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Cocal vesiculovirus envelope pseudotyped retroviral vectors

a technology of cocal vesiculovirus and pseudotyped retroviral vectors, applied in the field of immunology, virology, molecular biology, can solve the problems of difficult generation of stable packaging cell lines, limitations of clinical gene therapy applications, and disadvantages of using vsv-g to pseudotype retroviral vectors, etc., to achieve the effect of improving serum stability

Inactive Publication Date: 2012-06-28
FRED HUTCHINSON CANCER RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]The present invention addresses these and other related needs by providing, inter alia, Cocal vesiculovirus envelope pseudotyped retroviral vector particles that may be suitably employed for gene transfer applications including gene therapy and vaccines, and, in particular, for the ex vivo and in vivo delivery of a gene of interest to a wide variety of target cells. The Cocal vesiculovirus envelope pseudotyped retroviral vector particles disclosed herein exhibit high titers, broad species and cell-type tropism, and improved serum stability.

Problems solved by technology

There are, however, some disadvantages to using VSV-G to pseudotype retroviral vectors.
Toxicity is associated with the constitutive expression of VSV-G, which has made generation of stable packaging cell lines difficult (Ory et al., Proc. Natl. Acad. Sci. U.S.A.
The titers reported for RD114 / TR pseudotyped lentiviral vectors have, however, generally been lower than those obtained with VSV-G, which may limit their utility for clinical gene therapy applications.

Method used

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  • Cocal vesiculovirus envelope pseudotyped retroviral vectors
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  • Cocal vesiculovirus envelope pseudotyped retroviral vectors

Examples

Experimental program
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example 1

Materials and Methods

[0061]Construction of a Cocal Envelope Pseudotype Plasmid

[0062]The deduced open reading frame (ORF) from the published sequence of Cocal envelope (Bhella et al., Virus Res. 54:197-205 (1998); GenBank Accession No. AF045556; SEQ ID NO: 1) was used to generate a human codon-optimized polynucleotide (SEQ ID NO: 3) that was synthesized by Blue Heron Biotechnology (Bothell, Wash.) using GeneMaker technology according to the sequence specified with ClaI and StuI flanking restriction sites. The optimized Cocal ORF was subcloned by standard techniques into pMD2.G kindly provided by Didier Trono, Lausanne, Switzerland (Addgene Plasmid No. 12259) replacing the VSV-G ORF to create pMD2.CocalG.

[0063]Production of Pseudotyped Lentiviral Vector Preparations and Determination of Titer

[0064]The SIN HIV vector plasmids used were pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene Plasmid No. 12252) and pRRLSIN.cPPT.PGK-YFP.WPRE (kindly provided by Luigi Naldini, San Raffaele Telethon Institute f...

example 2

Construction of a Cocal Expression Plasmid and Pseudotyping of Lentiviral Vectors

[0079]A human codon optimized version of Cocal DNA was synthesized based on the amino acid sequence reported by Bhella et al., Virus Research 54:197-205 (1998). The optimized sequence was cloned into the plasmid backbone that expresses the Cocal envelope glycoprotein from a CMV promoter with a human β-globin intron and polyadenylation sequence (FIG. 1). This plasmid backbone is identical to the plasmid backbone for pMD.2G, a VSV-G plasmid routinely used for the production of VSV-G pseudotyped lentiviral vectors.

[0080]SIN lentiviral vectors that express the enhanced green fluorescent (EGFP) protein from a phosphoglycerate kinase (PGK) promoter were generated by transient transfection and pseudotyped with Cocal, VSV-G, or RD114 / TR envelope. Viral titers were determined on HEK 293 cells and also on human HT1080 fibrosarcoma cells. Protamine sulfate and polybrene are cationic polymers commonly used to enhan...

example 3

Cocal Pseudotyped Lentiviral Vectors have a Broad Tropism

[0083]The high titers of Cocal pseudotyped vector preparations suggested that they will be highly effective for many gene transfer and gene therapy applications. The efficiency of transduction was compared for a panel of cell lines and primary cells derived from several tissues that are important targets for gene therapy, and from several species commonly used as preclinical models for gene therapy. FIG. 3 shows the relative transduction efficiency of transformed human cell lines and primary cells derived from blood, retinal epithelia, lung fibroblasts, bone endothelia, skin fibroblasts, stroma from humans, three species of macaques, cow, dog, cat or rat. The Cocal envelope allowed for highly efficient transduction of cell types from all tissues and most species tested with the exception of rhesus (Macaca mulatta) and cynomolgous or crab-eating macaque (Macaca fascicularis) nonhuman primate cells where the transduction rates w...

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Abstract

Provided herein are Cocal vesiculovirus envelope pseudotyped retroviral vectors that exhibit high titers, broad species and cell-type tropism, and improved serum stability. Disclosed Cocal vesiculovirus envelope pseudotyped retroviral vectors may be suitably employed for gene therapy applications and, in particular, for the ex vivo and in vivo delivery of a gene of interest to a wide variety of target cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Patent Application No. 61 / 175,376, filed May 4, 2009, and which provisional patent application is incorporated by reference in its entirety herein.GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT[0002]This disclosure was made, in part, in the course of research sponsored by the National Institute of Health, grant numbers HL36444, DK47754, HL074162, DK07786, DK56465, HL53750, AI061839, and AI063959. The U.S. government has certain rights in this disclosure.SEQUENCE LISTING[0003]The present application includes a Sequence Listing in electronic format as a txt file titled “Sequence_Listing—04May10,” which was created on May 4, 2010 and which has a size of 21 kilobytes (KB). The contents of txt file “Sequence_Listing—04May10” are incorporated by reference herein.BACKGROUND OF THE DISCLOSURE[0004]1. Technical Field of the Disclosure[0005]The present disclosure relates, generally, to the field...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/33A61P43/00C12Q1/70C12N15/86C12N15/63C07K14/005
CPCC07K14/005C12N15/86C12N2810/6081C12N2740/16045C12N2760/20222C12N2740/16043A61P43/00A61K35/28A61K48/00A61K2035/124C12N2760/20243
Inventor TROBRIDGE, GRANT D.KIEM, HANS-PETER
Owner FRED HUTCHINSON CANCER RES CENT
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