High-efficiency AAV helper functions

a technology of aav and function, applied in the field of high-efficiency aav helper function systems, can solve the problems of inability to maintain the concentration of protein at physiological levels, the level of protein is usually abnormally high, and the difficulty in safely and persistently delivering effective quantities

Inactive Publication Date: 2002-08-22
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach results in high-titer rAAV production with reduced levels of toxic long Rep proteins, enhancing the safety and efficacy of gene therapy applications by maintaining optimal Rep protein levels, thereby improving the production of recombinant virions.

Problems solved by technology

However, despite significant progress in the effort to identify and isolate genes, a major obstacle facing the biopharmaceutical industry is how to safely and persistently deliver effective quantities of these genes' products to patients.
Intravenous injection of recombinant proteins has been successful but suffers from several drawbacks.
First, patients frequently require multiple injections in a single day in order to maintain the necessary levels of the protein in the blood stream.
Even then, the concentration of protein is not maintained at physiological levels--the level of the protein is usually abnormally high immediately following injection and far below optimal levels prior to injection.
Second, intravenous delivery often cannot deliver the protein to the target cells, tissues or organs in the body.
And, if the protein reaches its target, it is often diluted to non-therapeutic levels.
Third, the method is inconvenient and severely restricts the patient's lifestyle.
The adverse impact on lifestyle is especially significant when the patient is a child.
In general, transfection methods are not suitable for in vivo gene delivery.
Second, AAV can infect cells from different species.
Because the patient's cells lack the rep and cap genes and the adenovirus accessory function genes, the rAAV are replication defective; that is, they cannot further replicate and package their genomes.
Similarly, without a source of rep and cap genes, wild-type AAV cannot be formed in the patient's cells.
Moreover, the rAAV titers produced are usually not sufficient for therapy.
The long forms of Rep, however, have toxic effects on many cell types.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1--

Western Blot

[0092] 293 cells (available from ATCC, catalog number CRL-1573) in 10 cm dishes were transfected with 10 .mu.g each of pVlacZ, pladeno5, and various AAV helper function constructs, which are depicted schematically in FIG. 1. Rep isoforms were analyzed by western blot analysis (shown in FIG. 2) using an anti-Rep monoclonal antibody.

[0093] AAVlacZ vector was produced and titer as previously described. Western blot analysis of the freeze / thaw lysates from vector production was carried out by standard methods using an 8% SDS polyacrylamide gel, electrotransfer to a nitrocellulose membrane, a monoclonal antibody to the AAV Rep protein (American Research Products, Belmont, Mass., Mab226.7) and chemiluminescent detection.

[0094] FIG. 2 illustrates a Western Blot of lysates prepared from 293 cells transfected with various AAV helper function vectors and probed with anti-Rep antibody. Lane 1 contains lysate from a cell transfected with the pAAVAd vector. Lane 2 contains lysate fro...

example 2--

rAAV Titers

[0096] AAVlacZ vector was produced by transfecting 293 cells in 10 cm dishes with 10 .mu.g each of pVlacZ, pladeno5, and various rep and cap constructs, as described in Example 1, above. AAVlacZ virion preparations were titered on 293 cells in the presence of adenovirus. The results of the vector production are shown in the table below.

1TABLE 1 AAVlacZ Titer / Helper Function Vector 10 cm Plate Relative Production pAAVAd 7.3 .times. 10.sup.8 0.85 pRCM 8.6 .times. 10.sup.8 1.0 pW1909 3.0 .times. 10.sup.9 3.5 pRCM.kozak 9.0 .times. 10.sup.8 1.05 pRCM.polyA 8.5 .times. 10.sup.8 0.98 pRCM.globinpolyA 2.0 .times. 10.sup.9 2.32

[0097] pAAVAd and pRCM both contain rep and cap sequences with wild-type promoter and gene configurations. These plasmids produced similar amounts of AAVlacZ and expressed similar amounts of both the long and short forms of Rep protein. With the modified constructs, alterations that decreased expression of the long forms of Rep protein and increased expres...

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Abstract

The present invention provides methods and compositions for producing high titer preparations of recombinant AAV ("rAAV") virions. The compositions of the present invention include AAV helper function systems and host cells. The present invention also includes methods of using AAV helper function vectors that effect the production of only small amounts of the long forms of Rep protein, and rAAV virions produced by such methods.

Description

1. RELATED APPLICATIONS[0001] This application is a continuation of copending U.S. patent application Ser. No. 09 / 107,708 of Georges Natsoulis, Peter Colosi, and Gary Kurtzman filed Jun. 30, 1998 and entitled "High-Efficiency AAV Helper Functions," which is a continuation-in-part of U.S. patent application Ser. No.08 / 688,648 of Georges Natsoulis filed Jul. 29, 1996 and entitled "High Efficiency Helper System for AAV Vector Production," which is a continuation-in-part of U.S. patent application Ser. No. 08 / 510,790 of Georges Natsoulis filed Aug. 3, 1995 and entitled "High Efficiency Helper System for AAV Vector Production," which issued as U.S. Pat. No. 5,622,856 on Apr. 22, 1997. These prior applications are incorporated herein by reference.2. FIELD OF THE INVENTION[0002] The present invention relates to adeno-associated virus (AAV) helper function systems for use in recombinant AAV (rAAV) virion production and methods of using such systems. More specifically, the present invention ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C07K14/015C12N5/10C12N7/04C12N15/864
CPCC07K14/005C07K2319/00C12N7/00C12N15/86C12N2750/14122C12N2750/14143C12N2750/14152C12N2750/14162C12N2800/30C12N2840/20
InventorNATSOULIS, GEORGESKURTZMAN, GARYCOLOSI, PETER
OwnerGENZYME CORP