plasmid(s), the virus(es), or both can be liposome encapsulated. As plasmids are much smaller than viruses, they can have more favorable pharmacokinetic properties attributable to their decreased size, such as increased permeability across endothelium to increase bioavailablity. In addition, methods to target liposomes to specific tissues are well known in the art and these may be utilized to target these preparations to specific cell types. Liposome encapsulation of a population of these complementary plasmids would then result in the expression of both complementary genomes within a target cell and subsequent generation of the complementary viruses they encode with further propagation of the viruses and expression of the therapeutic genes they encode. Similar application of liposome encapsulation of a single class of plasmids encoding replication competent genomes and therapeutic genes would have similar beneficial properties of delivery and replication and gene delivery in their substituted regions. These single genome replication competent constructs can have a smaller gene delivery capacity compared to the multiple genome complementary plasmids where large regions of the genomes deleted for genes necessary for replication from one plasmid may be substituted with therapeutic genes as the genes needed for replication are provided by the complementary plasmids.

Following initial transfection with the above genome set, host cells function as miniature production facilities producing numerous secondary non-replicating or replicating viral vectors depending upon their composition. These secondary particles, in-turn, deliver the payload genome to numerous additional surrounding target cells.

The genomes for the initial transfection can be loaded onto gold or titanium particles, each particle containing copies of the complementary set of genomes. A "gene gun" or similar propulsion device can be used for delivery to the appropriate organism. Other modes of administration including, but not limited to, intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intracavitary, oral, rectal, mucosal, and intraperitoneal, that are useful for the delivery of vectors will have utility depending upon the desired clinical effect by one skilled in the art. These different modes of administration can also be useful for the other vector systems described in the other examples in this application.

A second approach would be to load liposomes with the genomes such that individual liposomes contained a copy of each genome. A third approach would be to have the different genomes contained within a single sequence that is cut into individual genomes after the initial transfection. A fourth approach would be to have the different genomes contained within a single DNA sequence that is transcribed into individual RNA genomes after the initial transfection. This last example would apply where the secondary vector is an RNA virus such as a retrovirus.

Dissemination/payload systems described above can be delivered using a variety of different techniques ranging from intramuscular injections of condensed DNA to "gene gun"-type systems. Other modes of administration including intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intracavitary, oral, rectal, mucosal, intraperitoneal that are useful for the delivery of vectors will have utility depending upon the desired clinical effect by one skilled in the art. Additionally, dissemination/payload systems can be contained within the genome of a different viral vector. For example, all the constituents required for a triple transfection of the type used in retrovirus production could be contained within a single gutted adenovirus. Helper-dependent adenovirus or parvoviruses systems could be contained within a herpes virus.

Dissemination/payload systems could also be nested within one another resulting in tertiary vectors and greater amplification.

Gene products encoded by the payload genome can be expressed or suppressed during the initial transfection/amplification.

This approach would offer several advantages over more conventional gene transfer technologies. These advantages include, but are not limited to:

Potency: With naked DNA (or even replication defective virus) one initial transfection event results in one cell expressing the desired transgene(s). The invention described herein is different in that each initial transfection event is amplified through a secondary infection of gutted or replication-defective viral vectors. Because the secondary vector is generated in situ, one initial transfection/transduction leads to numerous secondary transduced cells expressing the desired transgene(s).

Safety: In some clinical applications with replication competent viruses, it is a potential concern that systemically administered or sustained virus replication could result in undesirable effects particularly if the delivered genes have toxic effects. With some embodiments of this new technology, sustained virus replication is not possible because at no point would a replication competent virion be generated. In other embodiments, the virions are replication competent and can propagate and repeatedly disseminate the delivered genes for amplified expression and efficacy. The ability to generate these encapsulated constructs with either of these attributes has great versatility in enhancing efficacy and reducing toxicity. This versatility can be adapted by one skilled in the art to alter the biodistribution of the delivered genomes from virus characteristics to those of liposomal preparations yet to have the subsequent benefits of virus gene expression and virus gene delivery at the sites of the liposome delivered plasmids.

Tropism: The secondary viral vector can be designed to have a tropism for cell types different from those initially transfected transduced. This could have a number of advantages.

For vaccines, serial infection of two different cell types might have beneficial effects in raising protective immunity. It is known that exposure to pathogens/antigens results in different antibody and cell based immune responses depending on which body tissue is exposed.

Production Cost: Gutted (or replication defective) viruses are difficult and expensive to produce at the scale needed to treat or vaccinate against human disease. Manufacturing generally requires large-scale mammalian cell culture and virus culture. These processes tend to be expensive, inefficient, and non-robust.

In contrast the production of plasmid DNA does not require mammalian cell culture or virus culture. Manufacturing using an essentially virus-free/cell-free (bacteria), system would be inexpensive, efficient, and robust.

Where the primary vector is a virus rather than DNA, increased potency should still lead to a reduction in cost per patient dose.

Process Development Time And Cost (for non viral vectors): Process development for DNA is less complicated, less time consuming, and less expensive then for viruses. This is because production of DNA does not require mammalian cell culture and virus culture. Moreover, where process development for virus production is often product specific, process development for DNA production is generally applicable for multiple products.

Vector Development Time (for non viral vectors): Although vector development would not likely be less complicated, it would likely be faster and less tedious than viral vector development. This is because there would be a reduced need for cell and virus cultures as projects reach preclinical, in-vivo studies. Materials and methods adaptable to the practice of in situ generation of viral vectors as well as the various preparation(s) for practicing in situ generation of viral vectors are described herein. Variations of the materials and methods described herein are also known to persons skilled in the art.

EXAMPLE 2 Minimal Adenoviral Vectors (e.g., "Gutted" Adenoviral Vectors) and/or Complementary Adenoviral Vector Systems (e.g., Dual-Ad) Comprising Heterologous Genetic Sequences

Using the vectors and vector systems disclosed and described herein as well as in any of the related applications {see related applications supra), one can generate vectors and/or vectors systems comprising heterologous genetic sequences encoding antigenic determinants of a variety of disease-causing organisms and/or compounds {e.g., including, but not limited to, amyloid protein(s), prion protein(s), and the like), which can include, but are not limited to, bacteria, archebacteria, mycobacteria; mycoplasma, viruses, prions, fungi, chlamydia, cancer cells, and the like. Such vectors and vector systems can be used in therapeutic and/or prophylactic pharmaceutical compositions for vaccines and/or other medical treatments.

