Gene therapy for infantile malignant osteopetrosis: review of pre-clinical research and proof-of-concept for phenotypic reversal.

Infantile malignant osteopetrosis is a devastating disorder of early childhood that is frequently fatal and for which there are only limited therapeutic options. Gene therapy utilizing autologous hematopoietic stem and progenitor cells represents a potentially advantageous therapeutic alternative for this multisystemic disease. Gene therapy can be performed relatively rapidly following diagnosis, will not result in graft versus host disease, and may also have potential for reduced incidences of other transplant-related complications. In this review, we have summarized the past sixteen years of research aimed at developing a gene therapy for infantile malignant osteopetrosis; these efforts have culminated in the first clinical trial employing lentiviral-mediated delivery of TCIRG1 in autologous hematopoietic stem and progenitor cells.

Transcriptome sequencing identifies a noncoding, deep intronic variant in CLCN7 causing autosomal recessive osteopetrosis.

BACKGROUND: Over half of children with rare genetic diseases remain undiagnosed despite maximal clinical evaluation and DNA-based genetic testing. As part of an Undiagnosed Diseases Program applying transcriptome (RNA) sequencing to identify the causes of these unsolved cases, we studied a child with severe infantile osteopetrosis leading to cranial nerve palsies, bone deformities, and bone marrow failure, for whom whole-genome sequencing was nondiagnostic. METHODS: We performed transcriptome (RNA) sequencing of whole blood followed by analysis of aberrant transcript isoforms and osteoclast functional studies. RESULTS: We identified a pathogenic deep intronic variant in CLCN7 creating an unexpected, frameshifting pseudoexon causing complete loss of function. Functional studies, including osteoclastogenesis and bone resorption assays, confirmed normal osteoclast differentiation but loss of osteoclast function. CONCLUSION: This is the first report of a pathogenic deep intronic variant in CLCN7, and our approach provides a model for systematic identification of noncoding variants causing osteopetrosis-a disease for which molecular-genetic diagnosis can be pivotal for potentially curative hematopoietic stem cell transplantation. Our work illustrates that cryptic splice variants may elude DNA-only sequencing and supports broad first-line use of transcriptome sequencing for children with undiagnosed diseases.

Generation of gene-corrected functional osteoclasts from osteopetrotic induced pluripotent stem cells.

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.

Hematopoietic Stem Cell-Targeted Neonatal Gene Therapy with a Clinically Applicable Lentiviral Vector Corrects Osteopetrosis in oc/oc Mice.

Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by nonfunctional osteoclasts. Approximately 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. Gene therapy represents a potential alternative treatment to allogeneic stem cell transplantation for IMO. The oc/oc mouse is a model of IMO characterized by a 1,500 bp deletion in the TCIRG1 gene, severe osteopetrosis, and a life span of only 3 weeks. Here we show that the osteopetrotic phenotype in oc/oc mice can be reversed by hematopoietic stem cell-targeted gene therapy with a clinically applicable lentiviral vector expressing a wild-type form of human TCIRG1 under the mammalian promoter elongation factor 1α short (EFS-hT). oc/oc c-kit+ fetal liver cells transduced with EFS-hT were transplanted into sublethally irradiated oc/oc mice by temporal vein injection 1 day after birth. A total of 9 of 12 mice survived long term (19-25 weeks) with evidence of tooth eruption, uncharacteristic of oc/oc mice. Splenocytes were harvested 19-25 weeks after transplantation and differentiated into osteoclasts on bone slices to assess resorption and on plastic to assess TCIRG1 protein expression. Vector-corrected osteoclasts showed human TCIRG1 expression by Western blot. CTX-I release relative to that mediated by oc/oc-derived osteoclasts increased 8-239-fold. Resorption pits on bone slices were observed for osteoclasts derived from 7/9 surviving transplanted oc/oc mice. Histopathology of the bones of surviving animals showed varying degrees of rescued phenotype, the majority with almost full correction. The average vector copy number per cell in the bone marrow was 1.8 ± 0.5. Overall, 75% of transplanted mice exhibited long-term survival and marked reversal of the osteopetrotic bone phenotype. These findings represent a significant step toward the clinical application of gene therapy for IMO.

Targeting NSG Mice Engrafting Cells with a Clinically Applicable Lentiviral Vector Corrects Osteoclasts in Infantile Malignant Osteopetrosis.

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 a subunit of the osteoclast proton pump. The aim of this study was to develop a clinically applicable lentiviral vector expressing TCIRG1 to correct osteoclast function in IMO. Two mammalian promoters were compared: elongation factor 1α short (EFS) promoter and chimeric myeloid promoter (ChimP). EFS promoter was chosen for continued experiments, as it performed better. IMO osteoclasts corrected in vitro by a TCIRG1-expressing lentiviral vector driven by EFS (EFS-T) restored Ca2+ release to 92% and the levels of the bone degradation product CTX-I to 95% in the media compared to control osteoclasts. IMO CD34+ cells from five patients transduced with EFS-T were transplanted into NSG mice. Bone marrow was harvested 9-19 weeks after transplantation, and human CD34+ cells were selected, expanded, and seeded on bone slices. Vector-corrected IMO osteoclasts had completely restored Ca2+ release. CTX-I levels in the media were 33% compared to normal osteoclasts. Thus, in summary, evidence is provided that transduction of IMO CD34+ cells with the clinically applicable EFS-T vector leads to full rescue of osteoclasts in vitro and partial rescue of osteoclasts generated from NSG mice engrafting hematopoietic cells. This supports the continued clinical development of gene therapy for IMO.

