© 2021. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2021) 14, dmm048940. doi:10.1242/dmm.

048940

REVIEW

Autosomal recessive osteopetrosis: mechanisms and treatments

Sara Penna1,2, Anna Villa1,3, * and Valentina Capo1,3

ABSTRACT Pathogenesis of ARO

Autosomal recessive osteopetrosis (ARO) is a severe inherited bone ARO has an incidence of 1:250,000 live births and is fatal within the
disease characterized by defective osteoclast resorption or first decade of life if untreated (Sobacchi et al., 2013). Patients suffer
differentiation. Clinical manifestations include dense and brittle severe symptoms, including growth retardation, skull abnormalities
bones, anemia and progressive nerve compression, which hamper (macrocephaly, frontal bossing, choanal stenosis), hydrocephalus,
the quality of patients’ lives and cause death in the first 10 years of age. hypocalcemia (owing to the defective calcium mobilization activity
This Review describes the pathogenesis of ARO and highlights the of osteoclasts) and abnormal tooth eruption (Wu et al., 2017). The
strengths and weaknesses of the current standard of care, namely abnormal bone density causes an expansion of skeletal tissue into
hematopoietic stem cell transplantation (HSCT). Despite an marrow cavities at the expense of the bone marrow niche, leading to
improvement in the overall survival and outcomes of HSCT, severe anemia, bleeding, frequent infections and hepatosplenomegaly
transplant-related morbidity and the pre-existence of neurological due to increased extramedullary hematopoiesis. The increased
symptoms significantly limit the success of HSCT, while the susceptibility to infections leads to the development of dental caries
availability of human leukocyte antigen (HLA)-matched donors still and facial osteomyelitis, especially after dental surgery (Mikami
remains an open issue. Novel therapeutic approaches are needed for et al., 2016). The limited bone marrow niches cause the circulation of
ARO patients, especially for those that cannot benefit from HSCT. Here, high numbers of hematopoietic stem and progenitor cells (HSPCs;
we review preclinical and proof-of-concept studies, such as gene positive for CD34 marker) in the peripheral blood, especially in
therapy, systematic administration of deficient protein, in utero HSCT young patients with severe forms (Capo et al., 2021; Steward et al.,
and gene editing. 2005). However, the mechanisms underlying the spontaneous CD34+
mobilization from the bone marrow niche of ARO patients still
KEY WORDS: Osteopetrosis, Bone disease, Osteoclast, remain poorly characterized. Moreover, the encroachment of cranial
Hematopoietic stem cell transplantation, Gene therapy nerve foramina leads to blindness, deafness and nerve palsies (Wu
et al., 2017).
Introduction ARO disease originates from mutations in different genes that are
Osteopetrosis, or marble bone disease, was first described in 1904 involved in osteoclast functions (osteoclast-rich osteopetrosis)
by the German radiologist Albers Schönberg as a heritable disorder (Fig. 1) or differentiation (osteoclast-poor osteopetrosis) (Fig. 2)
characterized by increased bone density, typically described as (Penna et al., 2019).
‘bone within bone’ appearance (Stark and Savarirayan, 2009).
The impaired equilibrium of bone formation and remodeling leads Osteoclast-rich osteopetroses
to structural brittleness, predisposition to fractures, skeletal Among osteoclast-rich forms (Fig. 1), more than 50% of ARO cases
deformities and dental abnormalities (Sobacchi et al., 2013). The are caused by mutations in the T cell immune regulator 1 (TCIRG1)
disease is caused by defective osteoclast differentiation or function, gene, which encodes the V-type proton ATPase 116 kDa subunit a3
but is clinically and genetically heterogeneous, ranging in severity (OC116). It acidifies the bone resorption lacunae (see Glossary,
from benign to lethal in early childhood (Penna et al., 2019). Based Box 1), favoring the dissolution of hydroxyapatite crystals, which
on the pattern of inheritance, osteopetroses have been categorized form the bone mineral fraction, and the degradation of the organic
into autosomal recessive osteopetrosis (ARO), also known as bone matrix (Frattini et al., 2000). Moreover, OC116 plays a role in
infantile malignant osteopetrosis, and autosomal dominant the interaction between the actin cytoskeleton and microtubules,
osteopetrosis, an adult-onset more benign form (Palagano et al., which is essential for osteoclast ruffled border (Box 1) formation
2018). In this Review, we will focus on ARO and its pathogenesis, (Matsumoto et al., 2018). The TCIRG1 gene is expressed in most
as it represents the most severe osteopetrosis with a strong unmet tissues, in particular by osteoclasts and gastric parietal cells on the Disease Models & Mechanisms
clinical need. We will discuss the standard of care and the ongoing apical membrane. In the stomach, the V-ATPase proton pump
clinical trials, as well as innovative treatments that are currently maintains the low pH essential for dietary Ca2+ absorption; thus,
being developed and can be applied to ARO. ARO patients also present rickets or osteomalacia due to defective
calcium uptake (Schinke et al., 2009).
1 Milder forms of osteopetrosis can be diagnosed later in life,
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele
Scientific Institute, Milan 20132, Italy. 2Translational and Molecular Medicine accounting for a small number of patients with slower disease
(DIMET), University of Milano-Bicocca, Monza 20900, Italy. 3Institute of Genetic and progression (Howaldt et al., 2019). In particular, recent studies
Biomedical Research, Milan Unit, National Research Council, Milan 20090, Italy.
reported novel single-nucleotide changes in the TCIRG1 gene that
*Author for correspondence ([email protected]) cause aberrant splicing and exon skipping, thereby reducing the
splicing efficiency without completely abrogating the production of
S.P., 0000-0002-5036-3809; A.V., 0000-0003-4428-9013; V.C., 0000-0002-
1851-1429
the normal transcript (Palagano et al., 2015; Zirngibl et al., 2019).
The second most frequent form of osteopetrosis (17% of ARO
This is an Open Access article distributed under the terms of the Creative Commons Attribution cases) is caused by mutations in the chloride voltage-gated channel
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed. 7 (CLCN7) gene. The CLCN7 gene encodes H(+)/Cl(−) exchange

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REVIEW Disease Models & Mechanisms (2021) 14, dmm048940. doi:10.1242/dmm.048940

