- Email: [email protected]
1033. Cell-Mediated Gene Therapy for Ligament and Tendon Repair
GENE THERAPY FOR BONE, CONNECTIVE TISSUE AND SKIN TISSUES level gene expression in transfected cells. The introduction of an SV40 poly-adenylation signal cannot compensate for the deteriorative effects upon gene expression that result from a spectrum of U3 deletions. Among all the regulatory elements in the U3 region, we determined that the TATA box is the most important, as its deletion led to the most profound reduction in gene expression. Further analysis of gene expression in the transduced cells showed that optimal, inducible gene expression by a self-inactivating retroviral vector requires the preservation of the DR1 and the TATA box. These findings enabled us to identify the best way to design selfinactivating vectors through which high level expression of bone morphogenetic protein 4 (BMP4) can be turned on and off via the addition or withdrawal of doxycyclin. Muscle-derived stem cells transduced with this vector system elicited bone formation in normal animals only following administration of doxycycline. More importantly, these cells healed critical-sized skull defects only following administration of doxycycline. In contrast, no bone formation or bone healing was detected in the animals that did not receive the inducer. In conclusion, we have identified the optimal design for a self-inactivating, tet-on retroviral vector that can efficiently transduce muscle-derived stem cells and promote bone formation and bone healing through controllable BMP4 gene expression. We believe this vector system could prove integral to the development of more efficient, safer gene therapy techniques for the treatment of a variety of musculoskeletal diseases.
1032. Electro-Gene Therapy of CollagenInduced Arthritis by Using an Expression Plasmid for the Soluble p75 Tumor Necrosis Factor Receptor-Fc Fusion Protein Woong Hahn,1 Jong-Mook Kim,1 Seoun-Hyun Ho,1 Sujeong Kim,1 Jae-Gyun Jeong,2 Hwang-Jae Lee,1 Seung Shin Yu,1 Sunyoung Kim.2 1 ViroMed Co., Ltd., Seoul, Korea; 2School of Biological Science, Seoul National University, Seoul, Korea. Tumor necrosis factor (TNF) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, and antagonism of TNF may reduce the activity of the disease. Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. In this study, we attempted to treat collagen-induced arthritis (CIA) with anti-TNF gene therapy by transferring the plasmid encoding soluble p75 TNF receptor linked to the Fc portion of human IgG1 (sTNFR:Fc) using in vivo electroporation. DBA/1 mice were immunized with bovine type II collagen and boosted with the same antigen. Two days after boosting, the plasmid vector containing cDNA for the sTNFR:Fc was injected into the gastrocnemius muscle followed by electroporation. Serum levels of sTNFR:Fc reached 2.3 ng/ml on day 5 when gene expression reached its peak. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate to severe CIA in mice treated with sTNFR:Fc was significantly prevented on a significant level compared with the control mice (P < 0.05). The beneficial effect of sTNFR:Fc DNA transfer lasted for at least 18 days following treatment. In addition, both the synovitis and the erosion of cartilage in the knee joints were dramatically reduced in mice treated with sTNFR:Fc (P < 0.05). The expression of IL-1b and IL-12 in the paw was also decreased by sTNFR:Fc treatment (P < 0.01) while there was little change in the levels of IL-17 and vWF. These data showed that sTNFR:Fc expression plasmid was effective in the prevention of CIA, and in vivo electroporation-mediated gene transfer may provide a new approach to cytokine therapy in autoimmune arthritis.