Forced expression of human macrophage colony-stimulating factor in CD34+ cells promotes monocyte differentiation in vitro and in vivo but blunts osteoclastogenesis in vitro.

OBJECTIVES: Here, we tested the hypothesis that human M-CSF (hM-CSF) overexpressed in cord blood (CB) CD34+ cells would induce differentiation and survival of monocytes and osteoclasts in vitro and in vivo. METHODS: Human M-CSF was overexpressed in cord blood CD34+ cells using a lentiviral vector. RESULTS: We show that LV-hM-CSF-transduced CB CD34+ cells expand 3.6- and 8.5-fold more with one or two exposures to the hM-CSF-expressing vector, respectively, when compared to control cells. Likewise, LV-hM-CSF-transduced CB CD34+ cells show significantly higher levels of monocytes. In addition, these cells produced high levels of hM-CSF. Furthermore, they are able to differentiate into functional bone-resorbing osteoclasts in vitro. However, osteoclast differentiation and bone resorption were blunted compared to control CD34+ cells receiving exogenous hM-CSF. NSG mice engrafted with LV-hM-CSF-transduced CB CD34+ cells have physiological levels of hM-CSF production that result in an increase in the percentage of human monocytes in peripheral blood and bone marrow as well as in the spleen, lung and liver. CONCLUSION: In summary, ectopic production of human M-CSF in CD34+ cells promotes cellular expansion and monocyte differentiation in vitro and in vivo and allows for the formation of functional osteoclasts, albeit at reduced levels, without an exogenous source of M-CSF, in vitro.

Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis: Implications for Gene Therapy.

Infantile malignant osteopetrosis (IMO) is a rare, recessive disorder characterized by increased bone mass caused by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene encoding a subunit of the V-ATPase involved in the osteoclasts capacity to resorb bone. We previously showed that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression is unknown. Here we show that expression of TCIRG1 protein from a bicistronic TCIRG1/GFP lentiviral vector was only observed in mature osteoclasts, and not in their precursors or macrophages, in contrast to GFP expression, which was observed under all conditions. Thus, vector-mediated TCIRG1 expression appears to be post-transcriptionally regulated, preventing overexpression and/or ectopic expression and ensuring protein expression similar to that of wild-type osteoclasts. Codon optimization of TCIRG1 led to increased expression of mRNA but lower levels of protein and functional rescue. When assessing the functional rescue threshold in vitro, addition of 30 % CB CD34+ cells to IMO CD34+ patient cells was sufficient to completely normalize resorptive function after osteoclast differentiation. From both an efficacy and a safety perspective, these findings will clearly be of benefit during further development of gene therapy for osteopetrosis.

A comparison of osteoclast-rich and osteoclast-poor osteopetrosis in adult mice sheds light on the role of the osteoclast in coupling bone resorption and bone formation.

Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts.

Sortilin expression is essential for pro-nerve growth factor-induced apoptosis of rat vascular smooth muscle cells.

BACKGROUND: Sortilin, a member of the Vps10p-domain receptor family, has been demonstrated a key regulator in mediating cellular response to pro-neurotrophins. In the present study, we investigated the role of sortilin in the apoptotic pathway of vascular smooth muscle cells. METHODS AND PRINCIPAL FINDINGS: Immunohistochemistry revealed that sortilin was barely detectable in human and rat normal young vessels, while its expression was increased in human fibroatheromatous plaques. Sortilin immunodetection was also marked in the neointima of the rat aorta fifteen days after ballooning.In vitro, rat aortic intimal cells expressed higher sortilin levels than normal media SMCs; sortilin was distributed in the cytoplasm and in correspondence of the cell membrane. After 48 h, pro-nerve growth factor (proNGF) induced the strong dose-dependent increase of intimal cell apoptosis and the accumulation of sortilin protein. ProNGF was a more potent apoptotic inducer than equimolar or even higher concentration of NGF, whereas brain derived neutrotrophic factor was ineffective. Targeted interfering RNA-mediated sortilin reduction counteracted proNGF-induced apoptosis without affecting p75(NTR) expression. ProNGF-induced apoptosis was associated to NF-κB down-regulation and bax increase. Inhibition of NF-κB activity increased intimal cell apoptosis that did not further increase with the addition of proNGF. CONCLUSIONS: Our results indicate that sortilin expression characterizes human atheromatous lesions and rat aortic post-injury neointima, and suggest that sortilin represents an important regulator of proNGF-induced SMC apoptosis and arterial remodeling.

