534. Silk-Elastinlike Polymers for Matrix-Mediated Adenoviral Gene Delivery
Authors: Arash Hatefi, Hamid Ghandehari
Journal: Molecular Therapy (2006)

INTRODUCTION Matrix-mediated polymeric gene delivery can reduce administration frequency and toxicity while maximizing efficacy of cancer gene therapy. Here we report the release and bioactivity of Ad.CMV-GFP from silk-elastinlike polymers (SELPs) hydrogels. We hypothesize that by genetic engineering of biodegradable SELPs, it is possible to tailor-make delivery systems that are liquid at room temperature, mixed under mild conditions with adenovirus particles, form vector-laden hydrogels at body temperature and release viable vectors over a desired period of time with sustained high transfection efficiency. METHODS Preparation of virus-containing hydrogelsMixtures of a silk-elastinlike polymer composed of 4 silk (GAGAGS) and 8 elastin (GVGVP) units with one elastin unit containing a lysine residue, namely SELP-47K and Ad.CMV-GFP viruses were produced to yield 2.5 × 108 virus particles per 50ul gel with various concentrations. The polymer/virus suspensions were incubated at 37 °C for 4 h, and allowed to gel. Virus release from hydrogels Hydrogels were placed 800ul of sterile DPBS and incubated at 37°C in a shaking incubator for 28 days. Samples were taken at predetermined time points. The viral DNA was extracted and quantified using RT-PCR. A standard curve ranging from 102 to 1010 virus particles was obtained for each assay. In vitro bioactivity of viruses The day prior to transfection, HeLa cells were seeded at a density of 1 × 105 cells per well in 6 well plates. Hydrogels (4% v/v) containing 1.5 × 109 Ad.CMV-GFP infectious viruses were transferred into MEME supplemented with FBS (90:10 v/v) and incubated at 37°C. At predetermined time points samples were drawn and used to transfect HeLa cells. RESULTS AND DISCUSSION A method was established for quantization of virus release from SELP hydrogels. The results demonstrated that by changing polymer concentration it is possible to control virus delivery over 28 days. The 4% gel released 100% of the loaded viruses in one week, followed by more than 70% release from 6% gel and up to 20% from 8% v/v hydrogels. An increase in polymer concentration leads to increased hydrogel crosslinking density, decreased swelling and decreased release [1, 2]. The released viruses were able to transfect Hela cells over 20 days studied so far. These results are consistent with our previous observations showing transfection of HEK293 cells with Ad-GFP released from SELP-47K [1]. CONCLUSION Results demonstrate that by manipulation of polymer concentration it is possible to modulate adenoviral release. The next logical steps are to evaluate the influence of polymer structure on network properties, virus release, and on in vitro and in vivo bioactivity of released therapeutic viruses. REFERENCES [1] Megeed Z, Haider M, Li D, O’Malley BW, Jr., Cappello J, Ghandehari H. In vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy. J Control Release 2004;94(2-3):433-45. [2] Haider M, Hatefi A, Ghandehari H. Recombinant polymers for cancer gene therapy: a minireview. J Control Release 2005;109(1-3):108-19.