Non-limiting examples of such heterologous genetic sequences can be found throughout the relevant literature known to persons skilled in the art. Organisms from which these heterologous genetic sequences can be readily obtained include, but are not limited to:

hepatitis C virus (see e g , U S Patent App No 09/296,441, International Patent App

No PCT US97/05096, International Patent App No PCT/US00/25987, International Patent App No PCT/US00/ 15446, U S Patent App No 09/980,559, International Patent App No PCT/US00/15527, U S Patent No 6,153,421 , U S Patent App No 09/662,454, International

Patent App No PCT/US00/15293, U S Patent No 5,980,899, International Patent App No PCT/US99/18674, U S Patent App No 08/224,973, International Patent App No PCT/US95/03935, Seong, Y R et al (2001) "Immunogenicity of the E1E2 proteins of hepatitis C virus expressed by recombinant adenoviruses " Vaccine, vol 19, pp 2955-2964, Seong, Y R et al (1998) "Characterization of the structural proteins of hepatitis C virus expressed by an adenovirus recombinant " Virus Res , vol 55, pp 177-185, references in any of the foregoing, and

rotavirus (see e g , International Patent App No PCT/US99/17036, U S Patent App No 09/743,338, references in any of the foregoing, and the like),

measles (see e g , International Patent App No PCT/US00/33293, references therein, and the like),

HIV (see e g , U S Patent App No 60/274,434, Amara, R R et al (2001) "Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA MVA vaccine " Science, vol 292, no 5514, pp 69- 74, U S Patent No 5,770,210, U S Patent No 5,849,304, U S Patent No 6,039,957, U S Patent No 6,171,596, U S Patent No 6,174,666, U S Patent No 5,972,596, U S Patent No 5,965,726, U S Patent App No 60/245,113, International Patent App No PCT/US01/45624, U S Patent App No 60/173,036, International Patent App No PCT/US01/25721, International Patent App No PCT/US99/11082, International Patent App No PCT/US98/14336, International Patent App No PCT/USOO/00372, U S Patent No 5,750,332, U S Patent No 5,976,541 , U S Patent No 5,820,865, U S Patent No 5,562,905, U S Patent App No 08/455,685, U S Patent No 5,030,449, U S Patent No 5,081,226, U S Patent App No 08/060,988, U S Patent No 5,939,074, U S Patent No 5,932,218, U S Patent App No 08/899,081, U S Patent No 6,099,847, International Patent App No PCT/US01/46919, U S Patent No 5,674,720, U S Patent App No 60/339,751 , International Patent App No PCT/US00/06946, U S Patent App No 09/936,702, International Patent App No PCT/US99/24548, U S Patent App No 09/835,124, U S Patent No 5,206,353, U S Patent No 5,428,143, U S Patent No 5,587,455, U S Patent No 5,183,949, U S Patent No 5,529,765, U S Patent No 5,523,232, U S Patent App No 09/230,195, International Patent App No PCT/US97/ 12637, U S Patent No 5,637,488, U S Patent No 5,252,477, U S Patent No 5,354,683, International Patent App No PCT/US97/17704, International Patent App No PCT/US00/17327, U S Patent No 5,990,088, U S Patent No 5,847,096, U S Patent No 5,459,056, U S Patent No 4,981,790, U S Patent No 5,607,831 , International Patent App No PCT/US00/09355, U S Patent No 5,484,889, U S Patent App No 09/481,209, U S Patent No 5,462,873, U S Patent No 5,714,313, International Patent App No PCT/US97/08936, U S Patent No 4,925,800, U S Patent No 5,246,833, International Patent App No PCT/US00/29655, International Patent App No PCT/US98/00887, International Patent App No PCT/US96/18993, International Patent App No PCT/US98/25492, U S Patent App No 09/555,663, U S Patent No 5,665,345, U S Patent App No 09/177,437, International Patent App No PCT/US97/00956, International Patent App No PCT US97/09586, International Patent App No PCT/US98/06517, U S Patent No 5,374,506, U S Patent No 5,652,133, International Patent App No PCT/US98/15730, International Patent App No PCT/US00/00417, International Patent App No PCT/US98/16523, International Patent App No PCT/US00/23505, references in any of the foregoing, and the

pertussis (see e g , International Patent App No PCT/US01/20356, references therein, and the like),

human papillomavirus (see e g , U S Patent No 6,084,090, U S Patent No 6,277,980, U S Patent No 5,437,951 , U S Patent No 5,985,610, U S Patent No 5,871,998, U S Patent No 5,716,620, U S Patent No 5,744, 142, U S Patent No 5,756,284, U S Patent No 5,709,996, U S Patent No 5,855,891, U S Patent No 5,618,536, U S Patent App No 08/484,503, U S Patent App No 09/316,487, U S Patent App No 08/482,434, U S Patent App No 08/482,433, U S Patent App No 08/482,432, U S Patent App No 08/485,026, U S Patent App No 09/170,129, U S Patent App No 07/941,371 , U S Patent App No 08/481 ,055, 08/484,181, U S Patent App No 08/476,078, International Patent App No PCT/US97/121 15, references in any of the foregoing, and

borrelia burgdorferi (see eg , U S Patent App No 60/240,575, International Patent App No PCT/US01/142472, International Patent App No PCT/US00/26746, references in any of the foregoing, and the hke), hepatitis A virus (see e g , U S Patent No 4,532,215, U S Patent No 4,620,978, U S Patent No 4,636,469, U S Patent No 4,894,228, U S Patent No 5,478,746, U S Patent No 5,837,260, U S Patent No 5,476,658, International Patent App No PCT/US00/21429, U S Patent App No 60/339,109, references in any of the foregoing, and the like),

hepatitis E virus (see e g , U S Patent App No 08/316,765, U S Patent App No 08/471,971, U S Patent App No 08/809,523, U S Patent App No 08/470,246, U S Patent No 6,054,567, International Patent App No PCT/US98/14665, International Patent App No PCT/US00/32614, U S Patent App No 60/289,220, U S Patent App No 60/350,122, references in any of the foregoing, and the like),

flavivirus (see e g , U S Patent No 6,184,024, International Patent App No PCT/US99/02598, U S Patent App No 60/274,684, U S Patent App No 60/293,049, references in any of the foregoing, and the like),

mycobacteria (see e g , International Patent App No PCT/US98/ 14227, references therein, and the like),

encephalitis virus (see e g , International Patent App No PCT/US98/21308, U S Patent App No 60/181,490, International Patent App No PCT/US01/04460, references in any of the foregoing, and the like),