LRRK1 TRAF6

MITF Cl−

Nuclei

RELA NEMO HCO3−

SLC29A3

SNX10 H2O + CO2 H2CO3

CA2
Lysosome PLEKHM1

KINDLIN3
H+ H+

CTSK
CLCN7
OSTM1 TCIRG1
Integrin-β
Cl− 2Cl − H+ H+
H+
H+ H+ H+

Fig. 1. Schematic representation of genes involved in osteoclast-rich osteopetrosis. The figure shows genes involved in the bone resorptive activity of
osteoclasts with different functions, including acidification of resorption lacunae and pH regulation (TCIRG1, CLCN7, OSTM1 and CA2), vesicular trafficking and
sorting of protein complexes to the membrane (SNX10 and PLEKHM1), lysosomal nucleoside trafficking (SLC29A3), cytoskeletal rearrangement for ruffled
border formation (KINDLIN3, integrin-β and LRRK1) and lysosomal proteolytic cleavage for bone remodeling and resorption (CTSK). Moreover, genes that are
involved in different signal transduction pathways and essential for osteoclast function (MITF, TRAF6, RELA and NEMO) have been reported. Figure originally
created using Servier Medical Art (http://smart.servier.com/), licensed under a CC-BY 3.0 license, and re-drawn according to journal style.

transporter 7 (ClC-7), a 2Cl−/H+ antiporter that is expressed at the present CNS involvement (Di Zanni et al., 2021). Additionally,
osteoclast ruffled border and on the membrane of late endosomes CLCN7 mutations could account for the milder IAO, when patients
and lysosomes, and that is regulated by a voltage-gating mechanism suffer from mild symptoms and reach adulthood. Conversely, single-
(Leisle et al., 2011). Because this channel is associated with the allele CLCN7 mutations lead to autosomal-dominant osteopetrosis
V-ATPase, it plays a fundamental role in maintaining an acid pH at and are associated with milder symptoms and later onset (Li et al.,
resorption lacunae. It is also involved in vesicle trafficking in early 2019a; Sobacchi et al., 2013).
and recycling endosomes, regulating the luminal Cl− concentration Less frequent osteopetroses (5% of ARO cases) present
in the kidney and central nervous system (CNS) (Novarino et al., mutations in the osteoclastogenesis-associated transmembrane
2010). protein 1 (OSTM1) gene, encoding a highly glycosylated protein
Depending on the type of mutation, patients present with mild or at its N-terminus able to stabilize ClC-7 and complement its activity Disease Models & Mechanisms
severe forms characterized by a wide variety of clinical manifestations at the ruffled border (Chalhoub et al., 2003; Leisle et al., 2011).
(Kornak et al., 2001; Palagano et al., 2018; Pangrazio et al., 2010). In A similar frequency of ARO cases is reported for mutations in
particular, symptom presentation varies from ‘classical’ infantile SNX10, encoding the protein sortin nexin-10, targeting OC116 at
osteopetrosis with or without progressive neurodegeneration the ruffled border (Pangrazio et al., 2013). SNX10 deficiency
(displaying pathological electroencephalography), to intermediate causes abnormal osteoclastogenesis, leading to relatively
autosomal osteopetrosis (IAO) and even to benign autosomal variable clinical manifestations, mainly affecting bone tissue
dominant osteopetrosis. In terms of genetic characterization, (Aker et al., 2012).
biallelic mutations of the CLCN7 gene are reported to cause severe Extremely rare forms of osteoclast-rich osteopetrosis are caused
forms of osteopetrosis, showing bone damage, hematological failure by defects in the carbonic anhydrase 2 (CA2) (Sly et al., 1983),
and primary neurodegeneration, with symptoms resembling pleckstrin homology and RUN domain-containing M1
lysosomal storage defects (Kornak et al., 2001; Sobacchi et al., (PLEKHM1) (Van Wesenbeeck et al., 2007), leucine-rich repeat
2007). Neurodegeneration is probably due to the defective kinase 1 (LRRK1) and melanocyte-inducing transcription factor
localization of ClC-7 in lysosomal membranes, because patients (MITF) genes (Howaldt et al., 2020; Iida et al., 2016; Lu et al.,
bearing mutations that maintain correct membrane targeting do not 2014). The pathogenesis of these forms is poorly characterized due

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Monocytes

Pre-osteoblasts

RANKL
Pre-osteoclasts

Osteoblasts
Undifferentiated
precursors M-CSF

RANK Osteoid

New
bone

Old
Osteocytes bone

Fig. 2. Representation of proteins involved in osteoclast-poor osteopetrosis. Impaired crosstalk between osteoclasts and osteoblasts gives rise to deficient
bone remodeling. In osteoclast-poor osteopetrosis, the osteoclast differentiation pathway is impaired due to mutations in the TNFSF11 and TNFRSF11A
genes, encoding RANKL and its receptor RANK, respectively, or in the CSF1R gene, encoding M-CSF. As a consequence, osteoclast precursors are not able to
fuse and to differentiate into multinucleated resorbing osteoclasts. Mutated genes are indicated by a red cross. Figure originally created using Servier Medical
Art (http://smart.servier.com/), licensed under a CC-BY 3.0 license, and re-drawn according to journal style.

to the small number of identified patients. Other mutations have dysosteosclerosis, is associated with red violet macular atrophy,
been reported to cause osteopetrosis as secondary disease among platyspondyly and metaphyseal osteosclerosis.
other clinical manifestations. In particular, patients with leucocyte Atypical cases of osteopetrosis have been reported in two siblings
adhesion deficiency III can display high bone mass due to mutations affected by severe combined immunodeficiency (SCID), who carry
disrupting fermitin family homolog 3 [Kindlin-3 protein; encoded a mutation in the TNF receptor-associated factor 6 (TRAF6) gene,
by the FERM domain-containing kindlin 3 (FERMT3) gene] and the most important adaptor for the RANK/RANKL signaling
the integrin-β pathway, which cause the abrogation of podosome pathway (Weisz Hubshman et al., 2017). In addition, a
(Box 1) and sealing zone (Box 1) formation, which are required for heterozygous truncating mutation in the colony-stimulating factor
osteoclast bone resorption (Malinin et al., 2009; Schmidt et al., 1 receptor (CSF1R) gene, which encodes the macrophage colony-
2011). Additionally, osteopetrosis may be secondary to the bone stimulating factor 1 receptor (M-CSF), was reported in the
pathology arising from cathepsin K (CTSK) mutations, which is consanguineous parents of two deceased siblings showing
termed pycnodysostosis in humans (Gelb et al., 1996). osteopetrosis and brain malformations (Monies et al., 2017).

