Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-09-22
2001-03-27
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, C435S325000, C435S363000, C435S366000, C435S368000, C435S369000, C435S370000, C435S371000, C435S372000, C435S455000, C435S456000
Reexamination Certificate
active
06207455
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to improved viral vectors useful for the expression of genes at high levels in human cells. These vectors also find use in anti-viral, anti-tumor, and/or gene therapy. The improved vectors contain novel viral gene expression vectors and packaging vectors, that permit increased efficiency of packaging the recombinant viral genome and increased long-term gene expression. The improved vectors may also contain regulatory gene sequences derived from viral or human genomes, but lack viral gene expression coding genomes.
BACKGROUND OF THE INVENTION
Viral vectors have been used as vehicles for the transfer of genes into many different cell types including whole embryos, fertilized eggs, isolated tissue samples, and cultured cell lines. The ability to introduce and express a foreign gene in a cell is useful for the study of gene expression and the elucidation of cell lineages (J. D. Watson et al.,
Recombinant DNA,
2d Ed., W.H Freeman and Co., NY [1992], pp. 256-263). Retroviral vectors, capable of integration into the cellular chromosome, have also been used for the identification of developmentally important genes via insertional mutagenesis (J. D. Watson et al., supra, p. 261). Viral vectors, and retroviral vectors in particular, are also used in therapeutic applications (e.g., gene therapy), in which a gene (or genes) is added to a cell to replace a missing or defective gene or to inactivate a pathogen such as a virus.
1. Retroviruses
The members of the family Retroviridae are characterized by the presence of reverse transcriptase in their virions. There are several genera included within this family, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. They are integrated into the host DNA, and are capable of transmitting sequences of host DNA from cell to cell. This has led to the development of retroviruses as vectors for various purposes including gene therapy.
2. Gene Therapy
Gene therapy has been investigated as one method to cure disease. Viral vectors transduce genes into target cells with high efficiencies owing to specific virus envelope-host cell receptor interaction and viral mechanisms for gene expression. Factors affecting viral vector usage include tissue tropism, stability of virus preparations, genome packaging capacity, and construct-dependent vector stability. In addition, in vivo application of viral vectors is often limited by host immune responses against viral structural proteins and/or transduced gene products. This host immunity problem plus potential safety concerns about the possibility of generating replication-competent viruses have prompted much effort towards the development of non-viral vector systems, such as liposome-mediated gene transfer, naked DNA injections and gene gun technology. However, all of these non-viral gene transfer methods lack the ability to allow permanent integration of foreign genes into the host cell chromosomes. For long term expression of therapeutic genes in target cells, efficient means of transduction and genome integration are essential. Viral vectors such as retroviruses and adeno-associated viruses (AAV) transduce and integrate genes into different cell and tissue types. Thus, these viral vectors have been useful tools in current clinical gene therapy applications.
3. Retroviral Gene Therapy Strategies
Efficient and long term gene transfer is essential to clinical gene therapy application. Retroviral vectors derived from the amphotropic Moloney murine leukemia virus (MLV-A) use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See e.g., E. M. Gordon and W. F. Anderson, Curr. Op. Biotechnol., 5:611-616 [1994]; and A. D. Miller et al., Meth. Enzymol., 217:581-599 [1993]).
Other retroviruses, including human foamy virus (HFV) and human immunodeficiency virus (HIV) have gained much recent attention, as their target cells are not limited to dividing cells and their restricted host cell tropism can be readily expanded via pseudotyping with vesicular stomatitis virus G (VSV-G) envelope glycoproteins (See e.g., J. C. Bums et al., Proc. Natl. Acad. Sci. USA 90:8033-8037[1993]; A. M. L. Lever, Gene Therapy. 3:470-471 [1996]; and D. Russell and A. D. Miller, J. Virol., 70:217-222 [1996]). However, a useful lentiviral vector system has not been well established, mainly because of the lack of sufficient studies on lentiviral vectorology and safety concerns.
4. Gene Therapy Strategies For Inborn Errors Of Metabolism
In a few cases, gene therapy has been used to successfully correct inborn errors of metabolism using existing vector systems. For example, the adenosine deaminase gene has been introduced into peripheral blood lymphocytes and cord blood stem cells via retroviral vectors in order to treat patients with severe combined immunodeficiency due to a lack of functional adenosine deaminase (K. W. Culver et al., Human Gene Ther., 2:107 [1991]). Partial correction of familial hypercholesterolemia has been achieved using existing retroviral vectors to transfer the receptor for low density lipoproteins (LDL) into hepatocytes. However, it was estimated that only 5% of the liver cells exposed to the recombinant virus incorporated the LDL receptor gene with the vector utilized (M. Grossman et al., Nat. Genet., 6:335[1994]).
A number of single-gene disorders have been targeted for correction using gene therapy. These disorders include hemophilia (lack of Factor VIII or Factor IX), cystic fibrosis (lack of cystic fibrosis transmembrane regulator), emphysema (defective &agr;-1-antitrypsin), thalassemia and sickle cell anemia (defective synthesis of &bgr;-globin), phenylketonuria (deficient phenylalanine hydroxylase) and muscular dystrophy (defective dystrophin) (for review see A. D. Miller, Nature 357:455 [1992]). Human gene transfer trials have been approved for a number of these diseases.
5. Gene Therapy Strategies For Cancer
In addition to replacement of defective genes, it has been proposed that viral vectors could be used to deliver genes designed to stimulate immunity against or to otherwise destroy tumor cells. Retroviral vectors containing genes encoding tumor necrosis factor (TNF) or interleukin-2 (IL-2) have been transferred into tumor-infiltrating lymphocytes in patients (A. Kasid et al., Proc Natl Acad Sci USA. 87:473-477 [1990]; and S. A. Rosenberg, Human Gene Therapy 5: 140 [1994]). It is postulated that the secretion of TNF or IL-2 stimulates a tumor-specific immune response resulting in the destruction of the tumor or the recruitment of effective tumor infiltrating lymphocytes from nearby lymph nodes. Other proposed anti-tumor gene therapy strategies include the delivery of toxin genes to the tumor cell.
Applications of antisense genes or antisense oligonucleotides in inhibition of oncogenes and modulation of growth factors have the potential to reduce the mortality of cancer, in particular, human leukemia (For review see, A. M. Gewirtz, Stem Cells 3:96 [1993]; and L. Neckers and L. Whitesell, Amer. J. Physiol., 265:L1 [1993]).
6. Current Viral Vector Systems
In view of the wide variety of potential genes available for therapy, it is clear that an efficient means of delivering these genes is sorely needed in order to fulfill the promise of gene therapy as a means of treating infectious, as well as non-infectious diseases. Several viral systems including murine retrovirus, adenovirus, parvovirus (adeno-associated virus), vaccinia virus, a
Cooper Iver P.
Guzo David
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