Development of Novel Models for
Studying Osteoclasts
Christian Schneider Thudium
Doctoral Dissertation
By due permission of the Faculty of Medicine, Lund University, Sweden.
This thesis will be defended on October 10th, 2014, at 13.00.
Segerfalk lecture hall, BMC A10
Sölvegatan 17, Lund
Faculty opponent
Professor Ulf Lerner, DDS, Ph.D.
Department of Molecular Periodontology, Umeå University
Umeå, Sweden
1
Organization
Document name
LUND UNIVERSITY
Doctoral Dissertation
Division for Molecular Medicine and Gene Therapy
Date of issue 2014.10.10
Faculty of Medicine
Author(s) Christian Schneider Thudium
Sponsoring organization
Title and subtitle
Development of novel models for studying osteoclasts
Abstract
This thesis focuses on developing and characterizing novel models for studying osteoclasts with an emphasis on how
mutations abolishing osteoclastic acidification affect osteoclast signaling and bone remodeling, as well as how to treat
patients bearing these rare mutations.
Bone remodeling is under normal circumstances a tightly balanced process where resorption of bone by osteoclasts is
followed by adequate amounts of bone formation by osteoblasts.Mutations in the TCIRG1 gene lead to severe
autosomal recessive osteopetrosis (ARO) in both mice and man, characterized by lack of bone resorption as a result of
abolished acid secretion, an increased number of non-resorbing osteoclasts, but normal or even increased bone
formation.
The first two papers are based on studies in adult mice transplanted with stem cells from either osteoclast-rich
osteopetrotic mice (oc/oc) or osteoclast-poor osteopetrotic mice (RANK KO). The first paper focuses on characterizing
the bone and cellular phenotype in the adult osteopetrotic mouse model and find that bones, are both bigger and, in
contrast to the endogenous oc/oc mouse model, stronger in mice transplanted with osteopetrotic stem cells compared
to wild type transplanted mice. In the second paper we compare the osteoclast-rich osteopetrotic oc/oc transplanted
model to adult mice transplanted with stem cells from the osteoclast-poor RANK KO mouse, and find that
maintaining non-resorbing osteoclasts in vivo increases bone formation. In the third paper we investigate the role of
bone matrix, cell stage and resorptive function on osteoclast mediated anabolic signaling. In paper IV human stem cells
from patients with ARO were investigated and we provide the first proof-of-principle for lentiviral mediated correction
of resorptive function in CD34+ derived osteoclasts from ARO patients.
The results can be devided into two exciting branges. Data obtained in the adult osteopetrotic mouse models
encourage further development of molecules targeting the acidification process in osteoclasts when treating low bone
mineral density diseases. Furthermore, the correctional results in the ARO patient cells are very promising, and will
serve as basis for further development of clinical gene therapy of osteopetrosis.
Key words Osteoclast, Bone remodeling, Gene therapy, hematopoietic stem cells, Osteopetrosis, Osteoporosis
Classification system and/or index terms (if any)
Supplementary bibliographical information
Language English
ISSN and key title 1652-8220
ISBN 978-91-7619-040-1
Recipient’s notes
Number of pages
Price
Security classification
I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all
reference sourcespermission to publish and disseminate the abstract of the above-mentioned dissertation.
Signature
2
Date
Development of Novel Models for
Studying Osteoclasts
Christian Schneider Thudium
Division of Molecular Medecine and Gene Therapy
Department of Laboratory Medicine
Faculty of Medicine, Lund University
And Nordic Bioscience A/S, Denmark
Faculty opponent:
Ulf Lerner, DDS., Ph.D.