RSV (see e g , International Patent App No PCT/US96/15524, U S Patent App No 08/162,102, U S Patent No 5,762,905, U S Patent App No 08/920,100, U S Patent App No 09/446,959, references in any of the foregoing, and the like),

prion(s) (see e g , U S Patent App No 09/762,725, U S Patent No 6,211,149, U S Patent No 6,355,610, references in any of the foregoing, and the

chlamydia (see e g , U S Patent No 5,869,608, U S Patent App No 09/247, 137, references in any of the foregoing, and the like),

plasmodium (see e g , U S Patent No 5,130,416, U S Patent No 5,296,382, U S Patent No 5,476,785, U S Patent No 4,886,782, U S Patent No 5,028,425, U S Patent No 5,217,898, U S Patent No 5,853,739, U S Patent No 5,527,700, U S Patent No 5,753,238, U S Patent No 5,733,773, references in any of the foregoing, and the like),

ebola virus (see e , U S Patent App No 09/913,909, references therein, and the

neisseπa (see e g , U S Patent App No 09/626,003, International Patent App No PCT/US92/01796, references in any of the foregoing, and the like),

vibrio (see e g , U S Patent App No 09/653,433, references therein, and the

salmonella (see e g , U S Patent No 5,738,855, International Patent App No PCT/US98/25746, references in any of the foregoing, and the like),

E coh (see e g , International Patent App No PCT/US98/14976, U S Patent App No 09/744,289, references in any of the foregoing, and the like),

staphylococcus (see e g , U S Patent App No 60/328,017, references therein, and the

leishmania (see e g , U S Patent App No 60/299,391 , references therein, and the

shigella (see e g , International Patent App No PCT/US00/31792, references therein, and the like),

filovirus (see e , U S Patent App No 60/326,476, references therein, and the

Marburg virus (see e g , European Patent, Application No 97101506 0, U S Patent No 6,517,842, International Patent No WO 01/41784, European Patent, Application No 1092031 , International Patent No WO 00/00616 International Patent No WO 94/02125, U S Provisional Patent Application No 60/326,476, filed October, 1 , 2001 , references in any of the foregoing, and the like) EXAMPLE 3

Complementary Adenoviral Vector Systems Comprising

E 3 -substituted Adenovectors

It is known in the art that E3 is not required for adenoviral replication. Therefore, adenoviral vectors in which the E3 region has been deleted and heterologous genetic sequences inserted therefore can be used as complementary adenoviral vectors {i.e., adenovectors) in complementary adenoviral vector systems (e.g., Dual-Ad, and the like; see Cross Reference to Related Applications supra). Any E3 -substituted adenovector would compliment (i.e., provide helper virus functionality) to a second adenovector in which non-E3 regions have been deleted and/or substituted with heterologous genetic sequences {e.g., including, but not limited to, El -deleted substituted, E1+E2+E4- deleted/substituted, Max- Ad, MAXIMUM- Ad^®, mini- Ad, early generation non-E3- deleted/substituted adenoviral vectors, and the like). The heterologous genetic sequences inserted into these adenovectors in place of the deleted adenoviral genetic sequence can express any useful disease antigen and/or cancer antigen. These complementary adenovectors systems would be widely useful for vaccine and/or immunotherapy applications. A person skilled in the art would be able to tailor the inserted heterologous genetic sequence to treat the particular disease organism/agent and/or cancerous cell.

EXAMPLE 4

Vaccination and/or Immunotherapy and Gene Therapy Regimens Comprising

Liposome-Encapsulated and Non-Liposome-Encapsulated Adenovectors

Liposome encapsulation of adenovectors shields the vector(s) from neutralization by circulating anti -adenoviral antibodies {supra) and ensures target delivery and expression of the antigen(s) or therapeutic genes encoded by the heterologous genetic sequence(s) inserted into the adenovectors(s). On the other hand, it is well known in the art that introduction of non-encapsulated adenovectors into appropriate host organisms {e.g. , humans, primates, mammals, vertebrates, and the like) induces an immune response to the viral coat proteins. This immune response comprises, in part, the production of pro-inflammatory cytokines that can enhance the immune response. Such enhancement of the immune response leads to an augmentation of the immune response against the antigen(s) encoded by the heterologous genetic sequence(s) inserted into the adenovectors(s).

One or more vaccine and/or immunotherapy composition(s) comprising both liposome encapsulated adenovector(s) and non-liposome-encapsulated adenovector(s) would capture the advantages of both liposome encapsulated adenovector(s) and non- liposome-encapsulated adenovector(s). These compositions may be simultaneously or subsequently administered to the appropriate host organism depending on the result sought to be achieved by the administration and the judgment of the person skilled in the art overseeing the administration. The precise parameters of such administration regimens would be readily determinable by a person skilled in the art. These preparations can be administered as part of the prime boost strategies with adenoviral vectors and other genetic vaccines.

EXAMPLE 5

Priming and Boosting with Minimal Vector Systems and/or Complementary Vector Systems

Disclosed herein is the use of one or more complementary vector systems {e.g. , including, but not limited to, controlled vector/supplementary vector systems; Dual-Ad; and the like) with their ability to amplify target antigen expression through viral replication for priming immunizations, followed by the utilization of one or more minimal vector systems {i.e., those vectors that retain a minimal amount of native vector structure thereby allowing the incorporation of a maximum amount of heterologous material {e.g., including, but not limited to, mini-Ad; Max -Ad; MAXIMUM-AD^®; helper dependent adenoviral vectors; gutted adenoviral vectors; and the like)) that do not contain vector genes {e.g., including, but not limited to, adenoviral genes) for booster immunizations. Such an approach should focus the boosting response on the target antigen rather than vector antigens {e.g., including, but not limited to, adenoviral antigens).

