A novel method for targeted gene therapy in ischemic tissues through viral transfection of an expression cassette containing multiple repetitions of hypoxia response element.
BACKGROUND
Increased levels of the transcription factor hypoxia inducible factor (HIF)-1 occur only in hypoxic tissue. The authors propose a therapeutic strategy that relies on HIF-1, the enhancer hypoxia response element (HRE), and the delivery vector adeno-associated virus-2 (AAV2) to direct ischemia specific gene therapy to skin.


METHODS
An expression cassette containing the CMV promoter driving the reporter gene green fluorescent protein (GFP) was used to assess cutaneous tropism of AAV2. Transfection of dermal fibroblasts and immortalized keratinocytes (HaCat) was assessed with flow cytometry. Human embryonic kidney 293 (HEK) cells were used to produce vector stocks and test the authors' therapeutic strategy in quadruplicate. An expression cassette with nine repeats of HRE linked to beta-galactosidase (LacZ) within the AAV2 vector was constructed. HEK cells were transfected and exposed to normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. LacZ activity was measured by conversion of galactoside red-beta-D-galactopyranoside.


RESULTS
Approximately 50 percent of dermal fibroblasts and HaCat cells were transfected when treated with 1 x 10(4) genome copies/cell of AAV2-CMV-GFP. Using the same titration of AAV2-9HRE-LacZ, transfected HEK cells demonstrated LacZ activity of 0.496 +/- 0.068 U/microg in normoxia and 2.9 +/- 0.58 U/microg in hypoxia. Transfected cells exposed to 24 hours of hypoxia show greater than an 11-fold increase in LacZ activity (p < 0.05) compared with baseline normoxic controls.


CONCLUSIONS
The authors' results confirm that AAV2 has in vitro tropism for skin-derived cell lines. Furthermore, HRE will drive gene expression in ischemia but not normoxia. This is the first step toward the authors' goal of HIF-1-regulated gene therapy to prevent ischemia related skin injury.
Cross
Kevin J
KJ
New York, N.Y. From the Division of Plastic Surgery, the Department of Pediatrics, and the Children's Blood Foundation Laboratories, Cornell University Medical College.
Bomsztyk
Elan D
ED
Weinstein
Andrew L
AL
Teo
Esther H
EH
Spector
Jason A
JA
Lyden
David C
DC
D002478:Cells, Cultured; D000229:Dependovirus; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D007511:Ischemia / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D020218:Response Elements; D012867:Skin / Q000098:blood supply*; D014162:Transfection