Gene therapy strategy for classical Osteogenesis Imperfecta due to mutations in COL1A2 gene

Osteogenesis Imperfecta (OI), better known as brittle bone disease, is a rare genetic skeletal disorder that occurs in about 1 in 15’000-20’000 live births1. The major clinical manifestations of OI are liability to fractures throughout life, bone deformities, and growth retardation, and the disease severity can span from subclinical osteoporosis to intrauterine lethality. Classical forms of OI are caused by dominant mutations in collagen type I genes COL1A1 and COL1A2, coding for collagen α1 and α2 chains respectively, mainly leading to Glycine substitutions. This type of mutation leads to the synthesis of abnormal collagen molecules, resulting in qualitative defects often associated to severe outcomes, OI type II, III and IV. Nowadays no definitive cure is available for OI patients and mainly palliative treatments aiming to ameliorate the patients quality of life are available2,3. Since OI is a genetic disorder, only a gene therapy approach aimed to correct or suppress the expression of the mutant allele could be effective to cure patients. The main aim of this study was to develop a novel gene therapy approach for the treatment of classical OI with mutations in the COL1A2 gene. In humans null mutations in both COL1A2 alleles lead to a mild form of Ehler-Danlos Syndrome (EDS), a vascular disease without bone phenotype4. This finding suggested that a bone specific COL1A2 suppression will not be associated with a bone phenotype. To this purpose, we developed a siRNA gene therapy approach to suppress only in bone both Col1a2 alleles in the Amish/+ murine model of OI, carrying a G610C mutation in the Col1a2 gene, in order to convert a severe phenotype characterized by collagen structural defects to a mild OI due to collagen insufficiency. The second aim of this study was the characterization of the bone healing process in the Amish/+ mouse model of OI since OI bones are susceptible to fractures and the management of multiple fractures is one of the main problem affecting OI patients and very limited information are available on this process.Osteogenesis Imperfecta (OI), better known as brittle bone disease, is a rare genetic skeletal disorder that occurs in about 1 in 15’000-20’000 live births1. The major clinical manifestations of OI are liability to fractures throughout life, bone deformities, and growth retardation, and the disease severity can span from subclinical osteoporosis to intrauterine lethality. Classical forms of OI are caused by dominant mutations in collagen type I genes COL1A1 and COL1A2, coding for collagen α1 and α2 chains respectively, mainly leading to Glycine substitutions. This type of mutation leads to the synthesis of abnormal collagen molecules, resulting in qualitative defects often associated to severe outcomes, OI type II, III and IV. Nowadays no definitive cure is available for OI patients and mainly palliative treatments aiming to ameliorate the patients quality of life are available2,3. Since OI is a genetic disorder, only a gene therapy approach aimed to correct or suppress the expression of the mutant allele could be effective to cure patients. The main aim of this study was to develop a novel gene therapy approach for the treatment of classical OI with mutations in the COL1A2 gene. In humans null mutations in both COL1A2 alleles lead to a mild form of Ehler-Danlos Syndrome (EDS), a vascular disease without bone phenotype4. This finding suggested that a bone specific COL1A2 suppression will not be associated with a bone phenotype. To this purpose, we developed a siRNA gene therapy approach to suppress only in bone both Col1a2 alleles in the Amish/+ murine model of OI, carrying a G610C mutation in the Col1a2 gene, in order to convert a severe phenotype characterized by collagen structural defects to a mild OI due to collagen insufficiency. The second aim of this study was the characterization of the bone healing process in the Amish/+ mouse model of OI since OI bones are susceptible to fractures and the management of multiple fractures is one of the main problem affecting OI patients and very limited information are available on this process

Osteoblasts mineralization and collagen matrix are conserved upon specific Col1a2 silencing

International audienceClassical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), −3554, −3825 and −4125, selected at the 3′-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy

Early Fracture Healing is Delayed in the Col1a2 +/G610C Osteogenesis Imperfecta Murine Model

Osteogenesis imperfecta (OI) is a rare heritable skeletal dysplasia mainly caused by type I collagen abnormalities and characterized by bone fragility and susceptibility to fracture. Over 85% of the patients carry dominant mutations in the genes encoding for the collagen type I α1 and α2 chains. Failure of bone union and/or presence of hyperplastic callus formation after fracture were described in OI patients. Here we used the Col1a2+/G610C mouse, carrying in heterozygosis the α2(I)-G610C substitution, to investigate the healing process of an OI bone. Tibiae of 2-month-old Col1a2+/G610C and wild-type littermates were fractured and the healing process was followed at 2, 3, and 5 weeks after injury from fibrous cartilaginous tissue formation to its bone replacement by radiography, micro-computed tomography (µCT), histological and biochemical approaches. In presence of similar fracture types, in Col1a2+/G610C mice an impairment in the early phase of bone repair was detected compared to wild-type littermates. Smaller callus area, callus bone surface, and bone volume associated to higher percentage of cartilage and lower percentage of bone were evident in Col1a2+/G610C at 2 weeks post fracture (wpf) and no change by 3 wpf. Furthermore, the biochemical analysis of collagen extracted from callus 2 wpf revealed in mutants an increased amount of type II collagen, typical of cartilage, with respect to type I, characteristic of bone. This is the first report of a delay in OI bone fracture repair at the modeling phase

Osteoblasts mineralization and collagen matrix are conserved upon specific Col1a2 silencing

Abstract Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), −3554, −3825 and −4125, selected at the 3′-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy

Altered cytoskeletal organization characterized lethal but not surviving Brtl+/- mice: insight on phenotypic variability in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-β signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication