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Method for generating replication defective viral vectors that are helper free

a technology of defective viral vectors and gene therapy, applied in the direction of sugar derivatives, biochemistry apparatus and processes, animal husbandry, etc., can solve the problems of development of simple systems, and inability to produce large quantities of pure replication defective viral vectors, so as to prevent the replication and/or packaging of dhlpv

Inactive Publication Date: 2012-05-10
THE ROCKEFELLER UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new method for producing helper-free, fully defective cAAV and traditional AAV vectors with high titers. This method involves using a replication-defective helper viral vector that requires the expression and / or transcription of at least one exogenous nucleic acid to replicate and preferably package. The replication-defective helper viral vector and the defective viral vector are placed into a permissive cell that contains the exogenous nucleic acid(s) required to replicate and preferably package. This method allows for the efficient production of high-titer cAAV and AAV vectors.

Problems solved by technology

However, before gene therapy becomes a standard medical procedure, certain technical problems common to all methods of gene delivery must be overcome.
One key obstacle is the current inability to produce large quantities of pure replication defective viral vectors.
One of the great challenges in effectively applying gene therapy to human disease is the development of simple systems for rapidly generating high volumes of high titer viruses completely uncontaminated by potentially toxic helper viruses.
Current strategies for producing such vectors, however, rely on techniques which either limit viral titers or which are so labor and resource intensive that they severely limit the clinical and commercial viability of these promising systems.
In an attempt to overcome this critical problem, new approaches have been attempted, though heretofore with limited success.

Method used

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  • Method for generating replication defective viral vectors that are helper free
  • Method for generating replication defective viral vectors that are helper free
  • Method for generating replication defective viral vectors that are helper free

Examples

Experimental program
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Effect test

example 1

Production of Helper-Free dAAV Vectors

[0125]An hygromycin-sensitive cell line was obtained that expresses the ICP4 gene product. A hygromycin resistant plasmid containing the Epstein-Barr Virus (EBV) origin of replication and the EBNA gene was constructed so as to contain two essential AAV genes, Rep and Cap. This plasmid was then introduced into this cell line. A cell line expressing Rep / Cap and ICP4 was created (i.e., Rep+ / Cap+ / ICP4+ cells) by selecting cells that were hygromycin resistant. A second cell line was prepared in an analogous manner except the cell line did not express ICP4 (i.e., Rep+ / Cap+ / ICP4− cells).

[0126]Rep / Cap are expressed at low levels in the absence of adenovirus sequences, so they are stable within the cell prior to infection. Both the Rep+ / Cap+ / ICP4+ cells and the Rep+ / Cap+ / ICP4− cells were used in the study below.

[0127]A defective helper vector was prepared from an HSV virus having a deletion in both copies of the ICP4 gene. Into this viral vector a casset...

example 2

Production of Helper-Free “Gutless” Ad Vectors

[0129]The identical dHSV / Ad helper vector disclosed above, in Example 1 was used with a different cell line for packaging a “gutless” adenovirus (Ad) vector. The gutless Ad vector contains adenovirus termini (harboring origins of DNA replication) and a packaging signal, but no other adenovirus genes.

[0130]A cell line was created which contains a subset of the adenovirus genome inserted into the EBV / EBNA plasmid as described in Example 1 above, to create a stable cell line. These adenovirus sequences contain the adenovirus genome with the E1A, E1B, E2a, E4orf6, and VAI RNA sequences deleted. The deletions were performed in a manner which eliminated any overlap with sequences in the dHSV / Ad helper vector and thereby prevent any possible homologous recombination between the two. In order to retain the essential fiber protein in the cell line, the fiber gene was cloned by PCR, and after deletion of the E4 and part of E3 sequences (which nece...

example 3

Construction of Cell Lines

[0133]One of the most efficient means of producing recombinant AAV, in theory would be to employ a packaging cell line. Unfortunately, heretofore, development of such a cell line has been limited due to the toxicity of the genes required for AAV replication and virion assembly. As disclosed herein, these genes include the AAV rep and cap genes and the adenovirus transcription units: E1A, E2a, E4orf6 and VA RNA.

[0134]The prospects of producing a cell line with a minimal complement of genes appeared to improve with the report that only a subset of these genes (rep, cap, E1 and E4orf6) were sufficient for the generation of high AAV titers (Allen et al. (2000) Mol. Ther. 1(1): 88-95). However, despite extensive efforts, these results could not be confirmed. Indeed, when the rep, cap and E4orf6 coding regions were placed under the control of heterologous promoters, a very poor rAAV titer was obtained (about 0.006 IU / cell).

[0135]Importantly, the addition of a pla...

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Abstract

Sequences are provided that are capable of directing circular adeno-associated virus replication, useful in vectors for providing therapeutic agents to a subject in need thereof. The vectors of the invention are particularly useful in the treatment of acute medical conditions requiring rapid gene expression. Further provided are methods for producing packaged defective viral vectors.

Description

RELATIONSHIP TO OTHER PATENT APPLICATIONS[0001]This application claims priority to U.S. provisional applications 60 / 294,797 filed 31 May 2001, and 60 / 313,007 filed 17 Aug. 2001, both of which applications are herein specifically incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention provides a method of producing defective viral vectors for gene therapy that are completely free of helper viral vectors and helper viruses. The invention further provides new circular AAV vectors which are particularly useful for use in gene therapy and production stocks of packaged defective viral vectors.BACKGROUND[0003]Gene therapy is likely to become the most significant development in medicine of our time. However, before gene therapy becomes a standard medical procedure, certain technical problems common to all methods of gene delivery must be overcome. One key obstacle is the current inability to produce large quantities of pure replication defective viral v...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/7088C12N15/63A01K67/027C12N15/113A01K67/02A61K48/00C12N7/04C12N15/06C12N15/07C12N15/864
CPCC12N7/00C12N2750/14162C12N2750/14143C12N15/86
Inventor KAPLITT, MICHAEL G.MOUSSATOV, SERGEI
Owner THE ROCKEFELLER UNIV
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