3
Copyright © Christian Schneider Thudium
Division of Molecular Medicine and Gene Therapy
Lund University, Faculty of Medicine Doctoral Dissertation Series 2014:111
ISBN 978-91-7619-040-1
ISSN 1652-8220
Cover: Nicolai Bentsen
Printed in Sweden by Media-Tryck, Lund University
Lund 2014
En del av Förpacknings- och
Tidningsinsamlingen (FTI)
4
Table of Contents
Abbreviations
7
Articles included in this thesis
9
Introduction
11
Skeletal Function
13
Bone Remodeling and Coupling
15
Bone Modeling
15
Bone Remodeling
16
Coupling in Remodeling and Between Bone Cells
18
Osteoporosis
19
Osteoclasts
21
Hematopoiesis
21
Osteoclastogenesis
22
Bone Resorption
24
Osteoblast Lineage Cells
27
Osteopetrosis
29
Pathogenesis
29
Current Treatment for Osteopetrosis
30
Osteoclast-rich Osteopetrosis
31
Osteoclast-poor Osteopetrosis
36
Evidence for Osteoclast Derived Coupling Signals
39
Gene therapy
41
Targeting Hematopoietic Stem Cells
41
Lentiviral Vector Mediated Gene Transfer
42
Aims of the Present Study
43
5
Summary of findings
45
Paper I
45
Paper II
46
Paper III
47
Paper IV
48
General Discussion
49
Investigating Osteopetrosis in Adult Mice
49
Phenotype Penetrance in Adult Osteopetrotic Mice
50
Secretion of Coupling Factors is Dependent on Osteoclast Subtype
52
Level of Correction for Gene Therapy of ARO
53
Post-transcriptional Regulation of Vector Driven TCIRG1 expression
54
Future Perspectives
57
Identifying Osteoclast Derived Anabolic Factors
57
Targeting Acidification When Treating Bone Turnover Diseases
57
Critical Issues for Further Development of Clinical Gene Therapy
59
Popular Scientific Summary
63
Papers not included in this thesis
65
Acknowledgements
67
References
69
6
Abbreviations
ADA
ADO
AE2
ARO
BM
BMU
BRC
CAII
CGD
ClC-7
CM
DC-STAMP
FLC
GFP
HLA
HSC
IMO
KO
MAR
M-CSF
MMP
RANK
RANKL
SCID
SFFV
SIN
TCIRG1
TGF-β
TRAP
wt
adenosine-deaminase
autosomal dominant osteopetrosis
anion exchanger II
autosomal recessive osteopetrosis
bone marrow
basic multicellular unit
bone remodeling compartment
carbonic anhydrase II
chronic granulomatous disease
chloride channel 7
conditioned media
dendritic cell-specific transmembrane protein
fetal liver cell
green fluorescent protein
human leukocyte antigen
hematopoietic stem cell
infantile malignant osteopetrosis
knock-out
mineral apposition rate
macrophage colony stimulating factor
matrix metalloproteinase
receptor activator of nuclear factor κB
receptor activator of nuclear factor κB ligand
severe combined immunodeficiency
spleen focus forming virus
self-inactivating
T cell immune regulator 1
transforming growth factor β
tartrate resistant acid phosphate
wild type
7
8
Articles Included in this Thesis
I) Dissociation of bone resorption and bone formation in adult mice with a nonfunctional V-ATPase in osteoclasts leads to increased bone strength
Kim Henriksen, Carmen Flores, Jesper S. Thomsen, Annemarie Brüel, Christian S. Thudium,
Anita V. Neutzsky-Wulff, Gerling E. J. Langenbach, Natalie Sims, Maria Askmyr, Thomas J.
Martin, Vincent Everts, Morten A. Karsdal and Johan Richter
Plos One, 2011; 6(11): e27482
II). 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.
Christian S. Thudium, Ilana Moscatelli, Carmen Flores, Jesper S. Thomsen, Annemarie Brüel,
Natasja S. Gudmann, Ellen-Margrethe Hauge, Morten A. Karsdal, Johan Richter and Kim
Henriksen
Calcified Tissue International, 2014; 95(1):83-93
III) A specific subtype of osteoclasts secretes factors inducing nodule formation by
osteoblasts.