Osteoclast-poor osteopetroses Consensus guidelines for the treatment of osteopetrosis

Mutations affecting osteoclast differentiation are responsible for the Supportive treatments
osteoclast-poor forms of osteopetrosis (Fig. 2). In particular, loss- Without treatment, ARO is lethal in 70% of cases (Gillani and
of-function mutations impairing the expression of the receptor Abbas, 2017). Because osteoclasts originate from hematopoietic
activator of nuclear factor kappa-Β ligand (RANKL) cytokine or its precursors, hematopoietic stem cell transplantation (HSCT) is the
receptor, RANK, have been reported to cause osteopetrosis in 2% therapy of choice, resulting in improved bone remodeling and
and 4.5% of ARO cases, respectively. The TNF superfamily reversal of pancytopenia and extramedullary hematopoiesis. Disease Models & Mechanisms
member 11 (TNFSF11) gene encodes RANKL, the master However, HSCT is effective only in cases of intrinsic osteoclast
osteoclastogenic cytokine produced mainly by osteoblasts and defects and is not recommended for osteopetrosis caused by absent
stromal cells in bone (Lacey et al., 1998), whereas TNF receptor osteoclasts due to mutation in TNFSF11 (Even-Or and Stepensky,
superfamily member 11a (TNFRSF11A) encodes RANK, a receptor 2021; Schulz et al., 2015; Wu et al., 2017).
mainly expressed by osteoclast precursors and mature osteoclasts In cases of CNS involvement, such as in osteopetrosis caused by
(Nakagawa et al., 1998). The disruption of this pathway causes the mutations in CLCN7 and OSTM1, which are associated with
complete absence of mature osteoclasts in bone biopsies (Lo Iacono primary neuropathy, HSCT is proven to be ineffective and
et al., 2013; Pangrazio et al., 2012). Patients affected by RANKL contraindicated (Pangrazio et al., 2010; Schulz et al., 2015). By
deficiency present with severe osteopetrosis with slower contrast, patients affected by intermediate osteopetrosis, comprising
progression of the disease compared to classical ARO. both severe dominant forms with an early onset and recessive ones
Mutations in the solute carrier family 29 member 3 (SLC29A3) without CNS involvement, have a better prognosis and are
gene, encoding a lysosomal nucleoside transporter highly expressed candidates for HSCT (Pangrazio et al., 2010). For this reason,
in myeloid cells, also affect osteoclast differentiation (Campeau extensive neurological evaluation by computed tomography or
et al., 2012; Howaldt et al., 2019). SLC29A3 deficiency, also called magnetic resonance imaging and electroencephalography are

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required, especially in patients carrying unknown mutations in the