Also, contemplated is an immunization strategy that primes with a minimal vector system (e.g. , including, but not limited to, MAXIMUM-AD^®, Max-Ad, mini-Ad, and the like) and boosts with a complementary vector system {e.g., including, but not limited to, Dual-Ad).

As mentioned above, it is known in the art that priming immunizations and boosting immunizations comprised of different antigen preparations may result in a more robust immune response than priming and boosting with the same antigen preparation. A reason for this may be that since only the target antigen(s) are shared between the different vaccine preparations, booster responses to the target antigen(s) are thereby facilitated rather than responses against other components of the vaccine preparations.

Also disclosed herein is the use of other types of vaccination compositions, formulations, and agents as either priming immunizations or boosting immunizations in conjunction with the Max-Ad and Dual-Ad strategies described above {e.g., including, but not limited to, naked nucleic acid; liposomal formulations of vectors; pegylated nucleic acid and formulations of vectors; RNA vaccines; other viral vector vaccines such as vaccinia, lentiviral, partially deleted adenoviral (E3 deleted, El deleted, El and E3 deleted, etc.), pox viruses, alpha viruses, retroviruses, herpes viruses, adeno-associated viruses; protein vaccines; attenuated viral or bacterial vaccines; and the like).

One of skill in the art, with reference to general references, basic and applied (see generally Molecular Cloning: A Laboratory Manual (Sambrook, J. et al., Eds. (1989) Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY); Gene Expression Technology (Methods in Enzymology, Vol. 185, Goeddel, D., Ed. (1991) Academic Press: San Diego, CA); PCR Protocols: A Guide to Methods and Applications (Innis, et al, Eds., (1990) Academic Press: San Diego, CA); Culture of Animal Cells: A Manual of Basic Techniques (R.I. Freshney (1987), 2nd edition, Liss, Inc.: New York, NY); Antibodies: A Laboratory Manual (Harlow & Lane (1988) Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY); Guide to Protein Purification. Methods in Enzymology, vol. 182 (M.P. Deutscher, Ed. Academic Press: San Diego, CA); Kuby Immunology (Goldsby, R.A. et al. (2000) 4^th ed., W.H. Freeman & Co.); Current Protocols in Immunology (Coligan, J. et al. (1999) John Wiley and Sons); Immunobiology (Janeway, C. et al. (2001) 5^th ed., Garland Publishing); Cellular and Molecular Immunology (Abbas, A.K. et al. (2000) 4^th ed., W.B. Sanders Co.); Roitt's Essential Immunology (Roitt, I. & Delves, P.J. (2001) 10^th ed., Blackwell Science Inc.); Vaccines (Plotkin, S.A. et al. (1999) 3^rd ed., W.B. Sanders Co.); The Cytokine Handbook (Thomson, A. (1998) 3^rd ed., Academic Press); references in any of the foregoing and the like. Other suitable references beyond those listed here would also be known to the skilled artisan and are readily available.) will appreciate the possible array of formulations encompassed by this invention.

EXAMPLE 6 Specific Dual Ad Constructs for Vaccines and Cancer Applications: Universal El and IL-3 Expressing

In this approach, a complementary vector system {e.g., including Dual-Ad, and the like) is constructed wherein the first vector (e.g., including, but not limited to, a controlled ad (e.g. Max-Ad and the like)) contains an El gene under the control of a universal promoter that will work in all cell types {e.g., CMV promoter, RSV promoter, and the like) and an immunostimulatory gene {e.g., IL-3, others mentioned herein and the like). Other therapeutic genes may also be included.

Compositions, formulations and vectors useful for cancer and vaccine immunotherapy based upon this general universal promoter/El and universal promoter/IL-3 construct are developed. For cancer and vaccine applications, the second virus may be an early generation El or E3-substituted viral vector, wherein one or more therapeutic genes substituted into the El or E3 region has cancer therapeutic and/or vaccine activity. Other earlier generation adenoviruses may also be utilized as the second vector provided that the substituted adenoviral genes do not encode genes that are necessary for replication that are not incorporated into a controlled virus (i.e., virus #1). The invention disclosed herein also encompasses the concept that the genes necessary for viral replication that are not found in a controlled virus {i.e., virus#l) may be incorporated into the supplemental virus {i.e., virus #2) and vice versa.

For cancer applications, the gene substituted into the El or E3 region may encode a protein with tumor suppressor activity, anti-angiogenic activity, prodrug-converting (i.e., "suicide") activity (e.g., herpes simplex virus thymidine kinase (HSV-TK) and the like) as well as any of the other possibilities disclosed in related applications {see Cross Reference to Related Applications, supra). For vaccine applications, the second virus may contain antigens/epitopes to generate immune responses to viral, fungal, bacterial, prions, amyloid proteins, tumors, receptors, mycoplasma, chalmydia. as well as any of the other possibilities disclosed in related applications (see Cross Reference to Related Applications, supra).

EXAMPLE 7

Complementary Vector (e.g., Dual- Ad and the like) Systems

Incorporating Replication Competent Components

These systems are principally designed for cancer applications although other indications may also be appropriate. In these applications, one of the vectors (e.g., adenovirus vectors) is independently replication-competent and does not require adenoviral gene expression from a second vector to replicate. The second vector would require certain products from the genes expressed by the first vector for replication. The second vector will have a very large capacity for delivering therapeutic DNA {e.g., including, but not limited to, a minimal adenovirus vector (MAXIMUM-AD^® and the like) that is devoid of any vector genes. The first vector may be a wild type adenovirus, an E3 deleted adenovirus or a replication-conditional adenovirus. Examples of replication-conditional viruses include, but are not limited to, those with tissue-specific promoters, enhancers that regulate El expression {e.g., PSA enhancer for selective replication in prostate tumors and the like; see Cross Reference to Related Applications supra). For example, E lb-deleted adenoviruses that are replication-competent only in p53-mutated cells may be utilized as virus #1 providing for selective replication of a Dual-Ad in any p53-mutated malignancy.

