Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-04
2003-11-18
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S091100, C435S455000, C435S465000, C435S320100, C435S235100, C435S325000
Reexamination Certificate
active
06649373
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to cis- and trans-activation methods of modulating the persistence of expression of a transgene in non-Herpes vectors, more particularly in at least E4-deficient (E4&Dgr;) adenoviral vectors, as well as transactivation systems for use in such methods.
BACKGROUND OF THE INVENTION
Gene therapy relies upon the introduction of one or more recombinant nucleic acid molecules (i.e., vectors) comprising one or more transgenes into a host (e.g., a human), wherein the presence, and in most cases the expression, of the transgene produces some desired effect (e.g., production of a therapeutic protein). Thus, for gene therapy to be effective, vectors (particularly, gene transfer vectors) that provide persistent expression of the transgene are desired.
Using vector reporter gene constructs, it has been established that high levels of vector transgene expression can be obtained in a variety of animal models. However, it also has been established that the high level of transgene expression so obtained is transient, with reporter gene expression peaking within the first week after infection and becoming essentially undetectable about 80 days after infection. Recent studies have indicated that the limited persistence of gene expression in vivo is most likely due to an immune response of the host against virally infected cells. For example, gene expression can be maintained in immunologically privileged neuronal or retinal tissues for periods in excess of two months and in immunodeficient or immunologically naïve rodents for periods in excess of six months. Transgene expression in immune-competent animals, by contrast, rapidly declines to baseline levels within 2-3 weeks of infection due to immune activation.
Using a combination of mouse strains, which are defective in specific elements of the immune system, it has been shown that the immune response against cells infected with viral vectors involves both cellular and humoral components of the immune system. For example, immunodeficient mice, which lack mature T- and B-lymphocytes, express adenovirus-mediated transgenes beyond four months (Kass-Eisler et al., Gene Therapy 1: 395-402 (1994); Yang et al., Immunity 1: 433-442 (1994a); Yang et al., PNAS USA 91: 4407-4411 (1994b); Dai et al., PNAS USA 92: 1401-1405 (1995); Kay et al., Nat. Genet. 11: 191-197 (1995); and Yang et al., J. Immunol. 155: 2564-2570 (1995)). Similarly, transfer of CD8
+
and CD4
+
cytotoxic T-cells from adenoviral vector-infected mice to infected RAG-2 mice, which lack mature B- and T-lymphocytes, results in clearance of the vector and the transgene by apoptosis (Yang et al. (1994a), supra; and Yang et al. (1995), supra), whereas immune depletion of CD8
+
or CD4
+
cells in immunocompetent mice results in persistent transgene expression (Yang et al. (1994a), supra; Kay et al., Nat. Genet. 11: 191-197 (1995); Yang et al. (1995), supra; Kolls et al., Hum. Gene Ther. 7: 489-497 (1996); and Guerette et al., Transplantation 62: 962-967 (1996)). While pathways involving perforin and Fas are the major pathways responsible for T-cell cytotoxicity (Kojima et al., Immunity 1: 357-364 (1994); Henkart, Immunity 1: 343-346 (1994); Kagi et al., Science 265: 528-530 (1994); and Kagi et al., Eur. J. Immunol. 25: 3256-3262 (1995)), the perforin/granzyme pathway has been reported to mediate clearance of adenoviral gene transfer vectors by antigen-specific, cytotoxic T-cells (Yang et al., PNAS USA 92: 7257-7261 (1995)).
In addition to limiting the persistence of gene expression from viral vectors, the immune response inhibits successful readministration of viral vectors, which limits the period of gene expression. For example, adenoviruses are classified into 47 different serotypes and a number of subgroups, namely A through G, based on a number of criteria, including antigenic cross-reactivity. Following an initial administration of adenovirus, serotype-specific antibodies are generated against epitopes of the major viral capsid proteins, namely the penton, hexon and fiber. Given that such capsid proteins are the means by which the adenovirus attaches itself to a cell and subsequently infects the cell, such antibodies (i.e., neutralizing antibodies) are then able to block or “neutralize” reinfection of a cell by the same serotype of adenovirus. This necessitates using a different serotype of adenovirus in order to administer one or more subsequent doses of exogenous DNA to continue to express a given gene, such as in the context of gene therapy.