S398
1033. Cell-Mediated Gene Therapy for Ligament and Tendon Repair Gadi Pelled,1 Andrea Hoffmann,2 Peter Eberle,2 Yoram Zilberman,1 Gadi Turgeman,1 Zulma Gazit,1 Dan Gazit.1 1 Skeletal Biotechnology Laboratory, Hebrew UniversityHadassah Medical Center, Jerusalem, Israel; 2OsteoAngiogenesis Group, GBF, Braunschweig, Germany. Current reconstructive techniques to repair torn ligaments and tendons consist of autografts, allografts or synthetic prosthesis. Unfortunately, none of these surgical alternatives provide a longterm adequate solution. Adult mesenchymal stem cells (MSCs) are capable of differentiating into several lineages including ligament and tendon. So far it has been shown that the SMAD signaling pathway plays a major role in the differentiation process of MSCs to bone and cartilage. Therefore, we hypothesized that ligament/ tendon tissue could be formed using genetically engineered Mesenchymal Stem Cells over expressing a SMAD signaling molecule. Murine SMAD5 was cloned by RT-PCR from RNA isolated from the mesenchymal stem cell line C3H10T1/2, and SMAD8 was cloned from rat brain. With a similar strategy, SMAD5 and SMAD8-variants consisting of the linker and MH2 domain (L+M) were constructed. C3H10T1/2 cells were co transfected with BMP2 (C3H-BMP2) and the resulting clone was further transfected with the SMADs cDNA or their variants. The in vitro phenotype of the different clones was evaluated using RT-PCR, Histochemistry and morphology. In addition, 5x10^6 of C3H-BMP2-SMAD8L+M and C3H-SMAD8-L+M cells were injected into the sacral subcutaneous tissue and into the kidney capsule of female C3H/ HeN mice in order to assess the tissue formation in vivo. 3x10^6 C3H-BMP2-SMAD8 L+M cells, also expressing Lacz or Luciferase marker genes, were implanted on a Collagen sponge into a 3 mm partial defect of nude rats Achilles tendon. 4 weeks post implantation, the area of the injection or implantation was isolated and analyzed by light and Electron microscopy, histochemical staining and by RT-PCR. The cells survival in the implantation site was also demonstrated non-invasively in vivo by the detection of Luciferase bioluminescence with a CCCD system. Interestingly, the C3H-BMP2-SMAD8 L+M cells demonstrated a completely different morphology than the other clones. These cells had a spindle morphology not resembling the stellate structured phenotype of osteoblastic cells. In addition, they expressed the characteristic genes of ligament, SIX 1 and SIX 2. Analysis of the in vivo injected implants revealed the formation of a dense connective tissue with parallel organized fibres and spindle shaped cells, in the C3H-BMP2SMAD8 L+MH2 implants. Electron microscopic analysis showed tightly packed collagen fibres adjacent to fibroblast-shaped cells with active Endoplasmic Reticulum. We were also able to observe the repair of the operated Achilles tendon with the Lacz labelled cells. This is the first report showing that a particular SMAD signalling cascade is involved in tendon/ligament formation. Our findings may have considerable importance for ligament/tendon healing and used as a clinical gene therapy platform that would replace the current inefficient allograft or synthetic implants.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
GENE THERAPY FOR BONE, CONNECTIVE TISSUE AND SKIN TISSUES 1035. Biodistribution and Expression of AAVhuTNFR:Fc after Intra-Articular Injection to Cynomolgus Monkeys Dana B. Martin,1 Tara Allen,1 Ed Kelly,1 Ziv Sandalon,1 Linda Wilson,1 Lauren M. Tatalick,1 Carmel Lynch.1 1 Preclinical Biology, Targeted Genetics, Seattle, WA.