Box 1. Glossary CLCN7 gene (Schulz et al., 2015).
Autologous back-up transplantation: before conditioning, autologous In intermediate- and late-onset forms, HSCT may pose greater
hematopoietic stem cells (HSCs) are harvested and stored as back-up to risks than benefits, so the patient-specific situations must be
be potentially reinfused to the patient in case of graft failure or severe considered. When HSCT is not indicated or adequate donors are
graft versus host disease (GvHD). Autologous transplant does not cure
the disease but allows rescue of the patient’s bone marrow as a bridge
lacking, patients are empirically treated and receive conservative care
therapy to a second allogeneic transplant. based on multi-disciplinary approaches, according to clinical
Circulating CD34+ cells: peripheral blood CD34+ cells are a manifestations (Stark and Savarirayan, 2009). These interventions
heterogeneous population of hematopoietic stem/progenitor cells may include, among others, calcium and vitamin D supplementation,
(HSPCs) that circulate outside the bone marrow niche. In physiological corticosteroid administration, antimicrobial therapy, orthopedic
conditions, low numbers of CD34+ cells are found in peripheral blood, surgery, neurosurgery, transfusions and pain management (Wu
while the lack of bone marrow niches in osteopetrotic bones induces
et al., 2017).
increased egress from bone marrow.
C-terminal telopeptide of type I collagen (CTX-I): type I collagen is the
most abundant component of the organic matrix of bone. During the HSCT: strengths and weaknesses
bone remodeling process, type I collagen is degraded, and small peptide HSCT is the standard care for ARO patients and, although it usually
fragments, such as CTX-I, are excreted into the bloodstream. Serum improves patients’ condition, it does not fully rescue the disease
levels of these degradation products can be quantified as a marker of (Fig. 3) (Box 2). Despite full engraftment in more than 50% of
bone resorption.
transplanted ARO individuals, treated patients present progressive
Disease-free survival: the term identifies the time after HSC
transplantation (HSCT), during which the patients survive with signs of
visual loss in the early post-transplant period (Orchard et al., 2015).
osteoclast function (hypercalcemia, improvement by radiographs, Young age (less than 1 year), when disease progression and
improvement of hepatosplenomegaly). However, irreversible nerve irreversible neurological damage are still limited, is a strong
damage or bone deformities occurring before HSCT are still present. indication to perform HSCT. Moreover, the frequency of transplant
Donor chimerism: the percentage of donor cells that have durably failure increases in patients receiving HSCT after 10 months of age,
engrafted in the recipient. The chimerism is complete if 100% of bone particularly in a haploidentical HSCT (Box 1) setting (Schulz et al.,
marrow and blood cells are of donor origin, whereas it is mixed or partial if
recipient cells persist.
2015).
Exchange apheresis: the apheresis machine allows the separation and Bone marrow transplants from human leukocyte antigen (HLA)-
collection of CD34+ cells from the other blood components by identical donors allow 62-88% overall survival (Driessen et al.,
centrifugation, returning all other blood components to the patient. The 2003; Gerritsen et al., 1994; Orchard et al., 2015; Wynn and Schulz,
procedure allows the processing of a large volume of blood to collect 2019). In 2015, a large retrospective international study reported the
HSCs, limiting side effects of multiple blood drawings. outcomes of HSCT from related and unrelated donors for infantile
Haploidentical HSCT: an HSCT in which the source of HSCs derives
osteopetrosis in 193 patients, with a median follow-up of 7 years
from a human leukocyte antigen (HLA) half-matched donor, usually a
parent or sibling of the patient. (Orchard et al., 2015). This study reported overall survival of 62% at
Podosome: conical, actin-rich structure located on the outer surface of 10 years post-treatment after HLA-matched donor transplant. An
the plasma membrane. Podosomes play a crucial role in the bone improvement in survival rates has been observed, and, recently,
remodeling process, connecting osteoclast precursors to the bone 100% overall survival was reported in 31 patients transplanted from
surface. When the bone resorption process starts, they become a single matched donors after reduced intensity conditioning (RIC) (Box 1)
actin ring, delimiting the sealing zone of mature osteoclasts on the bone
(Shadur et al., 2018).
resorption site.
Reduced intensity conditioning (RIC): a conditioning regimen that
Survival drops to 40-70% in recipients of HLA-mismatched or
does not completely abolish host bone marrow, with decreased toxic HLA-haploidentical donors (Schulz et al., 2015). Alternative
effect on the bone marrow niche compared to the standard myeloablative strategies have been tested, such as T cell-depleted haploidentical
approach. HSCT (Box 1), with improved outcomes (Porta et al., 2015; Pronk
Resorption lacunae: also called Howship’s lacunae, are tiny et al., 2017; Schulz et al., 2002). This procedure allows an
depressions, pits or irregular grooves in bone that is being resorbed by acceptable long-term donor cell engraftment and significantly limits
osteoclasts.
Ruffled border: in close proximity to the bone, at the resorption lacunae,
the risk of graft versus host disease (GvHD) (Porta et al., 2015).
the osteoclasts’ membrane is characterized by finger-like processes Another promising option is haploidentical HSCT followed by
composing the ruffle border. This extensively folded border increases the post-transplant cyclophosphamide (PT-Cy) regimens in order to
cell surface able to host the components of the bone resorption eliminate alloreactive T cells and decrease the risk of GvHD (Even-
machinery of osteoclasts. Or and Stepensky, 2021; Neven et al., 2019). The use of cord blood, Disease Models & Mechanisms
Sealing zone: specialized cell–matrix adhesion structure formed at the another standard source of cells for HSCT, is currently no longer
junction between mature osteoclast and bone, that assembles during
recommended, however, because frequent primary graft failures
bone resorption. It is a dynamic actin-rich structure that defines the
osteoclast resorption area of the bone. have been recorded, resulting in poor overall survival at 3 years
Sleeping Beauty transposon: a system that allows the (Chiesa et al., 2016; Schulz et al., 2015).
transposition of a synthetic DNA sequence in the genome, through HSCT treatment of ARO patients is associated with frequent
a cut-and-paste process. The integration of transposon into host adverse events, including difficulty in achieving sustained long-
DNA guarantees long-term transgene expression in transgenic cells term donor engraftment, early transplant-related mortality,
and organisms.
pulmonary hypertension, and veno-occlusive disease (VOD) of
T cell-depleted haploidentical HSCT: an allogeneic transplantation in
which CD34+ cells are purified before the infusion in order to deplete T
the liver and sepsis (Schulz et al., 2015). Coupled with the severe
cells from the graft. In the non-identical setting, the use of T cell-depleted pre-existing clinical status of ARO patients, poor outcome and
transplants prevents GvHD. toxicities have also been associated with traditional myeloablative
UM171: a pyrimidoindole derivate that supports the expansion of HSPCs chemotherapy regimens, paving the way for research into new
while maintaining their stemness features and engraftment capacity. approaches for the conditioning of osteopetrotic patients to
ameliorate the HSCT outcome (Even-Or and Stepensky, 2021;

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Clinical trial gene therapy

CD34+
LV.EFS.TCIRG1

Patient’s
CD34+
collection
Ex vivo CD34+
Hematopoietic stem cell transplantation transduction
CD34+ harvest from
compatible donor

ARO patient
Conditioning and Experimental gene
CD34+ cell infusion therapy protocol
Conditioning
and infusion of
gene-corrected
CD34+ cells Expansion of
gene-corrected
CD34+ cells

Collection of
circulating
CD34+ cells from
peripheral blood
Ex vivo ARO CD34+ LV.PGK.TCIRG1
cell transduction

Fig. 3. Current therapeutic approaches for ARO and novel gene therapy strategies for TCIRG1-dependent osteopetrosis. Hematopoietic stem cell (HSC)
transplantation (beige box) is the current standard of care: CD34+ cells are collected from a compatible healthy donor and infused into the patient, who has
previously received myeloablative conditioning. Alternatively, gene therapy represents an innovative approach, allowing autologous HSC transplantation without
the risk of graft rejection. Currently, a Phase I clinical trial for TCIRG1-dependent ARO is ongoing (green box): the autosomal recessive osteopetrosis (ARO)
patient’s CD34+ cells are collected and transduced with a lentiviral vector (LV) carrying the curative gene under the control of the elongation factor 1α short (EFS)
promoter. The transduced stem cells are later re-infused into the patient after administration of a conditioning regimen. A different approach coupling circulating
ARO CD34+ cell expansion with LV gene correction has recently been proposed at a preclinical level by our group (blue box). In particular, circulating CD34+ cells
are collected from the peripheral blood of ARO patients and transduced with an LV carrying the TCIRG1 gene under the control of the phosphoglycerate kinase
(PGK) promoter. After transduction, CD34+ cells are expanded ex vivo, exploiting the UM171-based HSC expansion protocol, which is able to increase HSC
number while maintaining the cells’ stemness and engraftment capacity. The cell product is suitable for cryopreservation, thus providing the possibility to perform
multiple cycles of cell infusions. Finally, the transduced and expanded HSCs are re-infused into the patient after administration of a conditioning regimen.
Figure originally created using Servier Medical Art (http://smart.servier.com/), licensed under a CC-BY 3.0 license, and re-drawn according to journal style.

