ALBERT

Description: Background Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by non-functional osteoclasts and a fatal outcome early in childhood. About 50% of patients have mutations in the TCIRG1 gene. Methods IMO iPSCs were generated from a patient carrying a homozygous c.11279G〉A (IVS18+1) mutation in TCIRG1 and transduced with a lentiviral vector expressing human TCIRG1. Embryoid bodies were generated and differentiated into monocytes. Non-adherent cells were harvested and further differentiated into osteoclasts on bovine bone slices. Results Release of the bone resorption biomarker CTX-I into the media of gene-corrected osteoclasts was 5-fold higher than that of the uncorrected osteoclasts and 35% of that of control osteoclasts. Bone resorption potential was confirmed by the presence of pits on the bones cultured with gene-corrected osteoclasts, absent in the uncorrected IMO osteoclasts. Conclusions The disease phenotype was partially corrected in vitro, providing a valuable resource for therapy development for this form of severe osteopetrosis.

Description: Abstract 2058 PURPOSE: Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for the a3 subunit of a proton pump used by the osteoclast to acidify the resorption area. As a consequence of the lack of resorption, remodeling of bone is severely hampered, which results in dense and fragile bone. This, in turn, causes bone marrow failure followed by anemia and hepatosplenomegaly. The only curative treatment for IMO is HSC transplantation, but this form of therapy is associated with high mortality, especially when an HLA-identical donor is not available. IMO is thus a candidate disease for development of gene therapy because of its fatal outcome early in life if treatment with HSC transplantation is not possible. We have previously shown that the murine oc/oc disease model of osteopetrosis can be rescued by gene therapy targeting hematopoietic stem cells (Johansson et al, Blood 2007). The aim of the present study was to rescue the phenotype of human IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. METHODS AND RESULTS: CD34+ cells from peripheral blood of three IMO patients were isolated without need for mobilization as they have high levels (around 3%) of circulating blood progenitors (Steward et al, Biol Blood Marrow Transplant. 2005). These cells were cultured in SFEM medium with 50 ng/ml M-CSF, 30 ng/ml GM-CSF, 10 ng/ml IL-6, 200 ng/ml SCF and 50 ng/ml Flt3L for 2 weeks. During culture the cells expanded 500 fold and gradually lost CD34 expression while 50% became positive for CD14, a marker for osteoclast precursors. The cells were transduced with SIN lentiviral vectors expressing either endogenous or codon optimized TCIRG1, plus GFP, under a SFFV promotor. The transduction efficiency was approximately 40% at 2 weeks. Cells were then differentiated to mature osteoclasts by culturing for 10 days on bone slices with α-MEM containing 10% serum, 50 ng/ml M-CSF and 50 ng/ml RANKL. Expression of GFP was retained throughout differentiation. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1 compared to controls. Interestingly the protein appeared only at the end of the differentiation protocol suggesting regulation at the post-transcriptional level, a phenomenon that is under further investigation. Vector-corrected IMO osteoclasts generated increased Ca2+ release and bone degradation products such as C-telopeptide of type 1 collagen (CTX-1) into the media, while non-corrected IMO osteoclasts failed to resorb bone. Resorption per osteoclast (CTX-1/TRAP ratio) was 20–50% of that of osteoclasts derived from normal CD34+ cord blood cells and about 2–6 fold higher than that of osteoclasts derived from non-transduced IMO CD34+ cells. CONCLUSION: In conclusion we provide the first in vitro evidence of lentiviral-mediated correction of a genetic disease involving the osteoclast lineage, supporting further development of gene therapy of IMO and other diseases affecting these cells. Disclosures: Richter: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.