Kim Henriksen, Kim V. Andreassen, Christian S. Thudium, Karoline N.S. Gudmann, Ilana
Moscatelli, Catherine E. Crüger-Hansen, Ansgar S. Schulz, Morten H. Dziegiel, Johan
Richter, Morten A. Karsdal and Anita V. Neutzsky-Wulff
Bone, 2012; 51(3):353-61
IV) Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells
restores osteoclast function in infantile malignant osteopetrosis.
Ilana Moscatelli*, Christian S. Thudium*, Carmen Flores, Ansgar Schulz, Maria Askmyr,
Natasja Stæhr Gudmann, Nanna Merete Andersen, Oscar Porras, Morten Asser Karsdal, Anna
Villa, Anders Fasth, Kim Henriksen and Johan Richter
Bone, 2013; 57(1):1-9.
* These authors contributed equally to this work
9
10
Introduction
The osteoclast is an extraordinary cell type with the unique ability to degrade the
calcified bone matrix. The non-redundant nature of the osteoclast often makes cell
functionality the center of pathological disease states, such as osteoporosis and
osteopetrosis, as well as an interesting target for therapeutic development.
In contrast to the layman perception of the skeleton as rather inert, bone is a living
organ which is constantly degraded and rebuilt throughout life to maintain mineral
homeostasis and repair microdamage hereby ensuring skeletal integretry; a process
known as bone remodeling. This process involves the resorption of old bone by
osteoclasts, and the formation of new bone by the osteoblasts. Bone remodeling is
strictly regulated and characterized by a tight coupling between resorption and
formation so that under normal circumstances resorbed bone is always replenished
with equal amounts of new bone in the healthy adult. Hormonal changes or genetic
defects can skew the balance and lead to pathological changes in bone. Mutations
which affect osteoclastic acdidification, result in defective bone resorption and leads
to the severe heredetary disorder autosomal recessive osteopetrosis (ARO). These
individuals have a high number of non-resorbing osteoclasts, and despite lacking bone
resorption, have normal or increased bone formation, suggesting that the osteoclast,
but not necessarily bone resorption is important for ongoing bone formation.
The thesis focuses on developing in vivo and in vitro osteoclast models for
investigating how genetic defects in osteoclasts affect its regulation of bone
remodeling, as well how to rescue osteoclast function in ARO by gene therapy
mediated correction of mutational defects.
11
12
Skeletal Function
Bone, together with cartilage, makes up the human skeleton, and performs several
important functions in the human body. The skeleton itself acts as structural support
for the rest of the body and as attachment site for muscles and ligaments for
locomotion. It serves as a protective scaffold to shield vital organs, including the bone
marrow (BM). The skeleton also has a general metabolic function as a major source of
inorganic ions, actively participating in maintenance of mineral homeostasis and acidbase balance. Moreover, the bones serve as a large a reservoir of growth factors and
cytokines. Finally, the bones provide the environment for hematopoiesis within the
marrow cavities (Clarke, 2008)
In general the bones of the skeleton can be divided into two categories based on
anatomy: flat bones, such as the skull and the mandible (which will not be discussed
further), and long bones such as the femur, the tibia and the vertebrae (Clarke, 2008).
A schematic view of a long bone is given in Figure 1. Long bones are made up of two
different types of bone, namely cortical bone and trabecular bone (also known as
cancellous bone). (Clarke, 2008). Approximately 80% of the human skeleton consists
of cortical bone while 20% consists of trabecular bone; however, the ratio between
the two types varies greatly between skeletal sites (Seeman and Delmas, 2006). The
most prominent role of cortical bone is to ensure the mechanical properties of the
skeleton, while trabecular bone, in addition to its mechanic role, also serves various
metabolic functions (Seeman and Delmas, 2006). The varying roles of the two types
of bone tissue also naturally influence on their respective remodeling rates.