Wynn and Schulz, 2019). New transplant strategies such as the limiting the side effects of myeloablative conditioning. In particular,
Baltimore protocol of T cell-replete haploidentical transplantation, fludarabine is associated with improved T-cell chimerism and
involving PT-Cy administration post-transplant, may be exploited treosulfan with a decreased risk of VOD (Slatter et al., 2011).
for the treatment of children older than 10 months (Fuchs, 2012; Accordingly, promising data have recently been obtained in
Stepensky et al., 2019), having been successful in several cases of osteopetrotic patients younger than 6 years old, receiving a
re-transplantation after rejection or non-engraftment. transplant after RIC based on fludarabine, treosulfan, thiotepa and Disease Models & Mechanisms
The use of fludarabine, which has a more favorable toxicity antithymocyte globulin (Shadur et al., 2018).
profile than cyclophosphamide, significantly improves the outcome Despite the remarkable improvement in conditioning regimens,
of HSCT (Natsheh et al., 2016) and is currently recommended by severe side effects like VOD, pulmonary hypertension and
European Society for Immunodeficiencies (ESID) consensus hypercalcemia, as well as poor donor engraftment, still hamper
guidelines, resulting in a disease-free survival (Box 1) of 80% the transplantation outcome in ARO (Corbacioglu et al., 2006;
(Schulz et al., 2015). Of note, fludarabine-based conditioning Orchard et al., 2015; Schulz et al., 2015). Other frequent
has also been successfully employed in intermediate forms of complications in osteopetrotic patients are the development of
osteopetrosis, providing a new therapeutic approach for these forms Pneumocystic jirovecii pneumonia (PCP), probably due to slow
affecting adolescents or young adults (Stepensky et al., 2019). hematological and immunological reconstitution, although PCP can be
In recent years, HSCT outcomes have been significantly prevented with antifungal and PCP prophylaxis; CNS complications
improved by exploiting an RIC approach, which allows for the such as malformations of cranial bones or hydrocephalous, which are
creation of sufficient space for HSPCs in the bone marrow reversible after HSCT; and hypocalcaemia and the risk of convulsions
compartment with minimal chemotherapy administration, thus before the engraftment, as well as risk of hypercalcemic crisis upon
achieving acceptable levels of donor chimerism (Box 1) and successful engraftment (Schulz et al., 2015).

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The first attempt exploited a gammaretroviral vector, in which

Box 2. HSCT and conditioning regimens TCIRG1 expression is driven by the spleen focus-forming virus
Hematopoietic stem cell transplantation (HSCT) is the infusion of (SFFV) promoter, in vivo in the oc/oc mouse model (Tcirg1-
allogeneic stem cells to reestablish hematopoietic function in patients dependent ARO), which closely resembles the human TCIRG1-
with diseases affecting hematopoietic lineage. dependent ARO. In particular, oc/oc mice carry a spontaneous
To make room for donor hematopoietic stem cells (HSCs),
conditioning regimens by chemotherapy are required to eliminate
deletion of 1.6 kb in the 5′ end of the Tcirg1 gene, causing the
patients’ bone marrow completely (myeloablative conditioning) or absence of ruffled border formation and defective bone resorption.
partially (reduced intensity conditioning). The alkylating agent busulfan Homozygous mice usually die within 3 weeks after birth due to
is the most frequently used drug, because dosage can be measured and severe bone marrow fibrosis. They are characterized by growth
adjusted during treatment. It is usually combined with cyclophosphamide retardation, exhibiting radiological and histological features of
(an alkylating and immunosuppressive agent) or with the less toxic osteopetrosis, such as increased skeletal density and absence of
fludarabine (a highly immunosuppressive purine analog). Recently, the
marrow cavities, clubbed feet and circling behavior, owing to
substitution of busulfan by treosulfan (a myeloablative and
immunosuppressive alkylating agent) in reduced intensity conditioning cranial nerve compression and absent or delayed eruption of the
has been explored with limited success (Schulz et al., 2015; Shadur incisors (Scimeca et al., 2000).
et al., 2018). GT was able to improve bone defects and survival of 50% of the
In non-genoidentical transplants, thiotepa (an alkylating and treated oc/oc mice, despite the low number of available mice, which
immunosuppressive agent) and serotherapy are also administered to is due to the extremely severe phenotype of this model (Johansson
reduce the risk of rejection by suppressing the reaction of host T cells
et al., 2007). Subsequently, clinical trials of GT for
against the graft. Serotherapy usually consists of antithymocyte globulin
(ATG), a polyclonal antibody against human thymocytes or T-cell lines,
immunodeficiencies asserted the superiority of lentiviral vectors
or the monoclonal antibody alemtuzumab, specific for CD52. Both types (LVs) over retroviral vectors in terms of efficacy and safety
of serotherapy eliminate T cells, but they also target B and natural killer (Thrasher and Williams, 2017).
cells (Willemsen et al., 2015). Alternatively, T-cell depletion in the graft For these reasons, during the past decade, Richter’s group
prior to haploidentical infusion reduces graft versus host disease (GvHD) exploited an LV carrying the TCIRG1 gene under the control of the
but causes delayed immune recovery, making patients more susceptible elongation factor 1α short (EFS) promoter, and confirmed the
to infections (Bertaina et al., 2017).
For these reasons, in vivo T-cell depletion has been developed and
suitability of a GT approach by allowing nine out of 12 mice to
successfully employed, using post-transplant cyclophosphamide survive (Löfvall et al., 2019). In this study, transduced oc/oc c-Kit+
unmanipulated HSCT in osteopetrotic patients (Even-Or et al., 2021). (also known as Kit+) fetal liver cells were transplanted into
Depletion of both donor and recipient alloreactive cells promotes sublethally irradiated oc/oc pups by temporal vein injection, 1 day
engraftment and decreases risk of GvHD (Even-Or and Stepensky, after birth. Long-term (19-25 weeks)-surviving mice showed
2021). reversal of the osteopetrotic bone phenotype both in vitro, by
In osteopetrosis, myeloablative regimens are usually required to ensure
osteoclast culture, and in vivo, through the quantification of C-
proper donor engraftment. However, this needs to be balanced with the
higher risk of conditioning toxicity in such patients. Indeed, specific terminal telopeptide of type I collagen (CTX-I) (Box 1) in the serum
complications are frequent in the autosomal recessive osteopetrosis and histopathology analysis of the bones (Löfvall et al., 2019).
(ARO) setting. Prophylaxis or early therapy with defibrotide is Along the same lines, these authors previously demonstrated that
recommended to prevent venous occlusive disease of the liver, while this clinically applicable LV can also ex vivo correct circulating
antifungal prophylaxis may prevent post-transplant Pneumocystic jirovecii CD34+ cells (Box 1) of ARO patients, which rescues the resorptive
pneumonia. Acute pulmonary arterial hypertension often develops in ARO
capacity of ARO patient-derived osteoclasts in vitro (Moscatelli et al.,
patients in the first months after the transplant, and combined therapy with
bosentan (endothelin receptor antagonist) and sildenafil (a
2018). They also demonstrated that transduced ARO CD34+ cells are
phosphodiesterase type 5 inhibitor) should be initiated as soon as able to engraft in immunodeficient non-obese diabetic SCID Il2rγ−/−
possible. (NSG) mice with a rate similar to cord blood CD34+ cells.
Additionally, human CD34+ cells isolated from the bone marrow
and spleen of transplanted NSG mice are able to differentiate into
bone-resorbing osteoclasts in vitro (Moscatelli et al., 2018, 2013).
Innovative therapies for ARO Importantly, in preclinical GT studies, partial correction (in terms
In recent years, the outcome of HSCT has strongly improved as a of mixed donor chimerism or low vector copy number) seems to be
result of new drug combinations for conditioning and RIC sufficient to ensure osteoclast function and could possibly lead to
regimens. However, the availability of HLA-matched donors still clinical benefits (Johansson et al., 2006; Thudium et al., 2016).
remains an open issue, particularly for patients belonging to ethnic These observations are in accordance with the clinical data in Disease Models & Mechanisms
minorities. Because of the limited efficacy of HSCT, owing to the patients post-HSCT, which show that 5% donor chimerism (or even
severity of the disease and the short time window in which patients as low as 2% in some cases) allows sustained hematopoietic
can be treated, alternative therapeutic strategies have been recovery and normal bones at X-ray examination (Even-Or and
developed. Stepensky, 2021; Orchard et al., 2015). Thus, GT may provide a
cure for patients even if low numbers of transduced cells are
Preclinical and clinical studies transplanted and engraft in the bone marrow.
Lentiviral vector gene therapy The results achieved by Richter’s group paved the way for the
Gene therapy (GT), the therapeutic delivery of a healthy copy of the establishment of a new phase I clinical trial (Fig. 3), which started
mutated gene through viral vectors, has provided a successful cure enrolling ARO patients carrying TCIRG1 gene mutations
for many hematopoietic diseases in the past decades (Ferrari et al., (NCT04525352, https://clinicaltrials.gov/ct2/show/NCT04525352).
2021). Preclinical models of GT for TCIRG1-dependent Similar results have been obtained by our group, restoring the
osteopetrosis have been developed during these years, osteoclast activity of ARO patients ex vivo by using a clinically
demonstrating the efficacy and safety of this therapeutic approach optimized LV carrying the TCIRG1 gene under the control of the
as a valid alternative for patients lacking a compatible donor. phosphoglycerate kinase (PGK) promoter. Of note, we

