Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-10-21
2001-03-20
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091400, C435S091410, C435S091420, C435S320100, C435S455000, C435S456000, C435S457000, C435S325000
Reexamination Certificate
active
06203975
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of vectors useful in somatic gene therapy and the production thereof.
BACKGROUND OF THE INVENTION
Human gene therapy is an approach to treating human disease that is based on the modification of gene expression in cells of the patient. It has become apparent over the last decade that the single most outstanding barrier to the success of gene therapy as a strategy for treating inherited diseases, cancer, and other genetic dysfunctions is the development of useful gene transfer vehicles. Eukaryotic viruses have been employed as vehicles for somatic gene therapy. Among the viral vectors that have been cited frequently in gene therapy research are adenoviruses.
Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. Recombinant adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are currently being developed for gene therapy. Both Ad2 and Ad5 belong to a subclass of adenovirus that are not associated with human malignancies. Recombinant adenoviruses are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. High titers (10
13
plaque forming units/ml) of recombinant virus can be easily generated in 293 cells (the adeno virus equivalent to retrovirus packaging cell lines) and cryo-stored for extended periods without appreciable losses. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders [Y. Watanabe,
Atherosclerosis,
36:261-268 (1986); K. Tanzawa et al,
FEBS Letters,
118(1):81-84 (1980); J. L. Golasten et al,
New Engl. J. Med.,
309(11983):288-296 (1983); S. Ishibashi et al,
J. Clin. Invest.,
92:883-893 (1993); and S. Ishibashi et al,
J. Clin. Invest.,
93:1885-1893 (1994)]. Indeed, a recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved for use in at least two human CF clinical trials [see, e.g., J. Wilson,
Nature,
365:691-692 (Oct. 21, 1993)]. Further support of the safety of recombinant adenoviruses for gene therapy is the extensive experience of live adenovirus vaccines in human populations.
Human adenoviruses are comprised of a linear, approximately 36 kb double-stranded DNA genome, which is divided into 100 map units (m.u.), each of which is 360 bp in length. The DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication. The gene products are organized into early (E1 through E4) and late (L1 through L5) regions, based on expression before or after the initiation of viral DNA synthesis [see, e.g., Horwitz,
Virology,
2d edit., ed. B. N. Fields, Raven Press, Ltd. New York (1990)].
The first-generation recombinant, replication-deficient adenoviruses which have been developed for gene therapy contain deletions of the entire E1a and part of the E1b regions. This replication-defective virus is grown on an adenovirus-transformed, complementation human embryonic kidney cell line containing a functional adenovirus E1a gene which provides a transacting E1a protein, the 293 cell [ATCC CRL1573]. E1-deleted viruses are capable of replicating and producing infectious virus in the 293 cells, which provide E1a and E1b region gene products in trans. The resulting virus is capable of infecting many cell types and can express the introduced gene (providing it carries its own promoter), but cannot replicate in a cell that does not carry the E1 region DNA unless the cell is infected at a very high multiplicity of infection.
However, in vivo studies revealed transgene expression in these E1 deleted vectors was transient and invariably associated with the development of severe inflammation at the site of vector targeting [S. Ishibashi et al,
J. Clin. Invest.,
93:1885-1893 (1994); J. M. Wilson et al,
Proc. Natl. Acad. Sci., USA,
85:4421-4424 (1988); J. M. Wilson et al,
Clin. Bio.,
3:21-26 (1991); M. Grossman et al,
Som. Cell. and Mol. Gen.,
20 17:601-607 (1991)]. One explanation that has been proposed to explain this finding is that first generation recombinant adenoviruses, despite the deletion of E1 genes, express low levels of other viral proteins. This could be due to basal expression from the unstimulated viral promoters or transactivation by cellular factors. Expression of viral proteins leads to cellular immune responses to the genetically modified cells, resulting in their destruction and replacement with nontransgene containing cells.
There yet remains a need in the art for the development of additional adenovirus vector constructs for gene therapy.
SUMMARY OF THE INVENTION
In one aspect, the invention provides the components of a novel recombinant adenovirus production system. One component is a shuttle plasmid, pAd&Dgr;, that comprises adenovirus cis-elements necessary for replication and virion encapsidation and is deleted of all viral genes. This vector carries a selected transgene under the control of a selected promoter and other conventional vector/plasmid regulatory components. The other component is a helper adenovirus, which alone or with a packaging cell line, supplies sufficient gene sequences necessary for a productive viral infection. In a preferred embodiment, the helper virus has been altered to contain modifications to the native gene sequences which direct efficient packaging, so as to substantially disable or “cripple” the packaging function of the helper virus or its ability to replicate.
In another aspect, the present invention provides a unique recombinant adenovirus, an Ad&Dgr; virus, produced by use of the components above. This recombinant virus comprises an adenovirus capsid, adenovirils cis-elements necessary for replication and virion encapsidation, but is deleted of all viral genes (i.e., all viral open reading frames). This virus particle carries a selected transgene under the control of a selected promoter and other conventional vector regulatory components. This Ad&Dgr; recombinant virus is characterized by high titer transgene delivery to a host cell and the ability to stably integrate the transgene into the host cell chromosome. In one embodiment, the virus carries as its transgene a reporter gene. Another embodiment of the recombinant virus contains a therapeutic transgene.
In another aspect, the invention provides a method for producing the above-described recombinant Ad&Dgr; virus by co-transfecting a cell line (either a packaging cell line or a non-packaging cell line) with a shuttle vector or plasmid and a helper adenovirus as described above, wherein the transfected cell generates the Ad&Dgr; virus. The Ad&Dgr; virus is subsequently isolated and purified therefrom.
In yet a further aspect, the invention provides a method for delivering a selected gene to a host cell for expression in that cell by administering an effective amount of a recombinant Ad&Dgr; virus containing a therapeutic transgene to a patient to treat or correct a genetically associated disorder or disease.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
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patent: W
Chen Shu-Jen
Fisher Krishna J.
Weitzman Matthew
Wilson James M.
Guzo David
Howson and Howson
The Trustees of the University of Pennsylvania
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