1034. Gene Therapy with Decorin Reduces Diabetic Organomegaly in Rats N. S. Nahman, Jr.,1 A. Bringardner,1 U. Y. Bhatt,1 T. J. Sferra,2 M. Husa,1 T. Verrill,1 J. Vaughn.1 1 Medicine/Nephrology, The Ohio State University, Columbus, OH; 2 Pediatrics, The Ohio State University, Columbus, OH. Decorin is a small glycoprotein known to inhibit the biologic effects of the pro-sclerotic cytokine, TGF-b. Glucose-driven increases in TGF-b are thought to underlie accumulation of matrix protein in diabetes, causing glomerulosclerosis and nephromegaly in the kidney, and organomegaly of the liver and heart. On this basis, we theorized that decorin may retard matrix protein synthesis and organomegaly in diabetes. To test this question in vitro, we assessed the effect of decorin on glucose-driven fibronectin (FN) synthesis in cultured human mesangial cells (HMC). HMC were transduced with an adenovirus generating the human decorin transgene (rAdDec, a generous gift of P. Lowenstein, UCLA) or a control virus (rAd), and exposed to 10 or 50 mM glucose. After 24 hrs, FN gene transcription (quantitative PCR) from cells exposed to 50 mM and treated with rAdDec, was significantly lower than cells exposed to 50mM+rAd (41±0.04 % of 50mM+rAd, mean±SEM, p<0.05). This was similar to cells treated with 10mM+rAd (31±0.04 % of 50mM+rAd p<0.05). To assess the effect of decorin in vivo, 4 SD rats were rendered diabetic with streptozotocin and after 4 weeks, transduced with 109 rAdDec particles (DM-Dec) via carotid injection, as we have described (Kidney Int 58:1500). After 10 wks of diabetes (6 wk post virus injection), the animals were sacrificed and liver, kidney and heart weights determined. When compared to 10 week diabetics (DM) from our previous work (AJKD 23:105), decorin therapy significantly reduced liver, kidney, and heart weights (table). In 4 other rats, increased hepatic expression of the decorin transgene was demonstrated at 5 wks using quantitative PCR (7.4±9.3 fold increase in DM-Dec vs DM). CONCLUSION: Systemic injection of a decorin transgene reduces the organomegaly of diabetes, presumably by antagonizing TGF-b induced matrix protein synthesis. Decorin may be useful in controlling matrix accumulation in diabetes, and thus help reduce the systemic complications of the disease. Effect of decorin on organ weight (g/100g BW) Organ DM DM-Dec Liver 5.4+/-.23 4.3+/-.29* Kidney 0.63+/-.03 0.49+/-.03* Heart 0.59+/-.03 0.35+/-.04* * p < 0.05 vs DM
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
AAV-huTNFR:Fc is an AAV2 vector carrying a soluble TNF receptor. AAV-huTNFR:Fc is for use in the treatment of TNF-αmediated inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Intra-articular treatment would allow a high local TNFR:Fc expression level while minimizing systemic levels. The biodistribution and expression of AAV-huTNFR:Fc, was investigated after a single intra-articular injection in cynomolgus monkeys. Vector was administered to the right knee joint at a dose of 2.4 X1011, 3 X1012 and 3X1013 DRP of AAV-huTNFR:Fc. Vector was administered to an additional group of 2 animals at the high dose of 3X1013 IM injection to assess systemic expression. A human TNFR:Fc specific ELISA assessed vector expression in the serum and joint-lavage fluid on days 1 (prior to dosing), 30, 60 and 90. In addition, serum samples were also analyzed for anti-AAV neutralizing antibody titer and anti-huTNFR:Fc antibody titer. Animals were euthanized on day 90 and the injection site, iliac lymph node and spleen were collected for vector biodistribution and transgene expression analysis by vector specific DNA PCR and mRNA-specific RT-PCR, respectively. As expected, all animals developed a neutralizing antibody response to AAV2 by day 30 confirming exposure to vector. On day 30 circulating TNFR:Fc protein was detected in serum and joint lavage fluid samples of both monkeys given tgAAV-huTNFR:Fc by the IM route. However, assessing expression of huTNFR:Fc at day 60 and 90 were inconclusive due to inhibition of the spike recovery in the assay. This inhibition is likely secondary to an anti-huTNFR:Fc antibody response in the non-human primate model. Sporadic huTNFR:Fc transgene expression was detected in the serum and joint lavage fluid of animals dosed by the IA route and detection may be limited by anti-huTNFR:Fc antibody production. Vector was detected in the joint at day 90 in a dose-dependent manner. In conclusion, local delivery of AAV-huTNFR:Fc may be therapeutic in RA and PsA patients with a limited number of remaining painful joints despite current systemic therapy.