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REVIEW Disease Models & Mechanisms (2021) 14, dmm048940. doi:10.1242/dmm.048940

demonstrated that transduced and expanded circulating CD34+ cells Prenatal treatment
from ARO patients are bona fide long-term hematopoietic In utero transplantation of donor HSCs was tested to prevent early-
progenitors able to engraft in NSG mice and to repopulate onset irreversible bone defects and bone marrow fibrosis in the oc/
multiple lineages in secondary NSG recipients (Capo et al., 2021). oc mouse model. Pregnant females were injected in utero at
14.5 days post-coitum and transplant success was observed in 35%
HSC sources and collection of mutant oc/oc mice that survived past the expected lifespan of
As already mentioned, bone marrow harvest is not feasible in ARO oc/oc mice. Although smaller in size, they showed restored
patients due to dense bone marrow cavity sclerosis. However, these osteoclast resorption capacity, normalization of bone density and
patients present a high frequency of circulating CD34+ cells in their eruption of incisors, providing evidence that transplants have to be
blood, which have been exploited for autologous backup before performed very early in life to avoid the development of secondary
HSCT, to be reinfused in case of graft failure (Steward et al., 2005). irreversible defects (Frattini et al., 2005). These data were further
Steward et al. (2005) reported successful granulocyte colony- confirmed by a second study reporting results obtained from in utero
stimulating factor (G-CSF)-based HSC mobilization in two patients injection of allogeneic fetal liver cells, and this treatment showed a
with atypical osteopetrosis, having a poor CD34+ cell count in similar percentage of success (Tondelli et al., 2009). Importantly, in
peripheral blood, to guarantee an adequate autologous backup humans, in utero transplantation has already been successfully
before transplantation. However, there are no other reports of performed in cases of primary immunodeficiencies (Magnani et al.,
hematopoietic stem cell (HSC) mobilization in children with 2019) and could also prevent secondary neurological damage
classical severe ARO so far. If proven safe and effective, this caused by nerve compression. However, in utero transplantation
approach could favor the harvest of an adequate amount of CD34+ could be applicable only to pre-term diagnosis and may be
cells from the blood for autologous back-up transplantation (Box 1) challenging in humans.
or gene correction.
Conversely, patients affected by typical early-onset ARO present Novel xenograft model
a high number of circulating CD34+ cells in peripheral blood. These Recently, Palagano et al. (2020) developed an oc/oc (Tcirg1-
cells are easily accessible, although exchange apheresis (Box 1) or dependent) mouse model in the immunodeficient NSG background
multiple rounds of collection might be necessary to achieve an to study the osteoclast generation from patient cells in vivo,
adequate cell dose. In vitro CD34+ cell expansion has been performing xenotransplantation of ARO CD34+ cells in an
developed to increase cell number while maintaining hematopoietic osteopetrotic setting. This murine model displayed typical NSG
cell stemness and engraftment capacity. An HSC expansion features, such as the complete lack of B, T and NK cells, and, in
protocol, based on the small pyrimidoindole derivative UM171 parallel, osteoclast-rich osteopetrosis with severe growth
(Box 1), has recently been applied in an innovative clinical trial in retardation, short lifespan, defective tooth eruption and
the context of cord blood transplantation (NCT02668315, https:// extramedullary hematopoiesis. However, the short lifespan of
clinicaltrials.gov/ct2/show/NCT02668315; Cohen et al., 2020). these mice, resulting from the fast progression of the disease, did
This protocol was successfully used for ARO circulating CD34+ not allow a study of the corrective potential of human cells on the
cells after gene correction, showing long-term engraftment and bone phenotype. In addition, the NSG oc/oc model lacks human M-
multilineage reconstitution in NSG mice (Fig. 3) (Capo et al., 2021). CSF expression, which impedes human osteoclast differentiation,
HSC expansion, alone or in combination with mobilization, meaning that this cytokine is species specific. If implemented for
would allow the therapeutic limitations imposed by the reduced the expression of human M-CSF, this model could represent a
number of cells that can be retrieved in ARO patients to be valuable tool to study the homing and engraftment of ARO patients’
overcome, limiting the therapy burden, especially in very young and CD34+ cells, as well as the efficacy of GT, in the correct
severely affected patients. pathological context.