EXAMPLE 8

Complementary Vector (e.g., Dual Ad and the like) Systems that are

Conditionally Replication-Competent in p53-Mutated Cancers

In this system, an Ela gene of an adenovirus, rather than the complete El (Ela and Elb), is incorporated into vector #1 under the control of a universal promoter. This would leave considerable room for incorporating other therapeutic DNA into that vector. In a second vector, a typical early generation virus deleted or substituted in El would complement the first vector (Note: in this and in all of the Dual-Ad systems, therapeutic DNA may be incorporated into both vectors). However, the absence of an Elb gene would result in selective replication only in p53-mutated tumors. The Elb gene normally inactivates p53, which has an inhibitory effect on adenoviral replication. In normal cells, which have wild-type p53, adenoviral replication would be inhibited by p53, while in p53-mutated cells {e.g., certain malignancies/tumors and the like), replication of the vector would occur.

EXAMPLE 9 PEGylation of Vectors (e.g., Adenovirus Vectors and the like) to Improve Efficacy of Immunotherapy and Gene Therapy

PEGylation of vectors has been reported to improve the efficacy of early- generation vectors when administered systemically and locally (see generally Alemany, R. et al. (2000) "Blood clearance rates of adenovirus type 5 in mice." J. Gen Virol, vol. 81, pp. 2605-2609; Tsutsumi, Y. et al. (2000) "Site-specific chemical modification with polyethylene glycol [PEG] of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity." Proc. Natl. Acad. Sci. USA, vol. 97, no. 15, pp. 8548-8553; O'Riordan, C.R. et al. (1999) "PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo." Hum. Gene Ther., vol. 10, pp. 1349-1358; references in any of the foregoing; and the like). PEGylation of such vectors presumably inhibits inactivation by neutralizing antibodies.

Disclosed herein is the incorporation of PEGylation of minimal adenoviral vectors {e.g., MAXIMUM- AD^® and the like) and complementary vector systems (e.g., Dual-Ad and the like) into each of the applications disclosed above, as well as into all gene therapy, oncolytic and immunotherapy applications disclosed in any related patent application (see Cross Reference to Related Applications, supra). Combining PEGylation with the use of minimal adenoviral vectors (e.g., MAXIMUM-AD^® and the like) and/or complementary vector systems (e.g., Dual-Ad and the like) is novel and has not been previously described to the best of our knowledge.

In addition, PEGylated minimal adenoviral vectors (e.g., MAXIMUM- AD^® and the like) would represent an extremely, immunologically inert adenovirus - PEGylation would suppress the effects of humoral antibody responses, while the deletion of the adenoviral genes in the minimal adenoviral vectors would prevent cell-mediated immunity. This would permit the delivery of one or more transgenes without the negative effects of immune responses against vector components. To the best of our knowledge, PEGylation of adenoviruses has not as yet been employed in conjunction with adenoviral vaccines. This would be useful broadly for all vaccine preparations based on viral vectors, including, but not limited to, early generation adenovirus vectors, minimal adenoviral vectors {e.g., MAXIMUM-AD^® and the like), complementary vector systems (e.g., Dual-Ad and the like), vaccinia/pox virus vectors, herpes virus vectors, retro/lentivirus vectors, alpha virus vectors, and the like. PEGylation would permit repeated booster immunizations without the deleterious effects of antibody responses to viral envelope and coat proteins. In addition, the present inventors are unaware of any descriptions of minimal adenoviral vectors (e.g., MAXIMUM-Ad^® and the like), complementary vector systems (e.g., Dual-Ad and the like), vaccinia/pox virus vectors, heφes virus vectors, retro/lentivirus vectors, alpha virus vectors, and the like that have been utilized with PEGylation. These novel constructs can be utilized to enhance gene therapy applications for the treatment of genetic diseases (including, but not limited to, hemophilia with MAXIMUM-Ad^® vectors to deliver the gene for Factor VIII) by reducing the initial inflammatory responses associated with the administration of these agents and by permitting successful administration to individuals with neutralizing antibody titers to the vector.

EXAMPLE 10 Chemical Composition for a Cell Ly sing Agent

Often in the production of certain biological compositions, the species of interest is retained in the cytoplasm of the cells used for production. Cell disruption may be required in order to liberate the species of interest prior to purification and further processing.

Used in combination, tri-butyl phosphate and a non-ionic surfactant such as polysorbate-80 effectively lyse (disrupt) animal cells. These agents act rapidly and in a manner that is non-destructive to most protein structures and non-enveloped viruses. When used in applications involving adenovirus production, recovery is excellent

1 y and relative volumes are low, with virus concentrations exceeding 10 vp/ml (virus particles/milliliter). Unlike freeze/thaw methods, chemical lysis methods can be used in large scale applications.

In contrast to traditional freeze/thaw methods, as well as other chemical lysis techniques, treatment with polysorbate and tri-butyl phosphate yields non-viscous lysates that are relatively low in cell debris and DNA. Moreover, membrane-containing pathogens such as enveloped viruses and mycoplasma are destroyed upon exposure to these chemicals.

One formulation of a lysis agent stock disclosed and described herein comprises 23% tri-butyl phosphate and 77% polysorbate-80 (v/v). This mixture is diluted 1/100 into the media of cells to be lysed. Complete cell lysis occurs within about ten minutes.

It may be desirable to premix the tri-butyl phosphate and the polysorbate prior to contacting cells to be lysed with this mixture, however, the components of the mixture may be added separately to the cells to be lysed.

Other detergents and mixtures of detergents can be used instead of, or in addition to polysorbate-80 (e.g., those detergents/surfactants described in U.S. Patent No.

6,319,664; U.S. Patent No. 6,291,420; U.S. Patent No. 6,093,559; U.S. Patent No.

5,935,587; U.S. Patent No. 5,773,266; U.S. Patent No. 5,720,948; U.S. Patent No.

5,679,355; U.S. Patent No. 5,501,725; U.S. Patent No. 4,832,865; U.S. Patent No. 4,824,605; U.S. Patent No. 4,759,877; U.S. Patent No. 3,966,398; references in any of the foregoing; and the like).