1036. Platelet-Derived Growth Factor Gene Delivery Promotes Periodontal Tissue Repair Qi-ming Jin,1 Orasa Anusaksathian,1 Sarah A. Webb,1 William V. Giannobile.1 1 Center for Craniofacial Regeneration and Dept. of Periodontics, Prevention, and Geriatrics, University of Michigan, Ann Arbor, MI. Platelet-derived growth factor (PDGF) has potent effects on the wound repair including regeneration of tooth-supporting (periodontal) structures. Limitations of protein delivery to periodontal defects include transient biological activity of PDGFs and bioavailability of factors at the wound site. The objective of this investigation was to determine the ability of in vivo PDGF-B gene transfer to stimulate periodontal regeneration in large surgicallycreated periodontal alveolar bone wounds. Osseous defects were treated with a 2.6% collagen carrier or carrier containing recombinant adenoviruses encoding a control virus luciferase (Ad-luc; 5 x 108 particles (PN)/wound), or PDGF-B (Ad-PDGF-B at two dose levels (5 x 108 (low dose)/wound or 5 x 109 PN (high dose)/wound). Parameters of periodontal wound repair were measured and included bone length, bone area and length of new cementum using image analysis. The collagen carrier with or without genes was delivered to mandibular first and second molars (n=6 animals/group) in SpragueS399
1032. Electro-Gene Therapy of CollagenInduced Arthritis by Using an Expression Plasmid for the Soluble p75 Tumor Necrosis Factor Receptor-Fc Fusion Protein Woong Hahn,1 Jong-Mook Kim,1 Seoun-Hyun Ho,1 Sujeong Kim,1 Jae-Gyun Jeong,2 Hwang-Jae Lee,1 Seung Shin Yu,1 Sunyoung Kim.2 1 ViroMed Co., Ltd., Seoul, Korea; 2School of Biological Science, Seoul National University, Seoul, Korea. Tumor necrosis factor (TNF) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, and antagonism of TNF may reduce the activity of the disease. Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. In this study, we attempted to treat collagen-induced arthritis (CIA) with anti-TNF gene therapy by transferring the plasmid encoding soluble p75 TNF receptor linked to the Fc portion of human IgG1 (sTNFR:Fc) using in vivo electroporation. DBA/1 mice were immunized with bovine type II collagen and boosted with the same antigen. Two days after boosting, the plasmid vector containing cDNA for the sTNFR:Fc was injected into the gastrocnemius muscle followed by electroporation. Serum levels of sTNFR:Fc reached 2.3 ng/ml on day 5 when gene expression reached its peak. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate to severe CIA in mice treated with sTNFR:Fc was significantly prevented on a significant level compared with the control mice (P < 0.05). The beneficial effect of sTNFR:Fc DNA transfer lasted for at least 18 days following treatment. In addition, both the synovitis and the erosion of cartilage in the knee joints were dramatically reduced in mice treated with sTNFR:Fc (P < 0.05). The expression of IL-1b and IL-12 in the paw was also decreased by sTNFR:Fc treatment (P < 0.01) while there was little change in the levels of IL-17 and vWF. These data showed that sTNFR:Fc expression plasmid was effective in the prevention of CIA, and in vivo electroporation-mediated gene transfer may provide a new approach to cytokine therapy in autoimmune arthritis.
S398
1033. Cell-Mediated Gene Therapy for Ligament and Tendon Repair Gadi Pelled,1 Andrea Hoffmann,2 Peter Eberle,2 Yoram Zilberman,1 Gadi Turgeman,1 Zulma Gazit,1 Dan Gazit.1 1 Skeletal Biotechnology Laboratory, Hebrew UniversityHadassah Medical Center, Jerusalem, Israel; 2OsteoAngiogenesis Group, GBF, Braunschweig, Germany. Current reconstructive techniques to repair torn ligaments and tendons consist of autografts, allografts or synthetic prosthesis. Unfortunately, none of these surgical alternatives provide a longterm adequate solution. Adult mesenchymal stem cells (MSCs) are capable of differentiating into several lineages including ligament and tendon. So far it has been shown that the SMAD signaling pathway plays a major role in the differentiation process of MSCs to bone and cartilage. Therefore, we hypothesized that ligament/ tendon tissue could be formed using genetically engineered Mesenchymal Stem Cells over expressing a SMAD signaling molecule. Murine SMAD5 