Proof-of-concept studies Systematic administration of deficient protein

Novel therapeutic approaches are currently being studied, with the ARO caused by osteoclast-extrinsic deficiency, such as in the case
final aim to provide a safe and effective alternative treatment for of TNFSF11 mutations, requires a different approach, because
ARO patients who are not eligible for current standard care defective cells are not of hematopoietic origin. RANKL
(summarized in Table 1). replacement therapy has been explored in Tnfsf11−/− mice,

Disease Models & Mechanisms

Table 1. Proof-of-concept studies for the development of alternative therapeutic approaches to cure different ARO forms
Therapeutic strategy Osteopetrosis form Experimental model Reference
Prenatal treatment: in utero HSCT Tcirg1 oc/oc mouse model Frattini et al., 2005; Tondelli et al., 2009
Xenograft model Tcirg1 NSG oc/oc mouse model Palagano et al., 2020
RANKL replacement therapy Tnfsf11 Tnfsf11–/– mouse model Lo Iacono et al., 2012; Cappariello et al., 2015;
Menale et al., 2019
Transplant of osteoclast precursors Ctsk Ctsk–/– mouse model Jacome-Galarza et al., 2019
Csf1r Csf1r mouse models
iPSCs TCIRG1 oc/oc-derived iPSCs Neri et al., 2015
TCIRG1-defective iPSCs Chen et al., 2019; Xian et al., 2020
CLCN7 CLCN7-derived iPSCs Hennig et al., 2019; Rossler et al., 2018
Non-genotoxic conditioning To be tested in ARO – –
ARO, autosomal recessive osteopetrosis; HSCT, hematopoietic stem cell transplantation; iPSC, induced pluripotent stem cell; NSG, non-obese diabetic severe
combined immunodeficiency Il2rγ−/−; RANKL, receptor activator of nuclear factor kappa-B ligand.

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REVIEW Disease Models & Mechanisms (2021) 14, dmm048940. doi:10.1242/dmm.048940

starting early after birth. Subcutaneous delivery of recombinant et al., 2020) vector expressing the TCIRG1 gene and then induced to
soluble RANKL (sRANKL) for 1 month significantly improves differentiate into osteoclasts, partially restoring their bone-
bone defects and hematopoietic organ architecture, limiting nerve resorptive potential. Of note, gene editing in TCIRG1-defective
compression (Lo Iacono et al., 2012). ARO has been successfully demonstrated using a murine iPSC
Promising results have been obtained by exploiting the use of platform derived from the oc/oc mouse model, in which murine
biotechnological devices implanted subcutaneously, enabling the iPSC clones were genetically corrected using a bacterial artificial
release of sRANKL and allowing osteoclastogenesis in Tnfsf11−/− chromosome by a homologous recombination approach (Neri et al.,
mice. In particular, Cappariello et al. (2015) generated a bio-device 2015). These approaches could provide an additional therapeutic
harboring primary osteoblasts, cultured on 3D hydroxyapatite strategy for severe forms of osteopetrosis, although safety concerns
scaffolds carrying an immobilized metalloproteinase 14 catalytic still hamper their clinical use.
domain, which is able to cleave RANKL into the active sRANKL.
Implanted mice showed progressively improved weight and size and Non-genotoxic conditioning
rescued osteoclastogenesis in tibial histological sections when Successful HSCT, and autologous HSC transplantation after gene
multiple diffusion chambers were implanted (Cappariello et al., correction, requires vacating recipient HSC niches to allow donor
2015). cell engraftment and guarantee long-term hematopoietic and
Also in Tnfsf11−/− mice, Menale et al. (2019) exploited the use of immune functions. As already mentioned, significant
a 3D culture system based on a magnesium-doped hydroxyapatite/ improvements in conditioning regimens have been made so far,
collagen I biocompatible scaffold, which closely recapitulates bone starting with the use of RIC (Shadur et al., 2018). However, in
physicochemical characteristics. The scaffold was seeded with patients with pre-existing organ toxicity or in those younger than
Tnfsf11−/− mesenchymal stem cells, previously transduced with an 1 year of age, chemotherapy-based conditioning regimens are
LV overexpressing human sRANKL. Subsequently, one or two poorly tolerated and result in major morbidity and mortality. New
scaffolds were implanted subcutaneously in Tnfsf11−/− mice and approaches for treating these patients have recently emerged, based
extensive vascularization was observed after 2 months. This on the use of minimal-intensity conditioning to reduce the burden of
innovative strategy was able to drive the differentiation of TRAP conditioning regimens for patients affected by SCID. In particular,
(also known as ACP5)-positive osteoclasts, although osteopetrotic the use of monoclonal antibody-based conditioning specific for
features were still present in bones at sacrifice (Menale et al., 2019). CD45 (also known as PTPRC) or CD117 (also known as KIT)
The development of innovative biotechnological strategies taking allowed the specific depletion of the target HSPC population,
advantage of a bio-device delivering sRANKL, inducing avoiding a widespread toxic effect of the drug on the other cell
osteoclastogenesis in RANKL-deficient mice, supports the feasibility populations. Moreover, this approach seems to be well tolerated and
of novel approaches to treat systemic cytokine deficiencies. can achieve curative engraftment, even in patients with severe pre-
existing conditions (Agarwal et al., 2019; Straathof et al., 2009).
Transplant of osteoclast precursors A clinical trial exploiting an anti-CD117 antibody is currently
A recent elegant study found that osteoclasts are long-lived syncytia ongoing to treat SCID patients (NCT02963064, https://clinicaltrials.
that acquire new nuclei by iterative fusion of circulating blood gov/ct2/show/NCT02963064). In parallel, antibody drug conjugates
monocytes (Jacome-Galarza et al., 2019). In particular, the authors (ADCs), originally developed for cancer therapy, are an emerging
of this study abrogated RANK or CD115 expression in the bone class of non-myeloablative agents. Specifically, a monoclonal
marrow HSC progenitors (using Csf1rcre mice) or in bone marrow antibody is bound to a drug or a toxin, which is specifically
HSCs, and in embryonic erythro-myeloid progenitors (EMPs) internalized into the target cells, allowing targeted cell population
(using Flt3cre mice). They demonstrated that osteoclasts originating depletion (Abadir et al., 2019).
from EMPs are necessary for normal bone development, whereas The efficacy of CD45.2 ADC in HSCT conditioning was
osteoclasts originating from bone marrow HSC progenitors are demonstrated with a rat anti-mouse monoclonal antibody
essential for bone turnover in adulthood. As a consequence, conjugated to saporin (SAP), a catalytic N-glycosidase ribosome-
transplantation of monocytic cells is able to sustain inactivating protein that halts protein synthesis (Palchaudhuri et al.,
osteoclastogenesis and bone resorption in both early-onset ARO 2016). In mice, CD45.2–SAP preserved normal bone marrow
and adult-onset osteopetrotic mice, in the absence of HSC architecture compared to total body irradiation, which instead
engraftment (Jacome-Galarza et al., 2019). This approach would reduced vascular integrity and bone marrow cellularity. Mice
allow repeated infusions and could be exploited as a bridge therapy conditioned with CD45.2–SAP rapidly recovered their peripheral
before allogeneic HSCT or autologous GT. myeloid cells and had a survival advantage when exposed to Disease Models & Mechanisms
infections (Palchaudhuri et al., 2016). Additionally, conditioning
Gene-corrected induced pluripotent stem cells with CD45.2–SAP resulted in significant chimerism after
In the past decades, patient-derived induced pluripotent stem cells transplantation, even in a pathological mouse model (Castiello
(iPSCs) have been extensively applied to investigate the et al., 2021).
pathobiology of different diseases and to test innovative Another efficacious ADC is CD117–SAP immunotoxin coupled
treatments, thereby overcoming the limitations imposed by the with T cell-depleting agents. It has been proven to selectively
availability of specimens from patients. An iPSC line generated deplete more than 99% of host HSCs without causing clinically
from a CLCN7-defective ARO patient was used to test the significant side effects and to favor rapid and efficient donor HSC
integration of the Sleeping Beauty transposon (Box 1) carrying engraftment. Importantly, blood cell counts and immune cell
CLCN7 cDNA (Hennig et al., 2019; Rossler, 2018). function are preserved and treated mice effectively respond to both
More recently, iPSCs derived from ARO patients carrying a viral and fungal challenges (Czechowicz et al., 2019). The CD117
homozygous mutation in TCIRG1 were generated and corrected by ADC was also exploited successfully in MHC-mismatched
two different groups. Cells were transduced and genetically allotransplantation (Li et al., 2019b) and in hemophilia A GT
corrected by a retroviral (Chen et al., 2019) or lentiviral (Xian mice (Gao et al., 2019).

