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Method of targeted gene delivery using viral vectors

a viral vector and gene technology, applied in the field of targeted gene delivery, can solve the problems of low viral titers, difficult to reconstitute fusion function, and difficult to target viruses to particular cell types

Inactive Publication Date: 2007-01-25
CALIFORNIA INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] In some embodiments of the invention, a packaging cell line is transfected with one or more vectors encoding the retroviral elements, the gene of interest, the fusogenic molecule and the cell-specific binding determinant. Recom

Problems solved by technology

Targeting such viruses to particular cell types has proved to be challenging.
However, this manipulation adversely affects the fusion domain of env, resulting in low viral titers.
The unknown and delicate coupling mechanisms of binding and fusion make it extremely difficult to reconstitute fusion function once the surface domain of the same molecule has been altered.
The challenge to this approach is that env, once complexed with the one end of the bridge molecule, fuses inefficiently.
Since no practical strategies are available for targeted in vivo gene delivery, current gene therapy clinical trails are generally based on in vitro transduction of purified cells followed by infusion of the modified cells into the patient.
This in vitro approach is an expensive procedure with significant safety challenges.

Method used

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  • Method of targeted gene delivery using viral vectors
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  • Method of targeted gene delivery using viral vectors

Examples

Experimental program
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Effect test

example 2

Transduction of CD20-expressing Target cells and 293T Cells Utilizing the Retroviral Vector FUGW / αCD20+HAmu and FUGW / 60 CD20+SINmu

[0248] Next, the efficacy of a αCD20-bearing virus in transferring genes into cells expressing CD20 in a cell-specific manner was tested. GFP expression was used to measure the transduction efficiency. The supernatants containing virus bearing various surface proteins were incubated with CD20-expressing target cells and 293T cells served as a control. Four days post-transduction, the efficiency of targeting was analyzed by FACS. FIG. 5A (rightmost panel) shows that FUGW / αCD20+HAmu viral particles could specifically transduce 16% of 293T / CD20 cells. Panels to the left show that transduction required the presence on the virions of HAmu, but there was some background transduction with virions lacking αCD20, likely due to residual weak binding of HAmu to its ligand, sialic acid. The titer for FUGW / αCD20+HAmu (fresh viral supernatant, no concentration) was es...

example 3

Transduction of Primary Human B-Lymphoid Cells Using the Retroviral Vector FUGW / αCD20+SINmu

[0254] Having established the ability of the system to mediate CD20-specific transduction of artificially created cell lines, the specific transduction of primary human B-lymphoid cells, cells that naturally carry the CD20 antigen, was investigated. Fresh, unfractionated human peripheral blood mononuclear cells (PBMCs) were transduced with FUGW / αCD20+SINmu and then stimulated with lipopolysaccharide (LPS) to expand the B cell population. Four days later, the cells were stained for CD19 (a B cell marker), CD20 and GFP expression (FIG. 6A). Over 35% of cells were CD20+ B cells under the described culture condition. The majority of them were GFP+. On the contrary, virtually no GFP+ cells were detected among CD20− non-B cells, confirming that the transduction was strictly dependent on CD20 expression. In another control experiment, fresh PBMCs were transduced with FUGW / αCD20+SINmu followed by sti...

example 4

Demonstration of Transduction Utilizing Lentiviral Vectors CCMV / αCD20+SINmu and CPGK / αCD20+SINmu

[0255] To demonstrate that the targeting method is not limited to the lentiviral vector FUGW, two additional lentiviral vectors with different promoter configurations were evaluated. Kohn et al. have incorporated the immunoglobulin heavy chain enhancer (Eμ) with associated matrix attachment regions into lentivectors carrying either the human cytomegalovirus (CMV) promoter (CCMV) or the murine phosphoglycerate kinase promoter (CPGK) (C. Lutzko et al. J Virol. 77, 7341-51 (2003)). These two lentiviral vectors were then adapted into the system and recombinant lentiviruses CCMV / αCD20+SINmu and CPGK / αCD20+SINmu were prepared. Transduction of PBMC-derived B cells with these viral supernatants exhibited results similar to those observed previously with FUGW (FIG. 6A). Stable integration of the GFP-transgene was detected by genomic PCR amplification (FIG. 6B).

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Abstract

Methods and compositions are provided for delivering a polynucleotide encoding a gene of interest to a target cell using a virus. The virus envelope comprises a cell-specific binding determinant that recognizes and binds to a component on the target cell surface, leading to endocytosis of the virus. A separate fusogenic molecule is also present on the envelope and facilitates delivery of the polynucleotide across the membrane and into the cytosol of the target cell. The methods and related compositions can be used for treating patients having suffering from a wide range of conditions, including infection, such as HIV; cancers, such as non-Hodgkin's lymphoma and breast cancer; and hematological disorders, such as severe combined immunodeficiency.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 60 / 686,215, filed Jun. 1, 2005 and U.S. Provisional Application No. 60 / 738,078, filed Nov. 19, 2005, which are both herein expressly incorporated by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention relates generally to targeted gene delivery, and more particularly to the use of a recombinant virus comprising a fusogenic molecule and a distinct affinity molecule. [0004] 2. Description of the Related Art [0005] The delivery of functional genes and other polynucleotides into particular target cells can be used in a variety of contexts. For example, gene therapy can be used to prevent or treat disease. A particularly desirable gene delivery protocol would be able to precisely deliver a gene of interest to specific cells or organs in vivo. Certain viruses are naturally suited for gene delivery, and s...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N15/867C12Q1/70C12N7/00
CPCA61K2039/505C07K16/2887C07K2317/53C07K2317/622C07K2319/00C07K2319/03C12N2810/859C12N2740/16043C12N2740/16045C12N2799/027C12N2810/80C12N2810/851C12N15/86Y02A50/30C12N2740/15043
Inventor BALTIMORE, DAVIDWANG, PINGYANG, LILI
Owner CALIFORNIA INST OF TECH
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