Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-05-20
2002-10-22
Kunz, Gary L. (Department: 1647)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S069100
Reexamination Certificate
active
06469153
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
BACKGROUND OF THE INVENTION
Advances in understanding of human diseases at molecular level have led to possibility of treating human diseases by introducing genes into specific cells of patients. Gene therapy offers a great promise for modern medicine (Mulligan 1993) (Anderson 1984). The first step of gene therapy is to transduce specific target cells. There are two basic ways to deliver genes. In ex vivo therapy, cells from certain tissues, such as hematopoietic cells, are removed from patients and are infected with vectors which carry therapeutic genes. Cells expressing the therapeutic genes are then returned into patients. Vectors are directly administrated to patients in in vivo therapy. This approach is especially useful when it is difficult to isolate cells from tissues and to be infected in vitro. Advantages with retroviruses include long-term stability of therapeutic genes because viral genome is inserted into human chromosomes.
To engineer retroviruses for gene therapy, viral genes are deleted from viral genome, and an exogenous gene is inserted instead into viral genome. Plasmids containing a viral genome is introduced into a packaging cell line which provides viral components necessary to make viral particles (Cone and Mulligan 1984; Mann et al. 1983; Miller et al. 1985; Sorge et al. 1984; Watanabe and Temin 1983; Markowitz et al. 1988a; Markowitz et al. 1988b). Virions produced from the packaging cell line are used to transduce target cells. In the first step of viral infection, envelope proteins on retroviruses interact with receptors on target cells, which lead to a series of events resulting in fusion of viral membrane and cellular membrane. Viral core containing viral genomes with exogenous genes thus enters cells. Based on their host range, retrovirus vectors commonly used in human gene therapy are classified as ecotropic or amphotropic. Although ecotropic virus can only infect murine cells, modification of envelope protein, such as inserting a ligand epitope for a specific receptor, will expand or change host range, resulting in infection of human cells expressing that particular receptor (Kasahara et al. 1994). Amphotropic virus can infect both murine and human cells. One problem associated with retrovirus vectors is low transduction efficiency. For example, amphotropic virus has been used to infect hematopoietic cells, and transduction efficiency is low. To circumvent this problem, viruses pseudotyped with envelope proteins from other viruses have been tested. For example, Vesicular stomatitis virus (VSV) G protein and Gibbon ape leukemia virus (GALV) envelope protein have been studied in pseudotyping murine leukemia viruses (Hopkins 1993; Ory et al. 1996; Sharma et al. 1996; Wang et al. 1996) (Lam et al. 1996). In general, however, the titers of those vector stocks are still low, preventing successful clinical application of gene therapy in treating human diseases.
Gene therapy holds great promise in modern medicine. Advances in understanding of genetic bases of human diseases make it possible to treat human diseases by transferring normal genes into specific cells of patients. It has been proposed to use gene therapy to treat any human diseases, genetic and acquired (Anderson 1984; Mulligan 1993). Significant advances have been made to develop protocols to deliver exogenous genes into human cells. Vectors, vehicles used to deliver genes, include retroviruses and other viruses. Retroviral vectors, by far, the most extensively studied among viral vectors offer several advantages over other viral vectors, specially in ex vivo strategy of gene therapy. A retroviral vector can transduce any human cells, and lead to a long-term expression of exogenous gene.
To achieve therapeutic effects with gene therapy technology, vectors which express exogenous genes at a level sufficient to achieve therapeutic effects are required. A great deal of effort has been devoted to identify nucleic acid molecule sequences important for gene expression and to incorporate such nucleic acid molecules in vectors to thereby develop vectors which can express therapeutic genes at high levels (Leboulch et al. 1994; Leboulch et al. 1995; Takekosh et al. 1995). In some instances, regulated expression of the therapeutic genes is required (Cone et al. 1987). Retroviruses, offer an excellent choice to introduce exogenous genes into cells because of their ability to infect any kind of cells. In infected cells, retroviral genomes are inserted into host chromosomes, resulting in a long term expression of exogenous genes.
The second important aspect of gene therapy using a retrovirus as vector is the development of safe packaging cell lines (Cone and Mulligan 1984; Mann et al. 1983; Miller et al. 1985; Sorge et al. 1984; Watanabe and Temin 1983; Markowitz et al. 1988a; Markowitz et al. 1988b). In packaging cells, viral components are made and are able to assemble into viral particles. A vector carrying a therapeutic gene and a retroviral packaging signal is introduced into a packaging cell line and is packaged into viral particles. Retrovirus formed in such a way can be used to transduce target cells. To make safe packaging cell lines, viral proteins, gag and pol are expressed from a plasmid, and the envelope is expressed from another plasmid.
The chance of generating wild type virus through recombination is extremely low (Markowitz et al. 1988a; Markowitz et al. 1988b). Because a viral vector contains only a packaging signal, and does not encode any viral proteins, there is only one round of infection.
To successfully apply retroviral gene therapy in treatment of human diseases, several technical problems have to be solved. One of the difficulties involved is low transduction efficiency of retroviral stocks, which is addressed extensively in this study. Amphotropic retrovirus is widely used in gene therapy because of its ability to infect human cells. In transduction of human lymphocytes using retroviruses with amphotropic envelopes, however, transduction efficiency is relatively low. To improve gene therapy efficiency, different viral envelope proteins have been studied for their application in gene therapy. Gibbon Ape leukemia virus envelope virus envelope, for example, has been used to pseudotype murine leukemia viral vector (Lam et al. 1996). Vesicular stomatitis virus (VSV) G protein is another alternative envelope protein used in gene therapy (Hopkins 1993; Porter et al. 1996; Sharma et al. 1996; Wang et al. 1996). It has been shown that retroviral vectors pseudotyped with G protein can transduce human lymphocytes with much higher efficiency than amphotropic retroviral vectors.
In treating human diseases, sometimes specific targeting of certain cell types is required. Different strategies have been used to modify envelope proteins on vectors. For example, a ligand epitope is inserted into the envelope, which enables virus to infect specific cell type (Kasahara et al. 1994). In general, such modifications result in low infection efficiency.
Polybrene, a chemical compound, is often used to increase viral infectivity of retrovirus. In several cases, instead of boosting transduction efficiency, it actually decreases viral transduction efficiency. For example, plates coated with a fibronectin fragment are often used to isolate human stem cells. Polybrene, however, because of its negative charges, will decrease efficiency of fibronectin. Thus, alternatives are needed for enhancing retroviral infectivity in such circumstances.
Discovery of the Envelope-interacting Proteins (EIPs)
Understanding of the basic mechanism used by retroviruses to enter cells will facilitate application of retroviruses as vectors for gen
Goff Stephen P.
Li Xingqiang
Cooper & Dunham LLP
Kunz Gary L.
Landsman Robert S.
The Trustees of Columbia University in the City of New York
White John P.
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