Amplifiable adeno-associated virus (AAV) packaging cassettes for the production of recombinant AAV vectors

Abstract

High-efficiency AAV packaging constructs and methods for their use are provided in the present invention. These high-efficiency packaging constructs comprise an activating element (such as the P1 sequence located within the AAV S1 integration site of human chromosome 19) amplifiably linked to one or more AAV packaging genes. The constructs may be either integrated into a mammalian cell genome or maintained episomally. Use of the high-efficiency AAV packaging vectors of the invention provides for controlled amplifiable production of rAAV vector constructs.

Description

TECHNICAL FIELD [0001] This invention is in the field of viral constructs for gene delivery. More specifically, the invention is in the field of recombinant DNA constructs for use in the production of adeno-associated virus (AAV) vectors for gene delivery. BACKGROUND [0002] Vectors based on adeno-associated virus (AAV) are believed to have utility for gene therapy but a significant obstacle has been the difficulty in generating such vectors in amounts that would be clinically useful for human gene therapy applications. This is a particular problem for in vivo applications such as direct delivery to the lung. Another important goal in the gene therapy context, discussed in more detail herein, is the production of vector preparations that are essentially free of replication-competent virions. The following description briefly summarizes studies involving adeno-associated virus and AAV vectors, and then describes a number of novel improvements according to the present invention that ar...

AI Technical Summary

Benefits of technology

The present invention provides methods and compositions for generating recombinant adeno-associated virus (rAAV) vectors. By removing the AAV rep and cap genes from their normal environment and placing them in amplifiable linkage with activating elements, the AAV packaging genes can be controlled but highly amplifiable expressed in cells. This allows for the production of stable AAV producer cells that can support the production of a very large burst of rAAV particles upon infection with a helper virus or provision of helper functions. The invention also provides compositions and methods for producing high-titer stocks of rAAV vectors containing a foreign gene of interest, as well as cell lines comprising an AAV packaging cassette of the invention. The AAV packaging cassettes can be introduced into a host cell and propagated either episomally or integrated into the genome of a host cell.

Problems solved by technology

Vectors based on adeno-associated virus (AAV) are believed to have utility for gene therapy but a significant obstacle has been the difficulty in generating such vectors in amounts that would be clinically useful for human gene therapy applications.

This is a particular problem for in vivo applications such as direct delivery to the lung.

This belief is supported by the observation that AAV replication may occur at low efficiency in the absence of helper virus co-infection if the cells are treated with agents that are either genotoxic or that disrupt the cell cycle.

Due to the inhibitory effects of expression of rep on cell growth, constitutive expression of rep in cell lines has not been readily achieved.

The attainment of high titers of AAV vectors has been difficult for several reasons including preferential encapsidation of wild-type AAV genomes (if they are present or generated by recombination), and the difficulty in generating sufficient complementing functions such as those provided by the wild-type rep and cap genes.

Useful cell lines expressing such complementing functions have been especially difficult to generate, in part because of pleiotropic inhibitory functions associated with the rep gene products.

All of these approaches failed to prevent generation of particles containing replication-competent AAV DNA and also failed to generate effective high titers of AAV transducing particles.

Thus, in packaging systems where the production of wild-type AAV is eliminated, the yield of packaged vector may actually be decreased.

Thus the effectively useful titer of this vector was limited.

Furthermore, this study did not demonstrate that the actual titer of the vector preparation was any higher than those obtained previously by Hermonat & Muzyczka (1984).

However, rep is expressed from its homologous promoter and is negatively regulated and thus its expression is limited.

That is, although overlapping homology of AAV sequence is not present, the complete AAV sequence is contained within the two plasmids and the plasmids share a short (non-AAV) sequence that might facilitate recombination to generate replication-competent AAV, which is undesirable.

Given the problems of low titer, and the capability of generating wild-type recombinants, the system described by Samulski et al., 1989, does not have practical utility for human gene therapy.

This system was based on pSub201 and thus suffers from the defect described above for this plasmid.

Second, the vector and the packaging plasmid contained overlapping AAV sequences (the ITR regions) and thus recombination yielding contaminating wild-type virus is highly likely.

However, these authors did not report any titers of their AAV vector stocks.

However, the difficulty in generating sufficient amounts of AAV vectors has been a severe limitation on the development of human gene therapy using AAV vectors.

One aspect of this limitation is that there have been very few studies using AAV vectors in in vivo animal models (see, e.g., Flotte et al., 1993b; and Kaplitt et al., 1994, Nature Genetics 8:148-154).

One of the limiting factors for AAV gene therapy has been the relative inefficiency of the vector packaging systems that have been used.

The efficiency of this process is expected to be limited by the efficiency of transfection of each of the plasmid constructs, and by the low level of expression of Rep proteins from the packaging plasmids described to date.

This also appears to relate to the tendency of Rep to reverse the immortalized phenotype in cultured cells, which has made the production of cell lines stably expressing functional rep extremely difficult.

353-359) attempted to generate cell lines containing the AAV rep and cap genes expressed from the normal AAV promoters, but these attempts were not successful either because the vectors were contaminated with a 100-fold excess of wild-type AAV particles or because the vectors were produced at only very low titers of less than 4×103 infectious particles.

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