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1221. DNA Methylation Modulates Expression of Transgenes Transduced by Recombinant SV40 Vectors
GENE THERAPY APPLICATIONS TO SKIN, BONE AND CONNECTIVE TISSUE 1221. DNA Methylation Modulates Expression of Transgenes Transduced by Recombinant SV40 Vectors Martyn K. White,1 David S. Strayer.1 1 Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA. We observed that the level of expression of certain reporter transgenes (e.g., EGFP, dsRFP) that had been introduced into recombinant SV40 vectors (rSV40), was much lower when DNA was delivered to target cells by transduction rather than by transfection. Analysis of the DNA sequences that encode the wildtype proteins of SV40 (VP1-3 and T/t-antigens) revealed that they have an extremely low content of the dinucleotide CpG: 9 CpGs in a combined sequence length of 4,601 bps. In contrast, commonly used reporter genes are rich in CpGs: dsRFP, for example, has 63 CpGs in 757 bps. CpG is the target sequence for DNA methylation during gene silencing in eukaryotic cells. Therefore we hypothesized that methylation of the CpG-rich reporter transgene DNA might account for reduced transgene expression following rSV40 transduction of target cells. This hypothesis fits with available information. DNA methylation could occur during the viral growth period that is used to generate the rSV40 transducing virus in the replication-permissive Cos-7 monkey cell line. Three lines of evidence support this hypothesis. rSV40 genomes carrying dsRFP grown in E. coli are not CpG methylated and when transfected into Cos-7 elicit very strong cytosolic red fluorescence. rSV40 vector (SV-RFP) produced from these transfections by viral replication carry intact dsRFP DNA but deliver minimal levels of dsRFP fluorescence to transduced target cells. When SV-RFP was grown in the presence of 5-azacytidine, an inhibitor of CpG methylation, expression of dsRFP fluorescence was increased in target cells transduced by SV-RFP virus prepared in this manner. Restriction analysis of the dsRFP DNA in cells transduced with SV-RFP cut with a methylation-specific restriction endonuclease, showed CpG methylation of dsRFP DNA. However dsRFP DNA in cells transfected with the rSV40 genomes carrying dsRFP grown in E. coli, showed minimal CpG methylation. We are currently constructing a mutated form of dsRFP DNA to reduce CpG content without altering the amino acid sequence to see if removing CpG methylation targets leads to higher levels of expression as seen with wild-type dsRFP in transduced target cells. We conclude that the presence of CpGs within a rSV40 transgene may place a restriction on its expression by rSV40 vectors due to DNA methylation of the transgene occurring during rSV40 production. Removal of CpGs by mutagenesis may provide an important approach to enhancing the expression of certain rSV40 transgenes.
GENE THERAPY APPLICATIONS TO SKIN, BONE AND CONNECTIVE TISSUE 1222. A Truncated Form of Sonic Hedgehog Lacking the Cholesterol Modification Exhibits In Vivo Biological Activity and Increased Range of Acceleration of Hair Growth Following Adenovirus-Mediated Gene Transfer Howard Lou,1 Philip L. Leopold,1 Ronald G. Crystal.1 Weill Medical College of Cornell University, New York, NY.