was cloned by RT-PCR from RNA isolated from the mesenchymal stem cell line C3H10T1/2, and SMAD8 was cloned from rat brain. With a similar strategy, SMAD5 and SMAD8-variants consisting of the linker and MH2 domain (L+M) were constructed. C3H10T1/2 cells were co transfected with BMP2 (C3H-BMP2) and the resulting clone was further transfected with the SMADs cDNA or their variants. The in vitro phenotype of the different clones was evaluated using RT-PCR, Histochemistry and morphology. In addition, 5x10^6 of C3H-BMP2-SMAD8L+M and C3H-SMAD8-L+M cells were injected into the sacral subcutaneous tissue and into the kidney capsule of female C3H/ HeN mice in order to assess the tissue formation in vivo. 3x10^6 C3H-BMP2-SMAD8 L+M cells, also expressing Lacz or Luciferase marker genes, were implanted on a Collagen sponge into a 3 mm partial defect of nude rats Achilles tendon. 4 weeks post implantation, the area of the injection or implantation was isolated and analyzed by light and Electron microscopy, histochemical staining and by RT-PCR. The cells survival in the implantation site was also demonstrated non-invasively in vivo by the detection of Luciferase bioluminescence with a CCCD system. Interestingly, the C3H-BMP2-SMAD8 L+M cells demonstrated a completely different morphology than the other clones. These cells had a spindle morphology not resembling the stellate structured phenotype of osteoblastic cells. In addition, they expressed the characteristic genes of ligament, SIX 1 and SIX 2. Analysis of the in vivo injected implants revealed the formation of a dense connective tissue with parallel organized fibres and spindle shaped cells, in the C3H-BMP2SMAD8 L+MH2 implants. Electron microscopic analysis showed tightly packed collagen fibres adjacent to fibroblast-shaped cells with active Endoplasmic Reticulum. We were also able to observe the repair of the operated Achilles tendon with the Lacz labelled cells. This is the first report showing that a particular SMAD signalling cascade is involved in tendon/ligament formation. Our findings may have considerable importance for ligament/tendon healing and used as a clinical gene therapy platform that would replace the current inefficient allograft or synthetic implants.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
GENE THERAPY FOR BONE, CONNECTIVE TISSUE AND SKIN TISSUES 1035. Biodistribution and Expression of AAVhuTNFR:Fc after Intra-Articular Injection to Cynomolgus Monkeys Dana B. Martin,1 Tara Allen,1 Ed Kelly,1 Ziv Sandalon,1 Linda Wilson,1 Lauren M. Tatalick,1 Carmel Lynch.1 1 Preclinical Biology, Targeted Genetics, Seattle, WA.
1034. Gene Therapy with Decorin Reduces Diabetic Organomegaly in Rats N. S. Nahman, Jr.,1 A. Bringardner,1 U. Y. Bhatt,1 T. J. Sferra,2 M. Husa,1 T. Verrill,1 J. Vaughn.1 1 Medicine/Nephrology, The Ohio State University, Columbus, OH; 2 Pediatrics, The Ohio State University, Columbus, OH. Decorin is a small glycoprotein known to inhibit the biologic effects of the pro-sclerotic cytokine, TGF-b. Glucose-driven increases in TGF-b are thought to underlie accumulation of matrix protein in diabetes, causing glomerulosclerosis and nephromegaly in the kidney, and organomegaly of the liver and heart. On this basis, we theorized that decorin may retard matrix protein synthesis and organomegaly in diabetes. To test this question in vitro, we assessed the effect of decorin on glucose-driven fibronectin (FN) synthesis in cultured human mesangial cells (HMC). HMC were transduced with an adenovirus generating the human decorin transgene (rAdDec, a generous gift of P. Lowenstein, UCLA) or a control virus (rAd), and exposed to 10 or 50 mM glucose. After 24 hrs, FN gene transcription (quantitative PCR) from cells exposed to 50 mM and treated with rAdDec, was significantly lower than cells exposed to 50mM+rAd (41±0.04 % of 50mM+rAd, mean±SEM, p<0.05). This was similar to cells treated with 10mM+rAd (31±0.04 % of 50mM+rAd p<0.05). To assess the effect of decorin in vivo, 4 SD rats were rendered diabetic with streptozotocin and after 4 weeks, transduced with 109 rAdDec particles (DM-Dec) via carotid injection, as we have described (Kidney Int 58:1500). After 10 wks of diabetes (6 wk post virus injection), the animals were sacrificed and liver, kidney