EXAMPLE 11 Dynamic Banding: A Centrifugation Method for Achieving Rapid Isopycnic Separations with Spontaneously Forming Density Gradients

An isopycnic separation can be a powerful and reliable purification tool. However, these techniques generally take from about 18 to about 36 hours to perform. Disclosed and described herein are isopycnic separation techniques that can be completed in about two hours.

Substances to be separated are formulated with a gradient material such as cesium chloride and centrifuged. The opposing forces of centrifugation and diffusion act on the gradient material to form a concentration/density gradient. Substances to be separated migrate to their isopycnic point, the position within the gradient were the density of the solution matches the buoyant density of the substance being separated.

If the hydrodynamic cross-sectional area of the substance being separated is not large relative to its mass as compared to the gradient material, the substance will reach its isopycnic point before the density gradient can reach equilibrium. This phenomenon can be exploited to affect unusually rapid isopycnic separations. As disclosed and described herein this phenomenon and associated practical techniques exploiting this phenomenon are labeled "dynamic banding."

Traditional isopycnic separations are limited by the rate of formation of an equilibrium density gradient. Dynamic banding occurs prior to reaching a stable equilibrium and therefore requires substantially less time.

The power of this technique has been demonstrated through its use in the separation of a minimal adenovirus vector comprising human FVIII genetic sequences from a helper virus. Using conventional CsCl techniques, the two viruses resolve into distinct bands in approximately 20 hours. Equivalent resolution is accomplished using dynamic banding in 135 minutes, and calculations suggest that substantially faster separations are possible.

Specifically, if conditions for an equilibrium density gradient separation are known, and if the minimum time (t) required to achieve the separation is also known, it is possible to reduce processing time by increasing the angular velocity (ω). Importantly, elevated angular velocities cause optimal separation to occur early while an equilibrium density gradient is forming. Therefore, it is critical to stop the centrifuge or reduce the angular velocity before the density gradient becomes overly steep, thereby causing the materials being separated to band too close together. The optimal separation time (t') can be approximated using known parameters from an effective equilibrium density gradient separation, (ω) and (t), and a new elevated angular velocity (ω'):

This equation does not take diffusion into account and therefore, (f) is actually over-estimated. Additionally, the optimal concentration of the gradient material will be lower than that used in the equilibrium density gradient separation. These errors are small and can be corrected empirically by those skilled in the art without undue experimentation.

Preferably, this techniques can be performed as follows: An effective CsCl density equilibrium method was established to separate an adenovirus with a 31 kb genome from an adenovirus with a 37 kb genome. The procedure called for a crude virus preparation to be adjusted to a density of 1.330 g/ml and centrifuged in a Beckman VTi50 rotor at 20,000 φm for 18 to 24 hours. Using the previously described equation, it was calculated that at 48,000 φm, an effective separation would briefly occur at a run time of approximately 3 hours. This was confirmed experimentally. Conditions were refined empirically and a new rapid separation method was established. The new procedure calls for a crude virus preparation to be adjusted to a density of 1.320 g/ml and centrifuged in a Beckman VTi50 rotor at 48,000 φm for 120 to 150 minutes.

EXAMPLE 12

Dual Column Chromatography

Most liquid phase chemical purification techniques deliver products in solutions not directly compatible with subsequent processing steps or end use applications. A chromatographic technique has been developed whereby purified macromolecules and other structures such as viruses and the like can be eluted in solutions directly compatible with subsequent processing steps or end use applications. The technique is generally applicable to ion exchange chromatography and/or affinity chromatography. It can also be applied to other chromatographic techniques such as thiophilic interaction, dye ligand binding, and the like.

With respect to ion exchange and affinity chromatography, it is often the case that solutions directly compatible with subsequent processing steps or end use applications will cause the species of interest to adsorb to the chromatographic matrix. Therefore, solutions not directly compatible with subsequent processing steps or end use applications are generally present during product elution.

Dual column chromatography entails placing a size exclusion chromatography column downstream of an ion exchange, affinity, or other chromatography column. The columns are equilibrated in a manner appropriate for a purification procedure using the first column. Impure material is loaded onto the columns, and impurities are pre-eluted, also as would be appropriate for a purification procedure using the first column only. Next, the columns are re-equilibrated with a solution directly compatible with subsequent processing steps or end use applications. Finally, a solution with chemistry appropriate for eluting the species of interest is applied to the dual column system.

As the product elutes from first column it enters the size exclusion column. Once in the size exclusion column, the species of interest travels more rapidly than the elution buffer and thereby is exchanged into the solution used to equilibrate the system one step earlier (the solution directly compatible with subsequent processing steps or end use applications).

Results similar to those obtained using the above technique could, in principle, be obtained using an ion exchange, affinity, or other chromatography column, and then "buffer exchanging" the eluted material over a separate size exclusion column. However, the above technique employs one chromatography procedure rather than two. This reduces processing time as well as the time potentially labile products are in contact with non-optimal solution chemistries. Both of these variables may improve end-product specific activity. Moreover, reducing the processing steps by a factor of two decreases costs, documentation, opportunities for human error, equipment malfunction and the like. Finally, purified product is recovered at higher concentrations and with higher yields than is generally possible using two separate chromatography procedures. This is because material eluting from the first column enters the second column as a Gaussian peak rather than as a square plug.

The two chromatography resins contemplated by this disclosure and description need not be packed into separate columns. One column can be packed with both resins, provided the size exclusion resin is in the downstream position.

Dual column chromatography has been used successfully to purify and formulate adenovirus (serotype 5) from crude cell lysates.

In a representative procedure, two 5 ml Hi-Trap Desalting Columns (Pharmacia,

#17-1408-01) were connected downstream of one 5 ml Hi-Trap Q Column, (Pharmacia,

#17-1154-01). This in effect created a dual column system comprising a 5 ml anion exchange column followed by a 10 ml size exclusion column. The column system was attached to an automated chromatography system and equilibrated with a solution comprising 36% 10X phosphate buffered saline concentrate, (PBS, Gibco, #14080); 54% water, and 10% Glycerol, (Gibco, #15514011). This and all other chromatographic steps employed a flow rate of 1.5 ml/min. 2 ml of cell lysate comprising approximately 2 x 10 virus particles was then pumped over the column followed by an additional 30 ml of the above equilibration buffer. Next, 75 ml of final formulation buffer, 90% PBS, 10% Glycerol, (v/v) was pumped through the column system. Finally, virus was eluted by pumping 45% 10X PBS, 45% water, 10% Glycerol (v/v) through the columns. Purified virus was recovered as a single peak in the final formulation buffer.