1
Adenovirus-mediated transient dermal expression of Sonic hedgehog (Shh), a signaling protein involved in diverse embryonic developmental events during organ and tissue morphogenesis, stimulated the anagen growth phase of the hair cycle, resulting in accelerated hair growth. Active Shh is normally produced from the N-terminal of a precursor peptide by autocatalytic proteolysis of Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
the precursor followed by addition of a cholesterol molecule to the newly-formed C-terminal, a post-translational modification thought to be important for establishing local gradients of Shh during tissue patterning. Although cholesterol-bearing Shh is thought to be responsible for all known Shh biological activities, we hypothesized that truncation of the Shh coding sequence leading to expression of the N-terminal of Shh without the cholesterol modification might yield a signaling molecule capable of exhibiting the biological activity of acceleration of hair growth over a longer range than native Shh, thus enhancing its possible application to hair loss. To test this hypothesis, we constructed denovirus vectors expressing either a full length murine Shh cDNA (AdShh) or a cDNA encoding the Nterminal 198 residues of Shh (designated AdN-Shh). To characterize the construct, A549 lung epithelial cells were infected with AdNShh or AdShh. Western blot analysis demonstrated a 19 kD protein that stained with anti-Shh antibody in both AdShh and AdN-Shhinfected cells, but not in naive cells or cells infected with a control adenovirus (AdNull). However, while the Shh-immunoreactive band in AdShh-infected cultures was almost entirely cell-associated, the Shh-immunoreactive band was both cell-associated and secreted into the culture medium following infection with AdN-Shh, suggesting that the N-Shh could diffuse more easily. To study induction of melanogenesis and hair growth in vivo, 109 particle units of each vector were injected intradermally into the dorsal skin of postnatal day 19 C57Bl/6 mice. Melanogenesis was observed by day 7 and new hair growth became evident by day 14 in groups receiving AdN-Shh and AdShh but not in naive mice or mice injected with AdNull. Two wk after administration, quantitative assessment of the area of hair growth demonstrated that AdN-Shh induced a significantly larger area of new hair growth compared with AdShh (1.99±0.32 cm² vs 0.42±0.06 cm², p<0.001). Further, AdN-Shh was more effective than AdShh at either a 10-fold lower dose (108 particle units) yielding hair growth over 0.94±0.19 cm² compared to AdShh which yielded hair growth over 0.39±0.14 cm² (p<0.05), or at a 10-fold higher dose (1010 particle units) yielding hair growth over 1.96±0.40 cm² compared to AdShh which yielded hair growth over 0.98±0.17 cm² (p<0.05). These data at three different doses of vector suggest that N-Shh without covalent conjugation with cholesterol is biologically active and signals over a longer range than native Shh in accelerating hair growth. In the context that the modified Shh functions more effectively and over a larger range, the N-Shh transgene would be the preferred choice in gene transfer strategies to accelerate hair growth. Dr. Crystal has equity in, is a consultant to, and receives sponsored research funds from, GenVec, Inc., Gaithersburg, Maryland, a publicly-traded biotechnology company.
1223. Combination Gene Therapy with Different BMPs Increases Osteogenic Potency In Vitro and Enhances Spine Fusion In Vivo Wei Zhu,1 Kohei Goshi,1 Beth Ford,1 Jay Lieberman,2 Bernard Rawlins,1 Oheneba Boachie-Adjei,1 Ronald G. Crystal,3 Chisa Hidaka.1 1 Hospital for Special Surgery, New York, NY; 2University of California, Los Angeles, CA; 3Weill Medical College of Cornell University, New York, NY. Non-union is a major complication of spine fusion, occurring in up to 26% of cases. Treatments with recombinant bone morphogenetic proteins (BMPs), including BMP2 and BMP7, have been shown in experimental models and clinical studies to enhance bone formation in spine fusion, but super-physiologic doses are almost always required. While recombinant BMPs are homodimers, with 2 identical monomeric proteins, several studies have suggested that BMP heterodimers may be more potent than homodimers in supporting of bone formation. In this study, we examined whether S473
GENE THERAPY APPLICATIONS TO SKIN, BONE AND CONNECTIVE TISSUE 1222. A Truncated Form of Sonic Hedgehog Lacking the Cholesterol Modification Exhibits In Vivo Biological Activity and Increased Range of Acceleration of Hair Growth Following Adenovirus-Mediated Gene Transfer Howard Lou,1 Philip L. Leopold,1 Ronald G. Crystal.1 Weill Medical College of Cornell University, New York, NY.
