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Polymorphic Variation of the Comt Gene Influences Outcome in Patients Undergoing Surgical Treatment for Lumbar Degenerative Disc Disease
66S
Proceedings of the NASS 23rd Annual Meeting / The Spine Journal 8 (2008) 1S–191S
Friday, October 17, 2008 10:30–10:55 a.m. NASS Research Awards Molecular Therapy for Intervertebral Disc Degeneration with Growth and Differentiation Factor-5 Xudong Joshua Li, MD, PhD, Gary Balian, PhD; University of Virginia, Charlottesville, VA, USA BACKGROUND CONTEXT: Musculoskeletal disorders of the spine and low back pain are the leading source of disability in people under 45 years of age and result in national economic losses of over 90 billion dollars per year. Current treatment options, including both conservative measures such as bed rest, anti-inflammatories, analgesia, and physical therapy as well as surgical measures, target the clinical symptoms of IVD disease rather than addressing the early pathologic processes occurring in the course of degeneration. However, recent advancements in molecular biology including gene cloning and gene transfer technology have made it possible to contemplate treating the IVD at the molecular level to prevent or delay the progression of disc degeneration. PURPOSE: The aim of the present study was to investigate the effect of recombinant GDF-5 protein and GDF-5 cDNA on the metabolism of intervertebral disc cells in vitro and in vivo. METHODS: Mouse disc cells in vitro were treated with recombinant GDF-5 protein. Mouse GDF-5 cDNA was cloned into an expression vector and was used to transfect mouse disc cells in vitro. Therapy with Ad-GDF5, GDF-5 protein and cDNA was assessed by measuring cell proliferation, proteoglycan production, and extracellular matrix gene expression in vitro and evaluating histological changes, immunostaining and extracellular matrix production in a needle puncture-induced disc degeneration model. RESULTS: Biochemical assays revealed an elevated GAG/DNA ratio in mouse IVD cells that were cultured in the presence of various concentrations of mGDF-5 protein. Real-time RT-PCR demonstrated that treating the cells with GDF-5 protein increased the expression of the collagen type II and aggrecan genes in a dose dependent manner while MMP-3 gene expression decreased. Immunohistochemistry showed an increase in the aggregation of mouse IVD cells that were treated with mGDF-5 in culture compared to the control group. The mouse GDF-5 gene was successfully cloned into an expression plasmid vector and GDF-5 protein production was confirmed by Western blot analysis. Type II collagen and aggrecan gene expression by the cells increased significantly in the cells that were transfected by Nucleofection with the GDF-5 plasmid compared with cells that were transfected with a control plasmid. The production of exogenous genes could express and keep on certain strength at least 5 weeks after the injections. Quantitative measurements of %DHI and MRI image showed the Ad-GDF5 injection improves the water content and the restoration of disc height. The histology study demonstrated the chondroid cells proliferated from the annulus fibrosus (AF). In Ad-GF5 groups, the extensive and intense of cells proliferation are obviously stronger than Ad-Luc groups. The immunostaining showed more collagen II expressed in Ad-GDF5 groups. The biochemical analysis indicated the cell number of the IVD in Ad-Luc groups reduced since 4 weeks post operation and didn’t recover throughout the study. CONCLUSIONS: This is the first report to clone the mouse GDF-5 gene and use the Nucleofection method to transfer DNA into IVD cells. The data suggests that both recombinant protein and the cDNA forms of GDF-5 can increase the expression of genes for extracellular matrix proteins in mouse IVD cells. Compare with the GDF-5 protein injection, Ad-GDF5 could provide a stable source of GDF-5 and prolong the time of keeping an effective concentration of GDF-5 in the specific location. Our research confirmed the therapeutic effect of Ad-GDF5 for intervertebral disc degeneration in vivo. Future attempts at gene therapy to treat degenerative disc disease with a novel ex vivo gene transfer technique are needed to develop a therapy that would alleviate the condition of patients with clinically relevant axial spine pain.
FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. doi:10.1016/j.spinee.2008.06.154
Polymorphic Variation of the Comt Gene Influences Outcome in Patients Undergoing Surgical Treatment for Lumbar Degenerative Disc Disease David H. Kim1,2, Robert Banco1,2, Louis Jenis1,2, Scott Tromanhauser1,2, Julia Martha1, Mitchell Max3, Inga Peter2, Inna Belfer3, Carolyn Schwartz1,2; 1New England Baptist Hospital; 2Tufts University Medical School, Boston, MA; 3National Institutes of Health, Bethesda, MD BACKGROUND: The experience of pain appears to be genetically determined. The catecholamine-O-methyltransferase gene (COMT) codes for a critical enzyme in catecholamine metabolism and modulates dopamine, epinephrine, and norepinephrine-mediated neurotransmission as well as m-opioid system responses. COMT demonstrates a common functional genetic polymorphism (val158met) that, in human studies, appears to affect the response to sustained pain. METHODS: Prospective genetic analysis of 100 patients undergoing surgical treatment for lumbar DDD based on clinical evaluation and MRI. For each patient, genomic DNA was extracted from a blood sample and DNA sequence analysis was performed with respect to 5 single nucleotide polymorphisms (SNPs) in non-coding regions potentially associated with pain response. Clinical outcomes at one year were compared with preoperative baseline measures and the results were analyzed with respect to COMT genotype for each SNP. RESULTS: Clinical outcomes at one year varied significantly in relation to COMT haplotype. 2 loci, rs6269 and rs4633, both in strong linkage disequilibrium, were significantly predictive of postoperative improvement based on percentage change of Oswestry Disability Index scores (p-values of 0.040 and 0.039 respectively). The mean percentage improvement in ODI comparing homozygous carriers of each allele differed by approximately 100%. CONCLUSIONS: This study demonstrates that common genotypic variations of COMT predict outcome following surgical treatment of lumbar DDD. This is the first prospective study demonstrating a genetic predictor of outcome following spinal surgery and suggests that assessment of allelic variations in COMT may allow improved counseling with respect to anticipated results and possibly an overall improvement in surgical success rates. doi:10.1016/j.spinee.2008.06.155
Mesenchymal Stem Cell Injections for Treatment of Intervertebral Disc Degeneration James D. Kang, MD, Lars G. Gilbertson, PhD, Gianluca Vadala, MD, James III Larson, MD; Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA ABSTRACT: The degeneration of intervertebral discs (IVD) causes a great deal of morbidity, loss of work, and medical expense each year. Ongoing research is directed toward treatment modalities which slow the catabolic process in the degenerating disc with a focus on gene therapy and molecular therapy. While these avenues do have promise, they are plagued with problems including difficulty with persistence of growth factor production and the immunogenic and oncogenic properties of viral vectors. Mesenchymal stem cells (MSCs) on the other hand have a high capacity for adaptation and persistent survival in many tissues and are associated with none of the problems of viral-mediated gene therapy. In addition,
Proceedings of the NASS 23rd Annual Meeting / The Spine Journal 8 (2008) 1S–191S
Friday, October 17, 2008 10:30–10:55 a.m. NASS Research Awards Molecular Therapy for Intervertebral Disc Degeneration with Growth and Differentiation Factor-5 Xudong Joshua Li, MD, PhD, Gary Balian, PhD; University of Virginia, Charlottesville, VA, USA BACKGROUND CONTEXT: Musculoskeletal disorders of the spine and low back pain are the leading source of disability in people under 45 years of age and result in national economic losses of over 90 billion dollars per year. Current treatment options, including both conservative measures such as bed rest, anti-inflammatories, analgesia, and physical therapy as well as surgical measures, target the clinical symptoms of IVD disease rather than addressing the early pathologic processes occurring in the course of degeneration. However, recent advancements in molecular biology including gene cloning and gene transfer technology have made it possible to contemplate treating the IVD at the molecular level to prevent or delay the progression of disc degeneration. PURPOSE: The aim of the present study was to investigate the effect of recombinant GDF-5 protein and GDF-5 cDNA on the metabolism of intervertebral disc cells in vitro and in vivo. METHODS: Mouse disc cells in vitro were treated with recombinant GDF-5 protein. Mouse GDF-5 cDNA was cloned into an expression vector and was used to transfect mouse disc cells in vitro. Therapy with Ad-GDF5, GDF-5 protein and cDNA was assessed by measuring cell proliferation, proteoglycan production, and extracellular matrix gene expression in vitro and evaluating histological changes, immunostaining and extracellular matrix production in a needle puncture-induced disc degeneration model. RESULTS: Biochemical assays revealed an elevated GAG/DNA ratio in mouse IVD cells that were cultured in the presence of various concentrations of mGDF-5 protein. Real-time RT-PCR demonstrated that treating the cells with GDF-5 protein increased the expression of the collagen type II and aggrecan genes in a dose