EXAMPLE 13

A Plaque Purification Technique for Cloning the Components of a Dual-Ad Vector System

One and perhaps both viral components of a DualAd vector system are dependent upon the other for propagation. Therefore, traditional plaque-generating techniques can not be used to produce genetically pure vectors. The following technique allows for the formation of plaques comprising both viral components of the DualAd system. Either one of the two components can be cloned in the process. Cells are plated onto an appropriate surface, allowed to attach and establish themselves. Next, the two viral components of a DualAd vector system are added to the culture. Importantly, the first vector is introduced at a concentration sufficient to transduce a large portion of the cells. The second vector, the one to be cloned, is introduced at a concentration intended to infect only a very small portion of the cells. Plaque purification is conducted using standard techniques well known in the art.

If the diameter of a plaque is less then the average distance between cells infected with the second vector, the plaque is likely to be clonal for that vector. The process can be repeated to purify each DualAd component, one at a time.

A low concentration of the second virus is important because many infected cells will not actually give rise to plaques. However, the virus that these cells harbor may lie dormant until it is helped to replicate by the virus present in the advancing front of a plaque. Furthermore, a high concentration of the complimenting vector is important in order to assure that plaques can get started.

This technique was successfully implemented for the purification of MaxAdIL-3, a gutted Ad vector component which expresses the Ad El gene from a PSA promoter. The purification was achieved from a heterogeneous mix of MaxAdIL-3 components which was comprised of vectors comprising rearranged genomes as well as the component which comprised an unrearranged genome. The component containing the unrearranged genome was plaque purified.

LnCAP-uPAR cells were plated onto 6-well plates and allowed to expand to near confluence. The culture was then infected with helper virus at a ratio of 10 virus particles (vp) per cell. The MaxAd component comprising a PSA-driven El gene was then diluted and added to the plate wells in the amount of 10 virus particles per well. The wells were overlayered with agarose. The agarose is covered with media and the plate was cultured until visible plaques were observed.

Isolation of clonal unrearranged MaxAd was demonstrated by restriction enzyme analyses of the plaque-purified component. EXAMPLE 14

A Method for Improving Virus Culture Systems Through the

Introduction ofTransgenes into Host Cells

This technology addresses general issues associated with the production of viral vectors. These may include poor cell growth and expansion, low burst size, slow progression to CPE, low infectivity, or incompatibility with various growth medias or conditions. A specific issue encountered by the inventors herein relates to the need to culture PSA IL-3 Dual-Ad Vectors in a PSA-expressing cell line. The only PSA-positive cell readily available is LNCaP. Unfortunately, these cells are difficult to culture and expand. Moreover, when these cells are used as host cells for culturing adenovirus, virus yields tend to be low. Both issues appear to stem from the cells' poor adhesive characteristics.

Host cells can be stably transfected with recombinant DNA coding for proteins that facilitate cell and or virus culture. This invention does not describe the introduction of viral sequences to create complementing cell lines such as A549E1 and PER-C6. Rather, it involves introducing genes that facilitate cell growth, expansion, burst size, time to CPE, infectivity, or compatibility with various growth medias or conditions.

For example, the human prostate carcinoma cell line, LNCaP was stably transfected to express human urokinase type plasminogen activator receptor (uPAR). uPAR, which can function as a vitronectin receptor, imparts an adhesive nature to LNCaP cells. The relatively adherent LNCaP-uPAR cells expand more rapidly and consistently than the non-transfected parental cells. Moreover, the culturing of PSA IL-3 Dual-Ad vectors in LNCaP-uPAR cells results in consistently high yields relative to those observed with non-transfected parental cells.

EXAMPLE 15 Altering Adenovirus Surface Chemistry to Improve

Pharmacological, Biodistribution, and/or Infectious Properties of Therapeutic Vectors

Current adenoviral vectors intended for therapeutic applications often encounter limitations associated with neutralizing antibodies, half life, tropism, and toxicity. Therapeutic adenovirus particles can be treated in such a way as to shield antigenic sites from circulating antibodies, increase circulating half life, alter tropism, or reduce toxicity. These treatments can also reduce the acute inflammatory reactions associated with viral vector systems that limit the dose of vector that can be administered without undesirable side effects. Shielding antigenic sites permits successful administration in subjects with neutralizing titers of anti- vector antibodies and allows for multiple dosing and repeated administration. Treatments can include lipid encapsulation as well as covalent attachment of polymers such as polyethylene glycols (see EXAMPLE 5 supra) and hyaluronans. Viral vectors can include minimal adenoviral vectors (e.g., mini-Ad, MAXIMUM Ad^®, and the like), complementary adenoviral vector systems (e.g., Dual Ad and the like), early generation adenoviral vectors, retroviral vectors and vector systems, and the like, as well as into all gene therapy, oncolytic and immunotherapy applications disclosed in any related patent application {see Cross Reference to Related Applications, supra). Combining liposomal encapsulation with the use of minimal adenoviral vectors (e.g., MAXIMUM-Ad^® and the like) and/or complementary vector systems (e.g., Dual-Ad and the like) is novel and has not been previously described.

These novel encapsulated constructs can be utilized to enhance gene therapy applications for the treatment of genetic diseases such as hemophilia with MAXIMUM- Ad^® vectors to deliver the gene for Factor VIII by reducing the initial inflammatory responses associated with the administration of the unencapsulated agents and by permitting successful initial and repeated administration to individuals with neutralizing antibody titers to the vector.

