Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2002-05-30
2004-11-23
Guzo, David (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S320100, C435S455000, C435S456000, C435S457000, C435S325000, C435S069100, C424S093100, C424S093600
Reexamination Certificate
active
06821512
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates generally to the field of adenoviral vectors useful in delivering genes and methods of producing same.
Adenoviruses have been described as useful viral vectors for delivery of therapeutic genes into selected host cells. Due to interest in adenoviruses as delivery vehicles, several groups have investigated the mechanisms that allow selective packaging of the adenovirus (Ad) genome into viral capsids.
Within the left end of the Ad genome, a cis-acting packaging domain has been identified [M. Grable and P. Hearing,
J. Virol
. 64:2047-2056 (May 1990)]. Mutants of Ad serotype 5 (Ad5) lacking this region are nonviable but can be rescued by insertion of the left-terminal 355 nt at the right end of the viral genome. The Ad5 packaging domain has been found to function in an inverted orientation, and can be moved within several hundred base pairs from its original location without a reduction in virus yield [P. Hearing et al,
J. Virol
., 61:2555-2558 (August 1987)].
The Ad5 packaging domain consists of at least seven elements which are functionally redundant. Four of the first five elements contain an AT-rich repeated sequence motif termed the A repeat. The fifth element does not contain any obvious primary sequence homology to the A repeat aside from the fact that it is also AT rich. With reference to the published sequences of Ad5, A repeat I is located within nt 240-248; A repeat II is located within nt 260-268; A repeat III is located within nt 302-311; A repeat IV is located within nt 313-321; A repeat V is located within nt 337-346; A repeat VI is located within nt 363 and 368 of Ad5; A repeat VII is located within nt 370-375 of Ad5 [M. Grable and P. Hearing,
J. Virol
, 66(2):723-731 (February 1992)]. The literature reports efforts to determine the minimum portion of the packaging domain required to package adenovirus DNA into virions. This approach is consistent with on-going efforts to maximize safety of the vectors, as well as to provide adequate space in the viral genome for heterologous gene sequences which are delivered by the adenoviral vectors.
The art continues to search for methods of optimizing production and yield of adenoviral vectors.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods useful for efficiently packaging recombinant adenoviruses and producing high yields thereof. The invention involves engineering recombinant adenoviruses to contain multiple functional adenoviral packaging domains. Suitably, these vectors contain at least five of the “A” repeat elements of the adenoviral packaging domains, in duplicate. Most preferably, the vectors contain at least one intact adenoviral packaging domain and a second adenoviral packaging domain containing at least five “A” repeat elements.
Thus, in one aspect, the invention provides (a) an adenovirus 5′ inverted terminal repeat, (b) a first adenovirus packaging domain, (c) a second adenovirus packaging domain; (d) a selected transgene under the control of regulatory sequences directing expression of the transgene, and (3) a 3′ inverted terminal repeat. Suitably, the second packaging domain is located 5′ to the native E1 region and the selected transgene.
In another aspect, the invention provides a pharmaceutical composition comprising a recombinant adenovirus of the invention and a physiologically compatible carrier.
In still another aspect, the invention provides a method of delivering a transgene to a selected host cell by infecting said cell with a recombinant adenovirus of the invention.
In a further aspect, the invention provides a method of increasing the packaging and yield of a selected recombinant adenovirus. The method involves engineering the selected recombinant adenovirus vector to contain at least two adenoviral packaging domains.
In yet a further aspect, the invention provides a method of producing a recombinant adenovirus which lacks functional adenoviral early, intermediate and late genes. The method involves co-culturing in a host cell (a) a recombinant adenoviral plasmid comprising multiple adenoviral packaging domains and a selected transgene, said plasmid lacking functional adenoviral early, intermediate and late genes; and (b) a helper virus. The rAd plasmids and helper virus, together with the host cell, provide sufficient adenoviral gene functions to permit packaging of the recombinant adenoviral plasmid into an adenoviral capsid.
Other aspects and advantages of the invention will be readily apparent from a review of the following detailed description of the invention.
REFERENCES:
patent: 5882877 (1999-03-01), Gregory et al.
patent: WO99/53085 (1999-10-01), None
Kmiec, American Scientist, 1999, vol. 87, pp. 240-247.*
Verma et al., Nature, 1997, vol. 389, pp. 139-242.*
Fox, Nature Biotechnology, 2000, vol. 18, pp. 143-144.*
Anderson, Nature, 1998, vol. 392, pp. 25-30.*
Mountain, TIBTECH, 2000, vol. 18, pp. 119-128.*
Ross et al., Human Gene Therapy, 1996, vol. 7, pp. 1781-1790.*
P. Hearing et al, “Identification of a Repeated Sequence Element Required for Efficient Encapsidation of the Adenovirus Type 5 Chromosome”, J. Virol., 61(8):2555-2558 (Aug., 1987).
M. Grable et al, “Adenovirus Type 5 Packaging Domain is Composed of a Repeated Element that is Functionally Redundant”, J. Virol., 64(5):2047-2056 (May, 1990).
M. Grable et al, “cis and trans Requirements for the Selective Packaging of Adenovirus Type 5 DNA”, J. Virol., 66(2):723-731 (Feb., 1992).
S. Kochanek et al, “A New Adenoviral Vector: Replacement of All Viral Coding Sequences with 28 kb of DNA Independently Expressing Both Full-Length Dystrophin and &bgr;-galactosidase”, Proc. Natl. Acad Sci. USA, 93:5731-5736 (Jun., 1996).
M. Lanuti et al, “Evaluation of an E1E1-Deleted Adenovirus Expressing the Herpes Simplex Thymidine Kinase Suicide Gene in Cancer Gene Therapy”, Human Gene Therapy, 10:463-475 (Feb., 1999).
K. Hehir et al, “Molecular Characterization of Replication-Competent Variants of Adenovirus Vectors and Genome Modifications to Prevent their Occurrence”, J. Virol., 70(12):8459-8467 (Dec., 1996).
D. Von Seggern et al, “A Helper-Independent Adenovirus Vector with E1, E3, and Fiber Deleted: Structure and Infectivity of Fiberless Particles”, J. Virol., 73(2):1601-1608 (Feb., 1999).
S. Schmid et al, “Bipartite Structure and Functional Independence of Adenovirus Type 5 Packaging Elements”, J. Virol., 71(5):3375-3384 (May, 1997).
S. Schmid et al, “Cellular Components Interact with Adenovirus Type 5 Minimal DNA Packaging Domains”, J. Virol., 72(8):6339-6347 (Aug., 1998).
V. Sandig et al, “Optimization of the Helper-Dependent Adenovirus System for Production and Potency in vivo”, Proc. Natl. Acad. Sci., 97(3):1002-1007 (Feb., 2000).
Gao Guangping
Wilson James M.
Guzo David
Howson and Howson
The Trustees of the University of Pennsylvania
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