Characterization of adenovirus p21 gene transfer, biodistribution, and immune response after local ocular delivery in New Zealand white rabbits.
Previous studies suggest that local gene therapy with rAd-p21(WAF1/Cip-1) [. Arch. Ophthalmol. 120, (2002) 941-949] may provide an effective adjunctive anti-proliferative treatment to prevent glaucoma surgery failure. To further investigate rAd-p21 in this indication, we have characterized several parameters of local gene delivery to conjunctiva including, vector delivery and transgene expression in target tissue, inflammatory response, biodistribution to non-target tissues, and immune response. Quantitative PCR and RT-PCR assays were employed to evaluate rAd-p21 dissemination and gene transfer following a single subconjunctival injection. In target tissue, significant levels of rAd-p21 DNA were found in 6/6 animals 1 and 4 days after injection. rAd-p21 DNA and RNA could be detected in the un-injected contralateral eye but at levels that were 10000-100000 lower than in the injected eye. Expression of human p21 transgene in conjunctival fibroblasts was confirmed by immunohistochemistry. Biodistribution of rAd-p21 following subconjunctival injection was substantially limited to ocular tissue. In 1/6 rabbits, rAd-p21 DNA was found in whole blood, liver, and spleen at levels that were barely detectable. All non-target organs were negative on day 4. In contrast, in a rabbit injected intravenously as a positive control, all blood samples and tissues samples were positive. rAd-p21 delivery to conjunctiva followed by filtration surgery caused an early acute inflammatory response, which by day 14 was indistinguishable from placebo-treated eyes. Neutralizing anti-adenovirus antibodies were detected following administration of rAd-p21 to conjunctiva, however, vector delivery and transgene expression were unaffected in a subsequent administration to the contralateral eye in the same animal. These results show that local delivery to conjunctiva may be a suitable delivery mode for ocular gene therapy.
Wen
S F
SF
Canji Inc., 3525 John Hopkins Court, San Diego, CA 92121, USA.
Chen
Z
Z
Nery
J
J
Faha
B
B
D000256:Adenoviridae / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D003228:Conjunctiva / Q000502:physiology*; D004279:DNA, Viral / Q000032:analysis; D015870:Gene Expression / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D005901:Glaucoma / Q000601:surgery; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007249:Inflammation / Q000276:immunology; D007267:Injections; D008099:Liver / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013154:Spleen / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes / Q000235:genetics
D000914:Antibodies, Viral; D004279:DNA, Viral; D012367:RNA, Viral