534. Silk-Elastinlike Polymers for Matrix-Mediated Adenoviral Gene Delivery

DNA VECTOROLOGY Histidine (H) residues to facilitate escape from the endosomoal compartment. To improve the solubility and flexibility of the KH repeats and enhance DNA condensing efficiency, here we report the initial steps towards the construction of a biopolymer with the structure (KKKHHHHKKKG) 6-FGF2. The rationale for this structural modification is that lysine residues in clusters can condense DNA more efficiently and glycine (G) residues can increase the flexibility and solubility of KH repeats. METHODS Cloning of FGF2 into pET21b expression vector: Human FGF2 gene was amplified by PCR from a cDNA to introduce NdeI, Hind III and EcoRI restriction sites. The gene was double digested by NdeI and HindIII restriction endonucleases and cloned into pET21b expression vector (Figure 1). Design and cloning of (KHG)6 concatamer: The oligonucleotides encoding (KHG)6 were identified and designed to maximize the use of preferred codons in E. coli, while minimizing the codon repetition of the monomer gene. Restriction sites NdeI and EcoRI were also included for cloning into the pET21b expression vector. The (KKKHHHHKKKG) 6 gene was synthesized and cloned into pET21b along with FGF2 gene using standard recombinant DNA techniques (Figure 1). RESULTS AND DISCUSSION (KHG)6-FGF2 gene cloning: The insertion of (KHG)6-FGF2 gene in pET21b vector was confirmed by double digesting the vector with NdeI and HindIII and the fidelity of both sense and antisense strands were confirmed by DNA sequencing. The results revealed the successful gene construction and cloning of the (KHG)6-FGF2. CONCLUSION Construction of (KHG)6-FGF2 gene with repeating sequences in tandem for targeted systemic gene delivery is reported. Work is under way for large scale expression, purification, complexation with nucleic acids, and systematic evaluation of vector structure with transfection efficiency as described previously [1]. These results coupled with other reports [2] demonstrate the potential of recombinant techniques for tailor-making well-defined polymers for cancer gene therapy. REFERENCES [1] Hatefi A, Megeed Z, Ghandehari H. Recombinant polymerprotein fusion: a promising approach towards efficient and targeted gene delivery. J Gene Med 2006. [2] Haider M, Hatefi A, Ghandehari H. Recombinant polymers for cancer gene therapy: a minireview. J Control Release 2005;109(13):108-19.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

534. Silk-Elastinlike Polymers for MatrixMediated Adenoviral Gene Delivery Arash Hatefi, Hamid Ghandehari. 1 Centre for Nanomedicine and Cellular Delivery, Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, MD. 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 hydrogels Mixtures 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 x 108 virus particles per 50ul gel with various concentrations. The polymer/virus suspensions were incubated at 37 oC for 4 h, and allowed to gel. Virus release from hydrogels Hydrogels were placed 800ul of sterile DPBS and incubated at 37oC 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 x 105 cells per well in 6 well plates. Hydrogels (4% v/v) containing 1.5 x 109 Ad.CMV-GFP infectious viruses were transferred into MEME supplemented with FBS (90:10 v/v) and incubated at 37oC. 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 silkelastinlike 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(13):108-19. S205