Gutted adenoviral vectors for gene transfer to muscle.
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Adenoviridae↗
Biomedical subjects
Jeannine M Scott
Publications and source records attributed to Jeannine M Scott.
Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin.
Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25-30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.
Adenoviridae↗
Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin.
Gene therapy for Duchenne muscular dystrophy will require methods to deliver gene constructs encoding functional versions of dystrophin to the vast majority of a patient's musculature. Obstacles to achieving these goals include identifying which forms of dystrophin would be effective in a clinical setting and developing gene delivery shuttles capable of carrying and expressing dystrophin cassettes without toxic or adverse immunologic consequences. We review here recent work from our laboratory to identify sequences within dystrophin that are required to prevent development of dystrophic changes in muscle or which might be able to correct pre-existing damage. We also describe work aimed at developing viral shuttle vectors able to carry and express these dystrophin cassettes at high levels and in a muscle-specific fashion. While great challenges remain in developing methods for systemic gene delivery, we show that a variety of viral vectors are able to carry and express therapeutic levels of dystrophin when delivered directly to mouse skeletal muscle.
Adenoviridae↗
Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses.
BACKGROUND: Helper-dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high-titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo. METHODS: Replication-defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7-cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre-terminal protein, and with a cre-recombinase plasmid. RESULTS: We show that C7-cre cells allow efficient production of gutted Ad using deltaE1 + deltaE2b + deltaE3 helper viruses whose growth can be limited by cre-loxP-mediated excision of the packaging signal. Gutted Ad vectors carrying approximately 28 kb cassettes expressing full-length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%. CONCLUSIONS: These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b-deleted vectors display a profound reduction in viral gene expression.
Adenoviridae↗