dependent manner while MMP-3 gene expression decreased. Immunohistochemistry showed an increase in the aggregation of mouse IVD cells that were treated with mGDF-5 in culture compared to the control group. The mouse GDF-5 gene was successfully cloned into an expression plasmid vector and GDF-5 protein production was confirmed by Western blot analysis. Type II collagen and aggrecan gene expression by the cells increased significantly in the cells that were transfected by Nucleofection with the GDF-5 plasmid compared with cells that were transfected with a control plasmid. The production of exogenous genes could express and keep on certain strength at least 5 weeks after the injections. Quantitative measurements of %DHI and MRI image showed the Ad-GDF5 injection improves the water content and the restoration of disc height. The histology study demonstrated the chondroid cells proliferated from the annulus fibrosus (AF). In Ad-GF5 groups, the extensive and intense of cells proliferation are obviously stronger than Ad-Luc groups. The immunostaining showed more collagen II expressed in Ad-GDF5 groups. The biochemical analysis indicated the cell number of the IVD in Ad-Luc groups reduced since 4 weeks post operation and didn’t recover throughout the study. CONCLUSIONS: This is the first report to clone the mouse GDF-5 gene and use the Nucleofection method to transfer DNA into IVD cells. The data suggests that both recombinant protein and the cDNA forms of GDF-5 can increase the expression of genes for extracellular matrix proteins in mouse IVD cells. Compare with the GDF-5 protein injection, Ad-GDF5 could provide a stable source of GDF-5 and prolong the time of keeping an effective concentration of GDF-5 in the specific location. Our research confirmed the therapeutic effect of Ad-GDF5 for intervertebral disc degeneration in vivo. Future attempts at gene therapy to treat degenerative disc disease with a novel ex vivo gene transfer technique are needed to develop a therapy that would alleviate the condition of patients with clinically relevant axial spine pain.
FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. doi:10.1016/j.spinee.2008.06.154
Polymorphic Variation of the Comt Gene Influences Outcome in Patients Undergoing Surgical Treatment for Lumbar Degenerative Disc Disease David H. Kim1,2, Robert Banco1,2, Louis Jenis1,2, Scott Tromanhauser1,2, Julia Martha1, Mitchell Max3, Inga Peter2, Inna Belfer3, Carolyn Schwartz1,2; 1New England Baptist Hospital; 2Tufts University Medical School, Boston, MA; 3National Institutes of Health, Bethesda, MD BACKGROUND: The experience of pain appears to be genetically determined. The catecholamine-O-methyltransferase gene (COMT) codes for a critical enzyme in catecholamine metabolism and modulates dopamine, epinephrine, and norepinephrine-mediated neurotransmission as well as m-opioid system responses. COMT demonstrates a common functional genetic polymorphism (val158met) that, in human studies, appears to affect the response to sustained pain. METHODS: Prospective genetic analysis of 100 patients undergoing surgical treatment for lumbar DDD based on clinical evaluation and MRI. For each patient, genomic DNA was extracted from a blood sample and DNA sequence analysis was performed with respect to 5 single nucleotide polymorphisms (SNPs) in non-coding regions potentially associated with pain response. Clinical outcomes at one year were compared with preoperative baseline measures and the results were analyzed with respect to COMT genotype for each SNP. RESULTS: Clinical outcomes at one year varied significantly in relation to COMT haplotype. 2 loci, rs6269 and rs4633, both in strong linkage disequilibrium, were significantly predictive of postoperative improvement based on percentage change of Oswestry Disability Index scores (p-values of 0.040 and 0.039 respectively). The mean percentage improvement in ODI comparing homozygous carriers of each allele differed by approximately 100%. CONCLUSIONS: This study demonstrates that common genotypic variations of COMT predict outcome following surgical treatment of lumbar DDD. This is the first prospective study demonstrating a genetic predictor of outcome following spinal surgery and suggests that assessment of allelic variations in COMT may allow improved counseling with respect to anticipated results and possibly an overall improvement in surgical success rates. doi:10.1016/j.spinee.2008.06.155
Mesenchymal Stem Cell Injections for Treatment of Intervertebral Disc Degeneration James D. Kang, MD, Lars G. Gilbertson, PhD, Gianluca Vadala, MD, James III Larson, MD; Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA ABSTRACT: The degeneration of intervertebral discs (IVD) causes a great deal of morbidity, loss of work, and medical expense each year. Ongoing research is directed toward treatment modalities which slow the catabolic process in the degenerating disc with a focus on gene therapy and molecular therapy. While these avenues do have promise, they are plagued with problems including difficulty with persistence of growth factor production and the immunogenic and oncogenic properties of viral vectors. Mesenchymal stem cells (MSCs) on the other hand have a high capacity for adaptation and persistent survival in many tissues and are associated with none of the problems of viral-mediated gene therapy. In addition,