The scientific literature is rich with disclosures and descriptions of liposome encapsulating technologies as well as covalent coupling of polymers to biomolecules, viruses, antibodies and the like {see e.g., Yotnda, P. et al. (2002) "Bilamellar canonic liposomes protect adenovectors from preexisting humoral immune responses." Mol. Ther., vol. 5, no. 3, pp. 233-241; Fu, X. & Zhang, X. (2001) "Delivery of heφes simplex virus vectors through liposome formulation." Mol. Ther., vol. 4, no. 5, pp. 447-453; Croyle, M.A. et al. (2001) ""Stealth" adenovirus blunt cell-mediated and humoral immunity responses against the virus and allow for significant gene expression upon readministration in the lung." J. Virol, vol. 75, no. 10, pp. 4792-4801 ; Croyle, M.A. et al. (2000) "Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions." Human Gene Ther., vol. 1 1 , pp. 1713-1722; O'Riordan, C.R. et al. (1999) "PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo." Human Gene Ther., vol. 10, pp. 1349-1358; Chillon, M. et al. (1998) "Adenovirus complexed with polyethylene glycol and cationic lipid is shielded from neutralizing antibodies in vitro." Gene Ther., vol. 5, pp. 995-1002; references in any of the foregoing; and the like). The inventors herein contemplate the application of these technologies to the various vectors and vector systems disclosed and described herein as well as in any of the related applications (see Cross Reference to Related Applications supra). In addition, the inventors herein believe this concept to be novel as applied to early generation adenoviral vectors for use in vaccines.

Expanding on this concept, one can envision placing a replication competent viral genome in a cationic liposome. A host cell, once transfected with such a liposome would replicate the viral genome and produce numerous viral particles. Other non-adenoviral delivery systems are also envisioned. For example, a viral genome can be delivered to skin on gold particles delivered using a so-called "gene gun" or similar propulsion device, injected as naked, condensed DNA into muscle tissue, incoφorated into the genome of a larger viral vector such as a heφes or pox virus, and the like.

EXAMPLE 16 De Novo Recombinant Adenovirus Generation/Rescue Using A549E1 Cells and a Gutted Adenovirus Expressing El

In constructing recombinant viruses, there generally exists a step in which a new virus is "rescued" from its plasmid form. This step involves introducing the plasmid form of the virus into the cytoplasm of a host cell and allowing it to replicate and assemble into new viral particles. Recombinant ΔE1 (delta El) adenovirus is most often rescued in the human kidney-derived cell line, 293. ΔEl (delta El) adenovirus generation/rescue in 293 cells often leads to homologous recombination events between the vector and genome of the host cell, resulting in replication competent adenovirus (RCA).

The human lung carcinoma derived cell line, A549E1, tends not to facilitate RCA generation. Unfortunately, recombinant adenoviruses can not efficiently be generated and/or rescued in A549E1 cells. This is presumably a limitation associated with low El expression by the cells.

It is plausible that A549E1 cells can be used to generate and or rescue a recombinant ΔEl (delta El) adenovirus from its plasmid form provided that, in addition to transfection with the plasmid, the cells are also infected with one or more minimal adenovirus vector that constitutively expresses El .

A minimal adenovirus vector that constitutively expresses El under the operational control of an exogenous promoter such as TK or RSV can be propogated in A549E1 cells with the aid of an ancillary virus to supply other required viral genes in trans. Preferably, the ancillary virus will have a replication or packaging disadvantage with respect to the El expressing virus. For example, a helper with an attenuated packaging signal could be used (see Related Applications supra).

A linearized plasmid version of early generation adenovirus containing a transgene {i.e., β-Gal, p53, etc.) can be constructed via either the AdEasy system (Statagene) or a ligation-based method.

The linearized plasmid can then be transfected into A549E1 cells which have been or will be infected with the El -expressing minimal adenovirus vector (e.g., a "gutted" Ad). Presumably, under these conditions the new virus will emerge and propogate. The constructs described herein in Examples 1-16 contemplate the use of plasmids, combinations of plasmids, and combinations of plasmids and viruses, one skilled in the art would readily understand that combinations of viruses comprising the constructs would similarly be useful and beneficial.

The foregoing description of preferred embodiments of the invention has been presented for puφoses of illustration and description only and is not intended to be exhaustive or to limit the invention to the precise embodiments disclosed and described. The disclosure and description was selected to best explain the principles of the invention and the practical application of these principles in order to enable others skilled in the art to best utilize the invention in various embodiments and various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention not be limited by the disclosure and description herein, but defined by the claims set forth below.

Claims

CLAIMSWe claim:

1. A method for delivering two or more genomes to at least one cell in situ in such a manner so as to assure that the cell or cells receive at least one copy of each genome and the cell or cells are thereby transfected, transduced, or transfected and transduced.

2. A method according to claim 1, wherein the delivering is accomplished using at least one non-viral vector.

3. A method according to claim 2, wherein the non- viral vector is a plasmid.

4. A method according to claim 1, wherein the delivering is accomplished using at least one viral vector.

5. A method according to claim 2, wherein the at least one viral vector is an adenoviral vector.

6. A method according to claim 1, wherein one of the two or more genomes is a payload genome.

7. A method according to claim 1, wherein one of the two or more genomes is a dissemination genome.

8. A method according to claim 1, wherein at least one of the two or more genomes comprises at least one gene of interest and further comprises necessary cis- acting elements for its own replication and packaging into viral particles.

9. A method according to claim 6, wherein the payload genome comprises at least one gene of interest and further comprises necessary czs-acting elements for its own replication and packaging into viral particles.

10. A method according to claim 1, wherein at least one of the two or more genomes comprises necessary tr w^-acting elements for viral replication and packaging.

11. A method according to claim 7, wherein the dissemination genome comprises necessary .raws-acting elements for viral replication and packaging.

12. A method according to claim 10, wherein the at least one of the two or more genomes lacks cw-acting elements necessary for packaging.

13. A method according to claim 11, wherein the dissemination genome lacks cw-acting elements necessary for packaging.

14. A method according to claim 11, wherein the dissemination genome lacks cώ-acting elements necessary for its own replication.

15. A method according to claim 11, wherein the dissemination genome is of an inappropriate length for packaging.

16. A method according to claim 1, wherein the two or more genomes are complementary for vector propagation.

17. A method according to claim 1 , wherein the two or more genomes are in plasmids.

18. A method according to claim 1 , wherein the two or more genomes are encapsulated in liposomes.

19. A method according to claim 1 , wherein the transfected, transduced, or transfected and transduced cell produces secondary viral vectors.

20. A method according to claim 19, wherein the secondary viral vectors are non-replicating.

21. A method according to claim 19, wherein the secondary viral vectors are replicating.