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
2000-05-03
2001-08-07
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S455000, C435S320100, C435S457000, C435S325000, C435S462000, C435S463000, C435S456000
Reexamination Certificate
active
06270996
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of somatic gene therapy, and specifically to methods and compositions useful in the treatment of genetic disorders.
BACKGROUND OF THE INVENTION
Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types [see, e.g., M. S. Horwitz et al, “Adenoviridae and Their Replication”,
Virology,
second edition, pp. 1712, ed. B. N. Fields et al, Raven Press Ltd., New York (1990)]. Recombinant adenovirus (rAds) are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. 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 [K. F. Kozarsky et al,
Somatic Cell Mol. Genet.,
19:449-458 (1993) (“Kozarsky et al I”); K. F. Kozarsky et al,
J. Biol. Chem.,
269:13695-13702 (1994) (“Kozarsky et al II) and others]. The use of recombinant adenoviruses in the transduction of genes into hepatocytes in vivo has previously been demonstrated in rodents and rabbits [see, e.g., Kozarsky II, cited above, and S. Ishibashi et al,
J. Clin. Invest.,
92:883-893 (1993)].
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.
Adeno-associated virus (AAV) is an integrating human DNA parvovirus which has been proposed for use as a gene delivery vehicle for somatic gene therapy [B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168 (1990)]. This small non-enveloped virus contains a 4.6 kb single stranded (ss) DNA genome that encodes sets of regulatory and capsid genes called rep and cap. Rep polypeptides (rep78, rep68, rep62 and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5′ and 3′ ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures.
Recombinant forms of AAV (rAAV) have been developed as vectors by replacing all viral open reading frames with a therapeutic minigene, while retaining the necessary cis elements contained in the ITRs. [See, e.g., U.S. Pat. Nos. 4,797,368; 5,153,414; 5,139,941; 5,252,479; and 5,354,678; and International Publication Nos. WO 91/18088 published Nov. 28, 1991; WO 93/24641 published Dec. 9, 1993 and WO94/13788 published Jun. 23, 1994]. However, progress towards establishing AAV as a transducing vehicle for gene therapy has been slow for a variety of reasons. For example, the integrated provirus preferentially targets specific sites in chromosome 19. Additionally, difficulties surround large-scale production of replication defective recombinants. The cells employed to produce rAAV must also be infected with adenovirus or herpesvirus to provide the necessary helper functions, thereby producing problems in purifying recombinant AAV (rAAV) from contaminating virus in culture. Practical experience with purified recombinant AAV as a gene therapy vector has been disappointing, because the more purified the AAV is from co-infection with its helper virus in culture, the lower the gene transduction efficiencies that the rAAV displays.
There remains a need in the art for additional recombinant adenoviruses and rAAV, therapeutic compositions and methods which enable effective use of these recombinant viruses in the treatment of disorders and diseases by gene therapy.
SUMMARY OF THE INVENTION
In one aspect of this invention, a packaging cell line is provided which expresses adenovirus genes E1a, E1b and E4, or functional fragments thereof, e.g., the E4 open reading frame (ORF) 6.
In another aspect, the invention provides a rAd comprising the DNA of at least a portion of the genome of an adenovirus having functional deletions of the E1 and E4 gene regions; a suitable gene operatively linked to regulatory sequences directing its expression, and an adenovirus capsid, the rAd capable of infecting a mammalian cell and expressing the gene product in the cell in vivo or in vitro. The invention also provides a mammalian cell infected with the rAd described above.
In still another aspect, the invention provides a rAd shuttle vector comprising the DNA of at least a portion of the genome of an adenovirus having functional deletions of the E1 and E4 gene regions.
In a further aspect, the invention provides a method for producing the above-described recombinant Ad and a method for delivering a selected gene into a mammalian cell using the recombinant Ad described above.
In another aspect, the invention provides a method for enhancing the efficiency of transduction of a recombinant AAV into a target cell. The method operates, in brief, by infecting a target cell with a ss recombinant adeno-associated virus (rAAV) which comprises a transgene operatively linked to regulatory sequences directing its expression, and contacting the infected cells with an agent which facilitates the conversion of ss rAAV to its double stranded (ds) form. Conversion of ss rAAV to ds rAAV occurs in the target cell, resulting in enhanced transduction of the rAAV into the target cell. The agent may be a helper virus which carries a selected gene or functional fragment thereof encoding a polypeptide capable of enhancing the conversion of the ss rAAV to ds rAAV and which is co-infected into the same target cell. The agent may also be a drug or chemical composition which accomplishes the same function and is applied to the infected target cell. This method can operate both in an ex vivo setting and in vivo.
In yet another aspect, the invention provides a novel recombinant AAV, which contains both the transgene intended for use in treating a genetic disease or disorder and at least one additional gene operatively linked to inducible or constitutive regulatory sequences. The additional gene(s) encodes a polypeptide capable of facilitating, alone or in concert with other additional genes, the conversion of ss rAAV to its ds form upon expression. In a preferred embodiment, the additional gene is adenovirus E4 or a functional fragment thereof. Also disclosed is a method for enhancing the efficiency of transduction of the novel rAAV into a target cell.
The novel rAAV and methods of this invention are also useful in pharmaceutical compositions for use in ex vivo and in vivo gene therapy treatment protocols for treating inherited diseases, cancer, and other genetic dysfunctions.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
REFERENCES:
patent: 4797368 (1989-01-01), Carter et al.
patent: 4920209 (1990-04-01), Davis et al.
patent: 5139841 (1992-08-01), Muzyczka et al.
patent: 5139941 (1992-08-01), Muzyczka et al.
patent: 5173414 (1992-12-01), Lebkowski et al.
patent: 5252479 (1993-10-01), Srivastava
patent: 5354678 (1994-10-01), Lebkowski et al.
patent: 5436146 (1995-07-01), Shenk et al.
patent: 5543328 (1996-08-01), McClelland et al.
patent: 5604090 (1997-02-01), Alexander et al.
patent: 5622856 (1997-04-01),
Fisher Krishna J.
Gao Guang-Ping
Wilson James M.
Guzo David
Howson and Howson
The Trustees of the University of Pennsylvania
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