Compounds and methods to enhance rAAV transduction

Abstract

Agents and methods to alter rAAV transduction are provided.

Description

STATEMENT OF GOVERNMENT RIGHTS[0001]This invention was made at least in part with a grant from the Government of the United States of America (Contract No. HL51887 from the National Institutes of Health). The Government may have certain rights in the invention.BACKGROUND OF THE INVENTION[0002]Recombinant adeno-associated virus (rAAV) has several characteristics that underscore its potential as a gene therapy vector for numerous target organs and inherited diseases. rAAV vector systems potentially offer major advantages over adenovirus and retroviruses. These include the ability of rAAV to integrate into the genome of non-dividing cells, the lack of potential immune responses since all viral genes can be deleted, and the fact that rAAV can be concentrated to high titers.[0003]Adeno-associated virus type-2 (AAV-2) has been suggested to be a very promising vector for the gene therapy of cystic fibrosis (Conrad et. al., 1996; Flotte et al., 1993; Halbert et al., 1997). In vivo administr...

AI Technical Summary

Benefits of technology

[0013]As described hereinbelow, virus binding, e.g., the restricted distribution of viral receptors, and endocytosis of AAV-2 at the apical membrane of airway epithelia is not the major rate limiting step in transduction of this tissue type. In fact, differentiated human airway epithelia internalize rAAV-2 quite efficiently from the apical surface. Rather, endosomal processing and trafficking of internalized virus to the nucleus is the major obstacle encountered by AAV-2 following infection from the apical membrane of the airway. In contrast to basolateral infection which led to the efficient conversion of single stranded AAV DNA to circular form genomes, apical infection gave rise to persistent intracellular single stranded viral DNA in a transcriptionally inactive state for up to 50 days. Using proteasome inhibitors which increase the efficiency of endosomal processing of AAV-2 and intracellular routing to the nucleus, a significantly enhanced transduction from the apical surface of more than 200-fold was observed, to nearly that of transduction from the basolateral surface. It was also found that AAV capsid proteins are ubiquitinated following endocytosis, and that ubiquitin-mediated proteasome degradation of incoming virus can be blocked by treatment with either proteasome or ubiquitin ligase inhibitors.

[0014]Ubiquitination of the viral capsid thus appears to be a major barrier for altering the efficiency of trafficking the virus to the nucleus and / or nuclear processing events for conversion of the single strand DNA genome to a transcriptionally active state. Moreover, importantly, in vivo application of proteasome inhibitor in mouse lung augmented rAAV gene transfer from undetectable levels to a mean of 10.4+ / −1.6% of the epithelial cells in large bronchioles. Thus, the use of proteosome inhibitors to circumvent the major endosomal processing barriers to transduction in the airway may provide clinically useful strategies for in vivo AAV-mediated gene therapy of respiratory disorders such as cystic fibrosis, as well as for other tissues in which viral processing appears to be a rate limiting event.

[0039]Further, as described herein, the activity of agents that inhibit endosomal processing of virus may be enhanced by the addition of agents, such as EDTA or EGTA, which may alter molecules in pathways associated with endosomal processing, e.g., agents such as calcium chelators or, modulators of intracellular calcium levels. Thus, the invention also provides for compositions or kits comprising: 1) an inhibitor of endosomal processing; and 2) an agent which enhances the activity of the inhibitor. The inhibitor, or combination thereof, may be employed in the methods of the invention, or may be employed with an agent that enhances the activity of the inhibitor(s).

[0043]Circularized intermediates of recombinant adeno-associated virus impart episomal persistence to linked sequences in Hela cells, fibroblasts and muscle cells. Thus, in vivo persistence of recombinant adeno-associated virus can occur through episomal circularized genomes which may represent preintegration intermediates with increased episomal stability. Thus, recombinant adeno-associated virus is preferably prepared from a vector comprising at least one first DNA segment, a biologically active fragment or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability or persistence of the vector in a host cell; and a second DNA segment comprising a gene. Preferably, the second DNA segment encodes a therapeutically effective polypeptide.

Problems solved by technology

Rather, endosomal processing and trafficking of internalized virus to the nucleus is the major obstacle encountered by AAV-2 following infection from the apical membrane of the airway.

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