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Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions vectors

a technology of adeno-associated virus and a delivery method, which is applied in the direction of recombinant dna-technology, genetic material delivery route, botany apparatus and processes, etc., can solve the problems of low expression level, ineffective long-term gene therapy, and decrease the life of the transduced cell, so as to achieve efficient delivery of genes

Inactive Publication Date: 2008-03-20
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015] Accordingly, the present invention is based on the surprising and unexpected discovery that recombinant AAV (rAAV) virions provide for efficient delivery of genes and sustained production of therapeutic proteins in various muscle cell types. The invention allows for in vivo secretion of the therapeutic protein from transduced muscle cells such that systemic delivery of therapeutic levels of the protein is achieved. These results are seen with both in vivo and in vitro modes of DNA delivery. Hence, rAAV virions allow delivery of DNA directly to muscle tissue. The ability to deliver and express genes in muscle cells, as well as to provide for secretion of the produced protein from transduced cells, allows the use of gene therapy approaches to treat and / or prevent a wide variety of disorders.
[0016] Furthermore, the ability to deliver DNA to muscle cells by intramuscular administration in vivo provides a more efficient and convenient method of gene transfer.

Problems solved by technology

However, this mode of administration generally results in sustained but low levels of expression.
Despite these advantages, adenovirus vectors suffer from several drawbacks which make them ineffective for long term gene therapy.
In particular, adenovirus vectors express viral proteins that may elicit an immune response which may decrease the life of the transduced cell.
Furthermore, the adult muscle cell may lack the receptor which recognizes adenovirus vectors, precluding efficient transduction of this cell type using such vectors.
Thus, attempts to use adenoviral vectors for the delivery of genes to muscle cells has resulted in poor and / or transitory expression.
However, such methods require substantial tissue culture manipulation and surgical expertise, and, at best, show inconclusive efficacy in clinical trials.

Method used

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  • Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions vectors
  • Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions vectors
  • Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions vectors

Examples

Experimental program
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Effect test

example 1

Expression of rAAV-LacZ in Terminally Differentiated Adult Rat Cardiomyocytes

[0124] The ability of recombinant AAV virions to transduce terminally differentiated adult cardiomyocytes was established in vitro. Cardiomyocytes were harvested by coronary perfusion with collagenase of adult rat hearts (Fischer 344, Harlan Sprague Dawley, Indianapolis, Ind.). Cardiomyocytes were grown on laminin-coated glass coverslips and exposed to rAAV-LacZ virions for 4 hours. After 72 hours, the cells were stained for β-galactosidase activity. AAV expression was detected by blue staining of the binucleated cells. These studies demonstrated the ability of rAAV virions to transduce terminally differentiated cells. The transduction efficiency in vitro was 30% of adult cells at a multiplicity of infection (MOI) of 104 genomes per cell.

example 2

Stability of rAAV-LacZ Expression In Vivo

[0125] Adult Fischer rats were used to analyze expression of transgenes in vivo. Incremental doses of rAAV-LacZ virions were injected into the left ventricular apex of the heart using either a subxyphoid or lateral thoracotomy approach. More particularly, experimental animals were anesthetized with Metofane followed by a subxyphoid incision to expose the diaphragmatic surface of the heart. Apical cardiac injections were performed with a glass micropipette. Recombinant virion was diluted in normal saline and injected at a volume of 20-50 μL.

[0126] At varying times post-injection, hearts were harvested and examined for β-galactosidase production and for the presence of infiltrating mononuclear cells. For 5-Bromo-4-chloro-3-indolyl β-D-galactoside histochemical determination, frozen sections (6 μm) were fixed in 0.5% glutaraldehyde and stained for β-galactosidase activity as described (Sanes et al. (1986) “Use of Recombinant Retrovirus to Stud...

example 3

In Vivo Transduction of Murine Skeletal Muscle using rAAV-LacZ Virions

[0128] Recombinant AAV-LacZ virions were injected into muscle tissue of mice, and transduction assessed by β-gal activity. Particularly, in vivo transduction was performed by intramuscular (IM) injection of recombinant AAV virions into the skeletal muscle of healthy 6-8 week old Balb / c mice (Jackson Laboratories, Bar Harbor, Me., Simonsen Laboratories, Gilroy, Calif., or Harlan Laboratories) under either Metofane (Pitman-Moore, Mundelein, Ill.) or ketamine-xylazine anesthesia. The mid-portion of each tibialis anterior muscle was exposed via a 1 cm incision. Injections into the tibialis anterior were carried out using a micro-capillary tube attached to a Hamilton syringe to administer the following formulations at a depth of 2 mm: phosphate-buffered saline.(PBS) alone (negative control); or PBS containing either rAAV-LACZ virions or pW1909adhlacZ plasmids.

[0129] Tissue samples of tibialis anterior muscle, forelim...

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Abstract

The use of recombinant adeno-associated virus (AAV) virions for delivery of DNA molecules to muscle cells and tissue is disclosed. The invention allows for the direct, in vivo injection of recombinant AAV virions into muscle tissue, e.g., by intramuscular injection, as well as for the in vitro transduction of muscle cells which can subsequently be introduced into a subject for treatment. The invention provides for sustained, high-level expression of the delivered gene and for in vivo secretion of the therapeutic protein from transduced muscle cells such that systemic delivery is achieved.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of pending U.S. application Ser. No. 11 / 303,896, filed Dec. 15, 2005, which is a continuation of pending U.S. application Ser. No. 10 / 445,088, filed May 23, 2003, which is a continuation of U.S. application Ser. No. 09 / 969,204, filed Oct. 1, 2001 (U.S. Pat. No. 6,610,290), which is a continuation of U.S. application Ser. No. 09 / 406,362, filed Sep. 28, 1999 (U.S. Pat. No. 6,335,011), which is a continuation of U.S. application Ser. No. 08 / 784,757, filed Jan. 16, 1997 (U.S. Pat. No. 5,962,313), which is a continuation-in-part of U.S. application Ser. No. 08 / 588,355, filed Jan. 18, 1996 (U.S. Pat. No. 5,858,351), from which priority is claimed pursuant to 35 USC §120 and which applications are incorporated herein by reference in their entireties.TECHNICAL FIELD [0002] The present invention relates generally to DNA delivery methods. More particularly, the invention relates to the use of recombinant adeno-as...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/63C12N15/861C12N5/00C12N7/01A61K38/00A61K38/18A61K38/47C07K14/505C12N5/08C12N9/24C12N15/18C12N15/864
CPCA01K2217/05A61K38/1816A61K38/47A61K48/00A61K48/0075C07K14/505C12Y302/01023C12N15/86C12N2750/14143C12N2799/025C12N2830/42C12N2840/44C12Y302/0102C12N9/2408
Inventor PODSAKOFF, GREGORY M.KESSLER, PAUL D.BYRNE, BARRY J.KURTZMAN, GARY J.
Owner GENZYME CORP
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