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Ribozyme suppression of mutant type I collagen mRNA in cultured OI fibroblasts: Development of gene therapy for dominant disorders
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Abstracts
Skeleto-Hematopoietic Defects in Collagen X Transgenic Mice Z. Tao, C. Healy and O. Jacenko University of Pennsylvania Veterinary School, Philadelphia PA Variable, skeleto-hematopoietic defects were previously described in nqice transgenic for collagen X. Specifically, by 3 weeks after birth, mice developed growth plate compressions, trabecular reductions, osteopenia, marrow aplasia, lymphatic organ atrophy, and suppressed immunity. Analysis of the subset of mice with perinatal lethality revealed the most severe skeletal defects coupled with a depletion of marrow hematopoiesis. Histology, immunohistochemistry, and flow cytometry revealed thymic reduction with a paucity of cortical CD4+ and CDS+ immature T cells, and a decrease in apoptosis, consistent with the marrow~;s inability to replenish maturing cortical lymphocytes. Spleens ~vere small, discolored, had poorly organized lymphatic nodules, but an unaltered CD3/CD4/CDS+ T cell distribution. B220 staining revealed a ~50% depletion of B cells, but the existing B cells were functional and produced lgD/lgM. Altered splenic architecture was confirmed by disorderly MOMA-1 staining of macrophages surrounding the periarteriolar lymphatic sheath, and diffuse Ter119 staining of erythrocytes in the depleted red pulp; the latter correlated with erythrocyte filled vascular sinusoids in marrows. Our data suggest that endochondral sketetogenesis may contribute to the marrow stroma environment, and implicate the disruption of collagen X function in the observed skeleto-hematopoietic defects. (NIH AR43362, Arthritis Biomedical Grant, Univ. Penn. Research Foundation to OIL
type I collagen mRNA in vitro and in cultured cells derived from a patient with OI. Ribozynqes were designed to target a portion of the al(I) collagen mRNA which surrounds a naturally occurring human mutation (907G->U). This single base substitution generates a GUA cleavage site in the mutant RNA transcript and eliminates a BstN I restriction site. In an in vitro assay, ribozymes cleaved mutant RNA targets whereas normal RNA targets were left intact. For the in cellulo experiments, both active and inactive ribozyme templates were cloned into the pCl.neo and pHbAPr-l-neo vectors, under the control of the CMV and ~-actin promoters, respectively. Primary skin fibroblasts were stably transfected with the vectors using cationic-liposomes. RTPCR analysis confirmed the presence of ribozymes in the stably transfected cells up to at least several cell passages. Quantitative competitive RT-PCR analyses of 0~1(I) collagen normalized to [~-actin expression levels revealed that the level of mutant mRNA was decreased (approx. 50%) in the cells containing active ribozyme in comparison to untransfected cells. In the cells harboring inactive ribozymes, both normal and mutant mRNAs ,vere suppressed to 90% of their level in untransfected cells, indicating that the binding arms of the ribozyme have a minimal antisense effect. Individual clones were examined for the effect of active ribozyme on mutant cd(I) mRNA; the level of ribozyme expression correlated with the level of mutant mRNA suppression. These preliminary findings suggest that hmnmerhead ribozymes can be used for the allele-specific suppression of mutant collagen in cellulo.