Another approach to increasing the persistence of transgene expression in vector systems involves the introduction of substantial deletions in a viral vector so as to reduce or eliminate completely the production of viral antigens by the viral vector. In this regard, the deletion of E4 from adenoviral vectors is especially important for safe adenoviral vector design. Removal of the E4 region severely disrupts adenoviral gene expression in transduced cells. Removal of the E4 region also eliminates several viral products that interact with and antagonize cellular targets and processes. E4-ORF6 has been shown to block p53 function and to have oncogenic potential (Dobner et al., Science 272: 1470-1473 (1996); Nevels et al., PNAS USA 94: 1206-1211 (1997)). It also appears that E4-ORF1 has oncogenic potential (Javier et al., J. Virol. 65: 3192-3202 (1991); Javier et al., Science 257: 1267-1271 (1992); Javier et al., Breast Cancer Res. Treat. 39: 57-67 (1996); Javier et al., J. Virol. 68: 3917-3924 (1994); Weiss et al., J. Virol. 71: 4385-4393 (1997); Weiss et al., J. Virol. 71: 1857-1870 (1997); and Weiss et al., J. Virol. 70: 862-872 (1996)). ORF6 and ORF3 of the E4 region of adenovirus also have been shown to be involved in altering mRNA expression post-transcriptionally (Nordqvist et al., PNAS USA 87: 9543-9547 (1990); Nordqvist et al., Mol. Cell. Biol. 14: 437-445 (1994); Nordqvist et al., Mol. Biol. Rep. 14: 203-204 (1990); Ohman et al., Virology 194: 50-58 (1993); Sandler et al., J. Virol. 63: 624-630 (1989); and Sandler et al., Virology 181: 319-326 (1991)). E4 products are also involved in controlling E2F (Nevins, Virus Res. 20: 1-10 (1991)), E1A-induced p53-independent apoptosis (Marcellus et al., J. Virol. 70: 6207-6215 (1996)), the modulation of the phosphorylation status of cellular and viral proteins (Kleinberger et al. 67: 7556-7560 (1993); and Muller et al., J. Virol. 66: 5867-5878 (1992)), and the alteration of the nuclear transport of various proteins (Goodrum et al., J. Virol. 70: 6323-6335 (1996)). Elimination of the E4 region of adenovirus eliminates these negative effects. However, E4 elimination also adversely affects maintenance of transgene persistence.
Provision of E4 in trans has been proposed as a method of activating transgene expression from an E4&Dgr; adenoviral vector (Brough et al., J. Virol. 71(12): 9206-9213 (1997)). Supply of E4 products in trans has been demonstrated to allow persistent expression from the cytomegalovirus E4 promoter (Armentano et al., J. Virol. 71(3): 2408-2416 (1997)). One potential problem associated with any administration of E4 products is that a multiplicity of E4 containing vectors has been found to result in cell toxicity, particularly in endothelial cells. Co-expression of the adenoviral E2 preterminal protein from an adenoviral vector or in trans has been demonstrated to stabilize in vitro an adenoviral mini-genome, which is deficient in E1, E2 and E3 but not E4 (Lieber et al., Nature Biotech. 15: 1383-1387 (1997)). Expression of a transgene operably linked to the cytomegalovirus (CMV) immediate early promoter has been demonstrated to be dependent on the infected cell protein 0 in
Herpes simplex
vectors; based on such a showing, it was suggested that ORF3 of the E4 region of adenovirus could have the same effect on transgene expression in an adenoviral vector (Samaniego et al., J. Virol. 72(4): 3307-3320 (1998)).
Adenoviruses in many settings are advantageous as viral vectors because they are easy to use, can be pr
Brough Douglas E.
Kovesdi Imre
GenVec, Inc.
Leydig , Voit & Mayer, Ltd.
Mosher Mary E.
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