Osteoclasts are not crucial for hematopoietic stem cell maintenance in adult mice.

The osteoclast is vital for establishment of normal hematopoiesis in the developing animal. However, its role for maintenance of hematopoiesis in adulthood is more controversial. To shed more light on this process, we transplanted hematopoietic stem cells from two osteopetrotic mouse models, with lack of osteoclasts or defective osteoclast function, to normal adult mice and examined the bone phenotype and hematopoiesis in the recipients. B6SJL mice were lethally irradiated and subsequently transplanted with oc/oc, Receptor Activator of Nuclear Factor Kappa B knockout or control fetal liver cells. Osteoclasts derived from the recipient animals were tested in vitro for osteoclastogenesis and resorptive function. Bone remodeling changes were assessed using biomarkers of bone turnover and micro-CT. Hematopoiesis was assessed by flow cytometry and colony formation, and hematopoietic stem cell function by secondary competitive transplantations and cell cycle analysis. After transplantation, a donor chimerism of 97-98% was obtained, and by 15 weeks mild osteopetrosis had developed in recipients of cells from osteopetrotic mice. There were no alterations in the number of bone marrow cells. Colony formation was slightly reduced in Receptor Activator of Nuclear Factor Kappa B knockout recipients but unchanged in oc/oc recipients. Phenotypically, stem cells were marginally reduced in recipients of cells from osteopetrotic mice, but no significant difference was seen in cell cycle status and in competitive secondary transplantations all three groups performed equally well. Our results indicate that osteoclast function is not crucial for hematopoietic stem cell maintenance in adult mice.

Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells restores osteoclast function in infantile malignant osteopetrosis.

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34(+) cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca(2+) and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34(+) cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.

Leptin attenuates ischemia-reperfusion injury in the rat liver.

Leptin is an adipocytokine that reduces ischemic damage in several organs including brain and heart. STAT3 activation is a key step for the attainment of leptin effects in various tissues. We evaluated the possible effect of leptin on liver viability and STAT3 activation, in a rat model of ischemia-reperfusion injury. Rat livers, flushed and stored with Belzer solution (4° C for 24 h), were warmly reperfused (3.5 ml/min/g liver for 1 h at 37° C with O(2) ) with Krebs-Ringer bicarbonate. Treatment group underwent an identical protocol with the adjunct of Leptin (10 ng/ml). Liver effluent was harvested to assess LDH and AST output. Liver tissue was used for pSTAT3 expression (western blot and immunostaining), optical microscopy, TUNEL, and Cell Death Detection assays. The pSTAT3 expression was enhanced by administration of leptin. In parallel, LDH and AST output were reduced (P = 0.04 and P = 0.02 for LDH and AST, respectively). Optical microscopy, TUNEL, and Cell Death Detection assay results demonstrated increased viability in livers treated with leptin in comparison with others (Optical microscopy P = 0.02; TUNEL P = 0.01; Cell death Detection assay P = 0.003). In conclusion, cold storage and reperfusion with leptin reduce liver ischemia-reperfusion injury. This effect is associated with an increased expression of pSTAT-3.

A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts.

Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.

Nonablative neonatal bone marrow transplantation rapidly reverses severe murine osteopetrosis despite low-level engraftment and lack of selective expansion of the osteoclastic lineage.

Infantile malignant osteopetrosis (IMO) is caused by lack of functional osteoclasts leading to skeletal abnormalities, blindness owing to compression of the optic nerves, bone marrow (BM) failure, and early death. In most patients, TCIRG1, a proton pump subunit essential for bone resorption, is mutated. oc/oc mice represent a model for IMO owing to a deletion in Tcirg1 and die around 4 weeks of age. To determine if hematopoietic stem cell transplantation without prior conditioning can reverse osteopetrosis, neonatal mice were transplanted intravenously with lineage-depleted BM cells. More than 85% of oc/oc mice transplanted with 5 × 10(6) cells survived long term with an engraftment of 3% to 5% in peripheral blood (PB). At 3 weeks, engraftment in the BM was 1% to 2%, but the cellularity had increased 60-fold compared with untreated oc/oc mice, and RANKL and macrophage colony-stimulating factor (M-CSF) expression in the BM was normalized. Histopathology and micro-computed tomography revealed almost complete reversal of osteopetrosis after 4 weeks. In vitro studies showed that bone resorption by osteoclasts from transplanted oc/oc mice was 14% of transplanted controls, and immunofluorescence microscopy revealed that resorption was mainly associated with osteoclasts of donor origin. Lineage analysis of BM, PB, and spleen did not provide any evidence for selective recruitment of cells to the osteoclastic lineage. The vision also was preserved in transplanted oc/oc mice, as determined by a visual tracking drum test. In summary, nonablative neonatal transplantation leading to engraftment of only a small fraction of normal cells rapidly reverses severe osteopetrosis in the oc/oc mouse model.

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells.

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes.

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.