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TCIRG1 transgenic rescue of osteoclast function using induced pluripotent stem cells
Conclusion and future perspectives derived from patients with infantile malignant autosomal recessive osteopetrosis.
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the clinical management of this disease. Novel techniques, Outcomes after unrelated umbilical cord blood transplantation for children with
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and gene-targeted sequencing panels, are further revealing the
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of novel cellular therapies, including GT approaches based on the use Corbacioglu, S., Hö nig, M., Lahr, G., Stö hr, S., Berry, G., Friedrich, W. and
of third-generation LVs for TCIRG1 deficiency. The severity of the Schulz, A. S. (2006). Stem cell transplantation in children with infantile
disease and the increased frequency of severe side effects occurring in osteopetrosis is associated with a high incidence of VOD, which could be
prevented with defibrotide. Bone Marrow. Transplant. 38, 547-553. doi:10.1038/sj.
patients undergoing hematopoietic stem cell transplantation upon bmt.1705485
conventional conditioning are increasing safety concerns, and prompt Czechowicz, A., Palchaudhuri, R., Scheck, A., Hu, Y., Hoggatt, J., Saez, B.,
the development of safe depleting agents able to create room for HSC Pang, W. W., Mansour, M. K., Tate, T. A., Chan, Y. Y. et al. (2019). Selective
engraftment in the bone marrow niche while preserving tissue hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables
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the establishment of tailored treatment programs to guarantee a cure Di Zanni, E., Palagano, E., Lagostena, L., Strina, D., Rehman, A., Abinun, M., De
for all ARO patients, irrespective of the severity of their clinical Somer, L., Martire, B., Brown, J., Kariminejad, A. et al. (2021). Pathobiologic
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Acknowledgements Driessen, G. J., Gerritsen, E. J., Fischer, A., Fasth, A., Hop, W. C., Veys, P.,
We acknowledge the many authors whose original contributions in the field have not Porta, F., Cant, A., Steward, C. G., Vossen, J. M. et al. (2003). Long-term
been cited in this Review for the sake of brevity. outcome of haematopoietic stem cell transplantation in autosomal recessive
osteopetrosis: an EBMT report. Bone Marrow. Transplant. 32, 657-663. doi:10.
Competing interests 1038/sj.bmt.1704194
The authors declare no competing or financial interests. Even-Or, E. and Stepensky, P. (2021). How we approach malignant infantile
osteopetrosis. Pediatr. Blood Cancer 68, e28841. doi:10.1002/pbc.28841
Even-Or, E., NaserEddin, A., Dinur Schejter, Y., Shadur, B., Zaidman, I. and
Funding
Stepensky, P. (2021). Haploidentical stem cell transplantation with post-transplant
This research was supported by a grant from Fondazione Telethon (TGT16C05) to cyclophosphamide for osteopetrosis and other nonmalignant diseases. Bone
A.V. and by a fellowship from the European Calcified Tissue Society to V.C. Marrow. Transplant. 56, 434-441. doi:10.1038/s41409-020-01040-9
Ferrari, G., Thrasher, A. J. and Aiuti, A. (2021). Gene therapy using
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