and heart weights determined. When compared to 10 week diabetics (DM) from our previous work (AJKD 23:105), decorin therapy significantly reduced liver, kidney, and heart weights (table). In 4 other rats, increased hepatic expression of the decorin transgene was demonstrated at 5 wks using quantitative PCR (7.4±9.3 fold increase in DM-Dec vs DM). CONCLUSION: Systemic injection of a decorin transgene reduces the organomegaly of diabetes, presumably by antagonizing TGF-b induced matrix protein synthesis. Decorin may be useful in controlling matrix accumulation in diabetes, and thus help reduce the systemic complications of the disease. Effect of decorin on organ weight (g/100g BW) Organ DM DM-Dec Liver 5.4+/-.23 4.3+/-.29* Kidney 0.63+/-.03 0.49+/-.03* Heart 0.59+/-.03 0.35+/-.04* * p < 0.05 vs DM
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
AAV-huTNFR:Fc is an AAV2 vector carrying a soluble TNF receptor. AAV-huTNFR:Fc is for use in the treatment of TNF-αmediated inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Intra-articular treatment would allow a high local TNFR:Fc expression level while minimizing systemic levels. The biodistribution and expression of AAV-huTNFR:Fc, was investigated after a single intra-articular injection in cynomolgus monkeys. Vector was administered to the right knee joint at a dose of 2.4 X1011, 3 X1012 and 3X1013 DRP of AAV-huTNFR:Fc. Vector was administered to an additional group of 2 animals at the high dose of 3X1013 IM injection to assess systemic expression. A human TNFR:Fc specific ELISA assessed vector expression in the serum and joint-lavage fluid on days 1 (prior to dosing), 30, 60 and 90. In addition, serum samples were also analyzed for anti-AAV neutralizing antibody titer and anti-huTNFR:Fc antibody titer. Animals were euthanized on day 90 and the injection site, iliac lymph node and spleen were collected for vector biodistribution and transgene expression analysis by vector specific DNA PCR and mRNA-specific RT-PCR, respectively. As expected, all animals developed a neutralizing antibody response to AAV2 by day 30 confirming exposure to vector. On day 30 circulating TNFR:Fc protein was detected in serum and joint lavage fluid samples of both monkeys given tgAAV-huTNFR:Fc by the IM route. However, assessing expression of huTNFR:Fc at day 60 and 90 were inconclusive due to inhibition of the spike recovery in the assay. This inhibition is likely secondary to an anti-huTNFR:Fc antibody response in the non-human primate model. Sporadic huTNFR:Fc transgene expression was detected in the serum and joint lavage fluid of animals dosed by the IA route and detection may be limited by anti-huTNFR:Fc antibody production. Vector was detected in the joint at day 90 in a dose-dependent manner. In conclusion, local delivery of AAV-huTNFR:Fc may be therapeutic in RA and PsA patients with a limited number of remaining painful joints despite current systemic therapy.
1036. Platelet-Derived Growth Factor Gene Delivery Promotes Periodontal Tissue Repair Qi-ming Jin,1 Orasa Anusaksathian,1 Sarah A. Webb,1 William V. Giannobile.1 1 Center for Craniofacial Regeneration and Dept. of Periodontics, Prevention, and Geriatrics, University of Michigan, Ann Arbor, MI. Platelet-derived growth factor (PDGF) has potent effects on the wound repair including regeneration of tooth-supporting (periodontal) structures. Limitations of protein delivery to periodontal defects include transient biological activity of PDGFs and bioavailability of factors at the wound site. The objective of this investigation was to determine the ability of in vivo PDGF-B gene transfer to stimulate periodontal regeneration in large surgicallycreated periodontal alveolar bone wounds. Osseous defects were treated with a 2.6% collagen carrier or carrier containing recombinant adenoviruses encoding a control virus luciferase (Ad-luc; 5 x 108 particles (PN)/wound), or PDGF-B (Ad-PDGF-B at two dose levels (5 x 108 (low dose)/wound or 5 x 109 PN (high dose)/wound). Parameters of periodontal wound repair were measured and included bone length, bone area and length of new cementum using image analysis. The collagen carrier with or without genes was delivered to mandibular first and second molars (n=6 animals/group) in SpragueS399