Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1995-09-08
2001-03-27
LeGuyader, John L. (Department: 1633)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C424S093200, C435S320100, C435S455000, C514S04400A
Reexamination Certificate
active
06207457
ABSTRACT:
DESCRIPTION
1. Technical Field
The present invention relates generally to methods and compositions for nucleotide sequence delivery. More particularly, the invention relates to vector systems for use in gene delivery and which provide for targeting and integration of a selected nucleotide sequence into a recipient genome.
2. Background of the Invention
Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral based systems for gene transfer purposes have been described, such as retroviral systems which are currently the most widely used viral vector systems for this purpose. For descriptions of various retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989)
BioTechniques
7:980-990; Miller, A. D. (1990)
Human Gene Therapy
1:5-14; Scarpa et al. (1991)
Virology
180:849-852; Burns et al. (1993)
Proc. Natl. Acad. Sci. USA
90:8033-8037; and Boris-Lawrie and Temin (1993)
Cur. Opin. Genet. Develop.
3:102-109.
Retroviral-based systems offer the desirable features of being able to enter suitable host cells and integrate themselves into the host genome, thereby inserting a gene of interest into the host genome. However, retroviral vector systems suffer from several drawbacks. In particular, retroviral particles are relatively labile and hence unstable. Therefore, purification of recombinant viruses can lead to significant loss in titer. Retroviruses also have a limited host range and are unable to integrate into nonreplicating cells. Accordingly, cells which do not normally divide, such as mature neurons, or cells which replicate slowly, cannot be genetically altered using retroviral vectors unless stimulated to divide before infection. Additionally, and importantly, retroviruses are known to cause disease in certain animals, including humans, and thus pose a significant health risk when used in gene delivery methods. Finally, retrovirus vectors integrate into the host cell chromosome randomly, which may cause insertional mutagenesis by activating oncogenes or inactivating tumor suppressor genes.
A number of adenovirus based systems have also been developed for gene delivery. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro. Adenoviruses are also able to infect quiescent as well as replicating target cells. Adenovirus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses cause only low level morbidity and are not associated with human malignancies. A number of adenovirus-based gene delivery systems have been described. See, e.g., Haj-Ahmad and Graham (1986)
J. Virol.
57:267-274; Bett et al. (1993)
J. Virol.
67:5911-5921; Mittereder et al. (1994)
Human Gene Therapy
5:717-729; Seth et al. (1994)
J. Virol.
68:933-940; Barr et al. (1994)
Gene Therapy
1:51-58; Berkner, K. L. (1988)
BioTechniques
6:616-629; Rich et al. (1993)
Human Gene Therapy
4:461-476.
However, despite their advantages, adenovirus-based systems suffer from several drawbacks. Particularly, adenovirus vectors do not integrate their genetic material into the host genome and are thus only able to express proteins transiently in a host cell. Hence, as the host cells divide, the transferred gene is lost, giving rise to the need for repeated treatments when long term gene therapy is desired. Furthermore, adenovirus vectors express viral proteins that may elicit an immune response in a host, thereby decreasing the life of a transduced cell. This immunogenicity may also preclude subsequent treatments because of humoral and/or cellular immune responses by the host organism.
Adeno-associated virus (AAV) systems have also been used for gene delivery. AAV is a helper-dependent DNA parvovirus which belongs to the genus Dependovirus. AAV requires co-infection with an unrelated helper virus, either adenovirus, a herpesvirus or vaccinia, in order for a productive infection to occur. In the absence of such co-infection, AAV establishes a latent state by insertion of its genome into a host cell chromosome. AAV has a wide host range and is able to replicate in cells from any species so long as there is also a successful co-infection of such cells with a suitable helper virus. Thus, for example, human AAV will replicate in canine cells co-infected with a canine adenovirus. AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. Furthermore, integration of AAV into a host genome occurs at high frequency and is independent of cell replication. AAV particles are also relatively stable, and are known to be refractive to common physical purification techniques such as sonication and heat. For a detailed review of AAV, see Berns and Bohenzky (1987)
Advances in Virus Research
(Academic Press, Inc.) 32:243-307.
The AAV genome is composed of a linear, single-stranded DNA molecule which contains 4681 bases (Berns and Bohenzky, supra). The genome includes inverted terminal repeats (ITRs) at each end which function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for the viral proteins involved in replication and packaging of the virion. In particular, a family of at least four viral proteins are synthesized from the AAV rep region, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. (1992)
Current Topics in Microbiol. and Immunol.
158:97-129.
AAV is unique among eukaryotic viruses in that it is able to integrate site-specifically into the genome of host cells. Particularly, it is now known that the AAV integration locus (termed “AAVS1”) is human chromosome 19q13.3-qter. Samulski et al. (1991)
EMBO J.
10:3941-3950; Kotin et al. (1992)
EMBO J.
11:5071-5078. The AAVS1 region of chromosome 19 has been isolated, partially characterized and sequenced. See, Kotin et al. (1992), supra; Kotin et al. (1991)
Genomics
10:831-834; and Kotin et al. (1990)
Proc. Natl. Acad. Sci. USA
87:2211-2215. Further, AAV Rep recognition sequences have been identified on human chromosome 19 near sites of viral integration in AAVS1, and those sequences have been shown to have a repeating nucleotide motif similar to sequences within AAV ITRs that are also believed to be recognized by Rep. Weitzman et al. (1994)
Proc. Natl. Acad. Sci. USA
91:5808-5812.
Characteristics of AAV, such as the ability to integrate into a host cell genome, nonpathogenicity, and particle stability, have elicited an interest in the art to provide AAV-based vector systems for use in gene delivery. A number of recombinant AAV vectors have been described. See generally, U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Numbers WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988)
Molec. Cell. Biol.
8:3988-3996; Vincent et al. (1990)
Vaccines
90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992)
Current opinion in Biotechnology
3:533-539; and Kotin, R. M. (1994)
Human Gene Therapy
5:793-801.
Recombinant AAV (rAAV) virions are produced in a suitable host cell which has been transfected with both an AAV helper plasmid and an AAV vector. See, e.g., U.S. Pat. No. 5,436,146 to Shenk et al.; and International Publication Nos. WO 95/13392, published May 18, 1995 and WO 95/13365, published May 18, 1995. An AAV helper plasmid generally includes AAV rep and cap coding regions, but lacks AAV ITRs. Accordingly, the helper plasmid can neither rep
Natsoulis Georges
Surosky Richard T.
Avigen, Inc.
LeGuyader John L.
Nguyen Dave Trong
Robins & Associates
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