Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1993-08-31
2004-02-03
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S235100, C435S455000, C435S456000, C435S325000, C435S366000, C435S069100
Reexamination Certificate
active
06686200
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and gene transfer and particularly concerns recombinant adeno-associated virus (AAV). The invention provides novel methods and compositions, including cell lines, recombinant AAV and adenovirus or herpes virus vectors, for use in the efficient and large-scale production of adeno-associated virus. The AAV production methods described herein do not require a transfection step. The resultant AAV may be used in a variety of embodiments including, for example, for transferring exogenous genes into human cell lines and for use in human gene therapy regimens.
2. Description of the Related Art
There are currently more than 4,000 known genetic disorders which lack fully effective therapies. In recent years the prospect of using gene therapy to treat such diseases has become to be viewed as a realistic goal. The ultimate form of gene therapy requires the integration of a wild-type gene able to correct the genetic disorder into the host genome, where it can co-exist and replicate with the host DNA. The expression of the gene should be regulated at a level that can best compensate for the defective gene. In the most ideal circumstances, the disease would be cured for life by one or a few treatments, with no serious side effects.
There have been several experimental approaches to gene therapy proposed to date, but each suffer from their particular drawbacks (Mulligan, 1993). Firstly, there are basic transfection methods in which DNA containing the gene of interest is introduced into cells non-biologically, for example, by permeabilizing the cell membrane physically or chemically. This approach is limited to cells that can be temporarily removed from the body and can tolerate the cytotoxicity of the treatment, i.e. lymphocytes. Furthermore, the efficiency of gene integration is very low, on the order of one integration event per 1,000 to 100,000 cells, and expression of transfected genes is often limited to days in proliferating cells or weeks in non proliferating cells.
The retroviral vector approach capitalizes on the natural ability of viruses to enter cells, bringing their own genetic material with them. Retroviruses have the advantage that they can integrate into host genome and thus transfer the gene of interest into the genome of the target cell. However, major problems are associated with using retroviral vectors for gene therapy, for example, they only integrate efficiently into replicating cells and they are difficult to concentrate and purify.
Several DNA viruses, such as adenovirus, have also been engineered to serve as vectors for gene transfer. But many DNA viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect. Moreover, adenoviruses do not integrate their genetic material into the host genome and, due to the resultant transient expression, repeated exposures would be necessary. Unfortunately, the limited serotypes of adenovirus available for vector development and host immunity pose limitations on repetitive administration.
Another limitation on adenovirus vectors is the fact that recipient cells generally express at least a low level of viral proteins in addition to the therapeutic gene (Gregory et al., 1992; Rosenfeld et al., 1992), attracting immune responses and causing inflammation in the recipient organ. The co-transfer of viral genes into recipient cells also opens the possibility that the defective viral genome can be rescued by a wild-type virus infection which may propagate the genetically engineered virus and gene among the normal population. Furthermore, studies have shown that recombinant adenovirus which are meant to be replication-defective can in fact replicate slowly (Shenk et al., 1980), raising general safety concerns.
The properties of Adenoassociated Virus (AAV), a single-stranded DNA parvovirus endogenous to the human population, make it one of the most suitable gene therapy vector candidates. Firstly, AAV is not associated with any disease (Ostrove et al., amp 1981; Cukor et al., 1984), therefore it is safe for gene transfer applications. Secondly, AAV virions are resistant to physical treatments, such as sonication and heat inactivation, that are not tolerated by other viruses during purification (Samulski et al., 1989). Thirdly, like retroviruses, AAV integrates into the host cell genome upon infection (Kotin et al., 1990; Samulski et al., 1991) so that transgenes can be expressed indefinitely. Furthermore, integration of AAV into the cellular genome is independent of cell replication (Lebkowski et al., 1988). This is particularly important as AAV can thus transfer genes into quiescent cells—which make up the vast majority of cells in the human body.
However, AAV technology does have certain limitations which remain to be overcome. For example, benefits may be realized by developing strategies to accommodate larger recombinant inserts. The major problem limiting the practical use of recombinant AAV is that AAV production methods are inefficient and laborious (Lebkowski et al., 1988; Samulski et al., 1989; Muzyczka, 1992). In recombinant AAV, key viral genes (such as cap, lip and rep) are replaced by the exogenous gene of interest. Methods for producing recombinant AAV therefore rely on co-transfecting the AAV vector carrying the gene of interest, together with a helper AAV plasmid that expresses all of the essential AAV genes, into adenovirus- or herpes-infected cells which supply the helper functions necessary for AAV replication and the production of new viral particles.
The use of cells infected with helper adenovirus or herpes virus does not create a problem, it is the transfection of the essential AAV genes which is the limiting step for the production of high titre AAV virus. Transfection of DNA molecules into cells is known to be very inefficient and the transfection-based methods generally used for AAV production are, therefore, particularly inefficient as they rely on co-transfection. Even the most-recent studies in this area have only reported a 10% increase in efficiency (Page et al., 1993). Unfortunately, new procedures, such as those utilizing chimeric Epstein Barr/AAV plasmids (Lebkowsi et al., U.S. Pat. No. 5,173,414) and transduced cells with AAV vectors stably incorporated into the genome (Muzyczka et al., U.S. Pat. No. 5,139,941), still require transfection of a helper plasmid that provides AAV genes for packaging. The production of a significant quantity of AAV virions for various applications, including clinical uses, using the current methodology thus remains impractical and a new procedure for the efficient production of large quantities of recombinant AAV vectors would clearly be highly beneficial.
SUMMARY OF THE INVENTION
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing novel compositions and methods for use in the efficient, large-scale production of recombinant adeno-associated virus (AAV). The compositions of the invention include recombinant AAV vectors, AAV-producing cell lines including such vectors, and recombinant infective virus vectors capable of expressing essential AAV genes required for AAV virion assembly and genome packaging. The invention also provides advantageous methods for AAV production which utilize virus infection and do not require DNA transfection.
The following terms and definitions employed herein to refer to recombinant adenoviruses and AAV inserts follow the conventional type of nomenclature used in the art. AdAAV is used as a general term to refer to adenovirus that contains one or more AAV genes inserted in any region of the adenovirus genome, this includes replication defective and replication competent adenovirus. Adenovirus carrying a particular gene can be termed as a prefix, Ad-, when the letters representing the inserted DNA will follow the prefix. The location in the adenovirus where the exogenous DNA is inserted w
Dong Jian-yun
Frizzell Raymond A.
Guzo David
UAB Research Foundation
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