Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration.
First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a host cytotoxic T-cell mediated immune response against virally expressed genes. In addition, a limited cloning capacity of approximately 8 kb does not cater for the incorporation of large upstream sequences essential for regulated tissue-specific expression or inclusion of multiple gene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosome (EAM) from which all of the viral genes have been deleted. EAMs contain only the inverted terminal repeats required for replication and five cis -acting Ad encapsidation signals necessary for packaging. We have shown previously that EAMs can efficiently transduce a variety of cell types in vitro. In this study we demonstrate that EAMs can transduce and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affected with retinal degeneration (rd) allows prolonged transgene expression and rescue of rod photoreceptor cells. RT-PCR analysis from the injected retina indicates that transgene products are present for at least 18 weeks post-injection. Both the alpha and beta subunits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thickness showed significant differences up to 12 weeks post-injection. These results demonstrate an improved level of rescue over first-generation adenoviral vectors and suggest the possibility of successful EAM-mediated treatment of some retinal diseases in humans.
Kumar-Singh
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Jules Stein Eye Institute, School of Medicine and Molecular Biology Institute, UCLA, 100 Stein Plaza, Room B243, Los Angeles, CA 90095-7008, USA. rajendra@ucla.edu
Farber
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D000256:Adenoviridae / Q000235:genetics* / Q000648:ultrastructure; D000818:Animals; D002213:Capsid / Q000235:genetics* / Q000648:ultrastructure; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010786:Photoreceptor Cells / Q000378:metabolism / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics / Q000628:therapy; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D011993:Recombinant Fusion Proteins