Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-11-21
2003-04-01
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S235100, C435S325000, C435S440000, C435S455000, C435S091100, C435S091400, C435S091410, C424S190100, C424S093600, C424S093210, C424S199100, C514S172000
Reexamination Certificate
active
06541245
ABSTRACT:
FIELD OF INVENTION
The present invention is directed to improved adenoviral helper vectors that facilitate the production of pseudoadenoviral vectors (PAV), wherein the helper vectors themselves cannot be packaged into viral particles efficiently. While the helper vectors of the present invention are engineered, such that during PAV manufacture they are packaging defective, they may provide replication functions and viral structural proteins in trans for PAV. The helper vectors of the present invention comprise recombinase protein recognition sequences, wherein said recognition sequences are inserted into regions of the helper vector genome to allow for separation of the replication and packaging elements of the helper vector. Action by the cognate recombinase on such recombinase protein recognition sequences create an inversion or deletion of the genome upon recombination, thereby positioning the packaging elements such that the helper genome cannot be packaged. The invention is also directed to improved cell lines for the production of PAV which facilitate PAV stock production. The improved producer cell lines are stably transfected with a novel PAV. The combination of the novel helper vectors of the present invention, the novel PAVs and the improved cell lines facilitate PAV stock production and make the invention adaptable to large scale commercial production of PAV stocks.
BACKGROUND OF INVENTION
Adenoviral vectors for use to deliver transgenes to cells for applications such as in vivo gene therapy and in vitro study and/or production of the products of transgenes, commonly are derived from adenoviruses by deletion of the early region 1 (E1) genes (Berkner, K. L.,
Curr. Top. Micro. Immunol.
158:39-66, 1992). Deletion of E1 genes renders such adenoviral vectors replication defective and significantly reduces expression of the remaining viral genes present within the vector. However, it is believed that the presence of the remaining viral genes in adenoviral vectors can be deleterious to the transfected cell for one or more of the following reasons: (1) stimulation of a cellular immune response directed against expressed viral proteins, (2) cytotoxicity of expressed viral proteins, and (3) replication of the vector genome leading to cell death.
One solution to this problem has been the creation of pseudoadenoviral vectors (PAVs), which are adenoviral vectors derived from the genome of an adenovirus that contain minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (See, U.S. Pat. No. 5,882,877 which covers pseudoadenoviral vectors (PAV) and methods for producing PAV, incorporated herein by reference). Such PAVs, which can accommodate up to about 36 kb of foreign nucleic acid, are advantageous because the carrying capacity of the vector is optimized, while the potential for host immune responses to the vector or the generation of replication-competent viruses is reduced. PAV vectors contain the 5′ inverted terminal repeat (ITR) and the 3′ ITR nucleotide sequences that contain the origin of replication, and the cis-acting nucleotide sequence required for packaging of the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements, e.g. promoters, enhancers, etc.
Adenoviral vectors, such as PAVs, have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for delivery of nucleic acids to recipient cells. Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Hurwitz, M. S.,
Adenoviruses Virology,
3rd edition, Fields et al., eds., Raven Press, New York, 1996; Hitt, M. M. et al.,
Adenovirus Vectors, The Development of Human Gene Therapy
, Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York, 1999). The viral genes are classified into early (designated E1-E4) and late (designated L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation of these events is viral DNA replication. The human adenoviruses are divided into numerous serotypes (approximately 47, numbered accordingly and classified into 6 groups: A, B, C, D, E and F), based upon properties including hemaglutination of red blood cells, oncogenicity, DNA and protein amino acid compositions and homologies, and antigenic relationships.
Recombinant adenoviral vectors have several advantages for use as gene delivery vehicles, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L.,
Curr. Top. Micro. Immunol.
158:39-66, 1992; Jolly, D.,
Cancer Gene Therapy
1:51-64, 1994).
PAVs have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for gene delivery. While adenoviral vectors can generally carry inserts of up to 8 kb in size by the deletion of regions which are dispensable for viral growth, maximal carrying capacity can be achieved with the use of adenoviral vectors containing deletions of most viral coding sequences, including PAVs. See U.S. Pat. No. 5,882,877 of Gregory et al.; Kochanek et al.,
Proc. Natl. Acad. Sci
. USA 93:5731-5736, 1996; Parks et al.,
Proc. Natl. Acad. Sci. USA
93:13565-13570, 1996; Lieber et al.,
J. Virol.
70:8944-8960, 1996; Fisher et al.,
Virology
217:11-22, 1996; U.S. Pat. No. 5,670,488; PCT Publication No. WO 96/33280, published Oct. 24, 1996; PCT Publication No. WO 96/40955, published Dec. 19, 1996; PCT Publication No. WO 97/25446, published Jul. 19, 1997; PCT Publication No. WO 95/29993, published Nov. 9, 1995; PCT Publication No. WO 97/00326, published Jan. 3, 1997; Morral et al.,
Hum. Gene Ther.
10:2709-2716, 1998.
Since PAVs are deleted for most of the adenovirus genome, production of PAVs requires the furnishing of adenovirus proteins in trans which facilitate the replication and packaging of a PAV genome into viral vector particles. Most commonly, such proteins are provided by infecting a producer cell with a helper adenovirus containing the genes encoding such proteins. However, such helper viruses are potential sources of contamination of a PAV stock during purification and can pose potential problems when administering the PAV to an individual if the contaminating helper adenovirus can replicate and be packaged into viral particles.
It is advantageous to increase the purity of a PAV stock by reducing or eliminating any production of helper vectors which can contaminate preparation. Several strategies to reduce the production of helper vectors in the preparation of a PAV stock are disclosed in U.S. Pat. No. 5,882,877, issued Mar. 16, 1999; U.S. Pat. No. 5,670,488, issued Sep. 23, 1997 and International Patent Application No. PCT/US99/03483, incorporated herein by reference. For example, the helper vector may contain mutations in the packaging sequence of its genome to prevent its packaging, an oversized adenoviral genome which cannot be packaged due to size constraints of the virion, or a packaging signal region with binding sequences that prevent access by packaging proteins to this signal which thereby prevents production of the helper virus.
Other strategies include the design of a helper virus with a packaging signal flanked by the excision target site of a recombinase, such as the Cre-Lox system (Parks et al.,
Proc. Natl. Acad. Sci. USA
93:13565-13570, 1996; Hardy et al.,
J. Virol.
71:1842-1849, 1997, incorporated herein by reference). Such helper vectors reduce the yield of wild-type levels.
Another hurdle for PAV manufacturing, aside from the problems with obtaining helper vector-free stocks, is that the production process is initiated by DNA transfections of the PAV genome and the helper genome into a suitable cell line, e.g., 293 cells. After cytopathic effects are observed in t
Berthelette Patricia
Romanczuk Helen
Wadsworth Samuel C.
Dupre Jennifer L.
Genzyme Corporation
Li Bao Qun
Salimi Ali R.
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