1
Adenovirus-mediated transient dermal expression of Sonic hedgehog (Shh), a signaling protein involved in diverse embryonic developmental events during organ and tissue morphogenesis, stimulated the anagen growth phase of the hair cycle, resulting in accelerated hair growth. Active Shh is normally produced from the N-terminal of a precursor peptide by autocatalytic proteolysis of Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
the precursor followed by addition of a cholesterol molecule to the newly-formed C-terminal, a post-translational modification thought to be important for establishing local gradients of Shh during tissue patterning. Although cholesterol-bearing Shh is thought to be responsible for all known Shh biological activities, we hypothesized that truncation of the Shh coding sequence leading to expression of the N-terminal of Shh without the cholesterol modification might yield a signaling molecule capable of exhibiting the biological activity of acceleration of hair growth over a longer range than native Shh, thus enhancing its possible application to hair loss. To test this hypothesis, we constructed denovirus vectors expressing either a full length murine Shh cDNA (AdShh) or a cDNA encoding the Nterminal 198 residues of Shh (designated AdN-Shh). To characterize the construct, A549 lung epithelial cells were infected with AdNShh or AdShh. Western blot analysis demonstrated a 19 kD protein that stained with anti-Shh antibody in both AdShh and AdN-Shhinfected cells, but not in naive cells or cells infected with a control adenovirus (AdNull). However, while the Shh-immunoreactive band in AdShh-infected cultures was almost entirely cell-associated, the Shh-immunoreactive band was both cell-associated and secreted into the culture medium following infection with AdN-Shh, suggesting that the N-Shh could diffuse more easily. To study induction of melanogenesis and hair growth in vivo, 109 particle units of each vector were injected intradermally into the dorsal skin of postnatal day 19 C57Bl/6 mice. Melanogenesis was observed by day 7 and new hair growth became evident by day 14 in groups receiving AdN-Shh and AdShh but not in naive mice or mice injected with AdNull. Two wk after administration, quantitative assessment of the area of hair growth demonstrated that AdN-Shh induced a significantly larger area of new hair growth compared with AdShh (1.99±0.32 cm² vs 0.42±0.06 cm², p<0.001). Further, AdN-Shh was more effective than AdShh at either a 10-fold lower dose (108 particle units) yielding hair growth over 0.94±0.19 cm² compared to AdShh which yielded hair growth over 0.39±0.14 cm² (p<0.05), or at a 10-fold higher dose (1010 particle units) yielding hair growth over 1.96±0.40 cm² compared to AdShh which yielded hair growth over 0.98±0.17 cm² (p<0.05). These data at three different doses of vector suggest that N-Shh without covalent conjugation with cholesterol is biologically active and signals over a longer range than native Shh in accelerating hair growth. In the context that the modified Shh functions more effectively and over a larger range, the N-Shh transgene would be the preferred choice in gene transfer strategies to accelerate hair growth. Dr. Crystal has equity in, is a consultant to, and receives sponsored research funds from, GenVec, Inc., Gaithersburg, Maryland, a publicly-traded biotechnology company.
1223. Combination Gene Therapy with Different BMPs Increases Osteogenic Potency In Vitro and Enhances Spine Fusion In Vivo Wei Zhu,1 Kohei Goshi,1 Beth Ford,1 Jay Lieberman,2 Bernard Rawlins,1 Oheneba Boachie-Adjei,1 Ronald G. Crystal,3 Chisa Hidaka.1 1 Hospital for Special Surgery, New York, NY; 2University of California, Los Angeles, CA; 3Weill Medical College of Cornell University, New York, NY. Non-union is a major complication of spine fusion, occurring in up to 26% of cases. Treatments with recombinant bone morphogenetic proteins (BMPs), including BMP2 and BMP7, have been shown in experimental models and clinical studies to enhance bone formation in spine fusion, but super-physiologic doses are almost always required. While recombinant BMPs are homodimers, with 2 identical monomeric proteins, several studies have suggested that BMP heterodimers may be more potent than homodimers in supporting of bone formation. In this study, we examined whether S473