Growth Plate Compressions and Immunopathology in Collagen X Null Mice C. Healy, B. De Crombrugghe ~and O. Jacenko
Ribozyme Suppression of Mutant Type I Collagen mRNA in Cultured OI Fibroblasts: Development of Gene Therapy for Dominant Disorders
University of Pennsylvania Veterinary School, Philadelphia PA and ~University of Texas M.D. Anderson Cancer Center, Houston TX
P. A. Dawson and J. C. Marini
A variable skeleto-hematopoietic phenotype was observed in collagen X null mice, and mirrored the defects previously described for a subset of mice transgenic (Tg) for collagen X. Specifically, perinatal lethality at 3 weeks was seen in -13% of null mutants, and in another subset by 12 weeks. In these mutants, growth plates were compressed, trabecular bone reduced, and hematopoietic aplasia and erythrocyte filled vascular sinusoids appar-
Heritable Disorders Branch, NICHD, Bethesda, MD Ribozymes represent a potential approach to the gene therapy of dominant genetic disorders, such as osteogenesis imperfecta (OIL We report here the preliminary analysis of hammerhead ribozymes targeted to mutant
Abstracts
Skeleto-Hematopoietic Defects in Collagen X Transgenic Mice Z. Tao, C. Healy and O. Jacenko University of Pennsylvania Veterinary School, Philadelphia PA Variable, skeleto-hematopoietic defects were previously described in nqice transgenic for collagen X. Specifically, by 3 weeks after birth, mice developed growth plate compressions, trabecular reductions, osteopenia, marrow aplasia, lymphatic organ atrophy, and suppressed immunity. Analysis of the subset of mice with perinatal lethality revealed the most severe skeletal defects coupled with a depletion of marrow hematopoiesis. Histology, immunohistochemistry, and flow cytometry revealed thymic reduction with a paucity of cortical CD4+ and CDS+ immature T cells, and a decrease in apoptosis, consistent with the marrow~;s inability to replenish maturing cortical lymphocytes. Spleens ~vere small, discolored, had poorly organized lymphatic nodules, but an unaltered CD3/CD4/CDS+ T cell distribution. B220 staining revealed a ~50% depletion of B cells, but the existing B cells were functional and produced lgD/lgM. Altered splenic architecture was confirmed by disorderly MOMA-1 staining of macrophages surrounding the periarteriolar lymphatic sheath, and diffuse Ter119 staining of erythrocytes in the depleted red pulp; the latter correlated with erythrocyte filled vascular sinusoids in marrows. Our data suggest that endochondral sketetogenesis may contribute to the marrow stroma environment, and implicate the disruption of collagen X function in the observed skeleto-hematopoietic defects. (NIH AR43362, Arthritis Biomedical Grant, Univ. Penn. Research Foundation to OIL
type I collagen mRNA in vitro and in cultured cells derived from a patient with OI. Ribozynqes were designed to target a portion of the al(I) collagen mRNA which surrounds a naturally occurring human mutation (907G->U). This single base substitution generates a GUA cleavage site in the mutant RNA transcript and eliminates a BstN I restriction site. In an in vitro assay, ribozymes cleaved mutant RNA targets whereas normal RNA targets were left intact. For the in cellulo experiments, both active and inactive ribozyme templates were cloned into the pCl.neo and pHbAPr-l-neo vectors, under the control of the CMV and ~-actin promoters, respectively. Primary skin fibroblasts were stably transfected with the vectors using cationic-liposomes. RTPCR analysis confirmed the presence of ribozymes in the stably transfected cells up to at least several cell passages. Quantitative competitive RT-PCR analyses of 0~1(I) collagen normalized to [~-actin expression levels revealed that the level of mutant mRNA was decreased (approx. 50%) in the cells containing active ribozyme in comparison to untransfected cells. In the cells harboring inactive ribozymes, both normal and mutant mRNAs ,vere suppressed to 90% of their level in untransfected cells, indicating that the binding arms of the ribozyme have a minimal antisense effect. Individual clones were examined for the effect of active ribozyme on mutant cd(I) mRNA; the level of ribozyme expression correlated with the level of mutant mRNA suppression. These preliminary findings suggest that hmnmerhead ribozymes can be used for the allele-specific suppression of mutant collagen in cellulo.
Growth Plate Compressions and Immunopathology in Collagen X Null Mice C. Healy, B. De Crombrugghe ~and O. Jacenko
Ribozyme Suppression of Mutant Type I Collagen mRNA in Cultured OI Fibroblasts: Development of Gene Therapy for Dominant Disorders
University of Pennsylvania Veterinary School, Philadelphia PA and ~University of Texas M.D. Anderson Cancer Center, Houston TX
P. A. Dawson and J. C. Marini
A variable skeleto-hematopoietic phenotype was observed in collagen X null mice, and mirrored the defects previously described for a subset of mice transgenic (Tg) for collagen X. Specifically, perinatal lethality at 3 weeks was seen in -13% of null mutants, and in another subset by 12 weeks. In these mutants, growth plates were compressed, trabecular bone reduced, and hematopoietic aplasia and erythrocyte filled vascular sinusoids appar-
Heritable Disorders Branch, NICHD, Bethesda, MD Ribozymes represent a potential approach to the gene therapy of dominant genetic disorders, such as osteogenesis imperfecta (OIL We report here the preliminary analysis of hammerhead ribozymes targeted to mutant