Determining the Optimal Viral Vector for Dermal Gene Therapy
Esther H. Teo, BS, Kevin J. Cross, MD, Elan D. Bomsztyk, BS, David C. Lyden, MD, PhD, Jason A. Spector, MD.
Weill Cornell Medical College, New York, NY, USA.
The skin is an ideal target for gene therapy because it is readily accessible, easily monitored, and affected by a variety of pathologic processes. Development of an optimal skin-targeted gene delivery mechanism has significant potential impact in promoting chronic wound healing and improved flap survival during reconstructive procedures. Dermal fibroblasts, in particular, are promising targets for skin-directed gene therapy because of their central role in response to tissue injury and skin wound healing. Viral vectors are the most commonly used gene delivery method to date. However, use of these vectors in skin-directed gene therapy has been disappointing because the efficiency and duration of vector transduction has not been extensively characterized in dermal fibroblasts. This study evaluates a broad panel of viral vectors to determine the optimal therapeutic gene delivery vehicle to dermal fibroblasts.
Viral vectors were made containing a recombinant green florescent protein (GFP) reporter gene with a constitutively active cytomegalovirus promoter. Viral subtypes included adenovirus type 5 (Ad-5), adeno-associated serotype 2 and 5 (AAV2, AAV5), MMLV-derived retrovirus (RV), and HIV-1-derived lentivirus (LV). Titer of viral stock was determined in HeLa cells using flow cytometry to detect GFP expression. Next, low passage primary human dermal fibroblasts were transduced with each vector by a single incubation with viral stock diluted to 10 infectious particles/cell. Retroviral and lentiviral transduction was done in the presence of polybrene. Viral media was removed after 72 hours. Cells were maintained in culture and passaged weekly for 5 passages. At each passage post-transduction, cells were analyzed with flow cytometry to evaluate GFP expression. Propidium iodide (PI) staining was used to measure cell death. Parallel studies were performed in NIH-3T3 embryonic mouse fibroblasts and HaCaT immortalized human keratinocytes.
Initially following viral transduction, human dermal fibroblasts demonstrated the following percentage of GFP-positive cells for each vector: Ad-5=72.7%, AAV2=33.1%, AAV5=16.4%, MMLV retrovirus=0.01%, and HIV-1 lentivirus=94.7%. <0.05% of uninfected controls demonstrated GFP positivity. GFP expression fell to <2% by the third passage for all vector types except lentivirus. Over 90% of the cell population transduced with the lentivirus maintained GFP-positivity over the 5-week duration of the experiment. Flow cytometry with PI of first passage cells showed increased cell death in Ad-5 treated cells, but no significant differences in other viral types. Similar trends were seen in NIH-3T3 and HaCaT cell lines.
HIV-1-based lentiviral vector resulted in the highest percentage of transduction in all cell lines. Greater than 90% of cells expressed the reporter gene over several passages following a single exposure to the lentivirus. Adenovirus and adeno-associated vectors had relatively high initial levels of transduction, but reporter gene expression fell dramatically after the first passage of cells. This study demonstrates that lentiviral vectors are the delivery mechanism of choice for long-term therapeutic gene expression in dermal fibroblasts, whereas adenoviral or AAV vectors may be preferred for short-term expression of a target gene.