13
Growth plate
Cancellous bone
Periosteum
Marrow cavity
Diaphysis
Cortical bone
Endosteum
Metaphysis
Epiphysis
Figure 1. Schematic drawing of a long bone
A schematic view of the structure and composition of a long bone. The bone is divided in to epiphysis,
metaphysis, and diaphysis. The bone consists of two kinds of bone, trabecular (cancellous), and cortical
bone. The inner and outer parts of the bone are called the endosteum and the periosteum, respectively.
The bone marrow is located in the cavity at the center of the bone. Adapted from (www.bbc.co.uk,
2014).
14
Bone Remodeling and Coupling
Frost described in what he termed intermediary organization separate distinct
functions of bone cells in the skeleton such as growth, modeling, remodeling and
fracture repair (Frost, 1983).
The forming and shaping of bone and maintenance and repair throughout life is
carried out by the combined cellular efforts of bone modeling and remodeling. These
two processes shape the skeleton and adapt it to the incoming load and strain by
optimizing bone strength, minimizing mass and maintaining mechanical competence.
Growth and fracture repair is largely outside the scope of the thesis and will not be
tread upon, while modeling is briefly described, and remodeling gone through
thoroughly.
Bone Modeling
Bone modeling is the physiological process by which the bones are shaped during
development and adapted in response to physiological changes or external mechanical
stimuli. The modeling process occurs mainly during growth.
Modeling incidences can occur as bone formation without prior bone resorption on
the periosteal surfaces of bone to increase bone size and modify bone size and shape
according to genetic programming or as an adaptation response to prevailing loading
circumstances (Clarke, 2008; Martin and Seeman, 2008; Wang and Seeman, 2013).
Importantly, during bone modeling, resorption and formation are processes occuring
independently of each other at different locations, so the two are in essence
uncoupled (Clarke, 2008). As modeling incidences are limited during adulthood,
where remodeling is more prominent, the process is outside the scope of this thesis
and will not be discussed further.
15
Bone Remodeling
Bone remodeling is a lifelong process and, on average, the entire skeleton is replaced
every 10 years. The process involves replacing old bone with new to mend microfractures and maintain bone strength, as well as contributing to optimal control of
mineral homeostasis, all processes important for maintaining a healthy skeleton. As
bone ages, micro damage causes the bones to become fragile which compromises the
weight bearing function of the skeleton. Bone remodeling secures adequate
maintenance of bones ensuring their functional capacity (Seeman and Delmas, 2006).
Bone remodeling is taking place in both cortical and trabecular bone, however, the
remodeling rates in the two compartments are quite different. The estimated
remodeling rate in trabecular bone is 28% per year, while only being an estimated 4%
per year in cortical bone (Manolagas, 2000).
Spatial orchestration of bone cells
The bone remodeling process is carried out by the coordinated efforts of a lineup of
cells (osteoclasts, osteoblasts, osteocytes, and bone lining cells) which together
comprise the basic multicellular units (BMUs)(Martin and Sims, 2005; Martin and
Seeman, 2008; Parfitt, 2002; Sims and Gooi, 2008). Approximately 1- 2 million
active BMUs are estimated to be present at any given time in the human skeleton
(Martin and Seeman, 2007). Bone remodeling in trabecular bone in humans, has
been shown to take place in specialized bone remodeling compartments (BRCs)
(Andersen et al., 2009; Hauge et al., 2001; Eriksen et al., 2007). In these
compartments, a canopy of cells create an optimal microenvironment for bone
remodeling, screening the remodeling site from the surrounding BM compartment,
while perforating blood vessels supply progenitor cells and regulatory factors for the
BRC (Andersen et al., 2009; Eriksen et al., 2007; Hauge et al., 2001).
The bone remodeling cycle
The remodeling process is carried out in a sequence of events involving the
engagement from a quiescent phase to an activation phase followed by a resorption
phase, reversal phase, formation phase, and termination phase (Parfitt, 2002; Parfitt,
2006; Seeman and Delmas, 2006). Normally, these events occur in a sequential
manner where resorbed bone is always replaced by adequate amounts of new bone
and complete replacement, a process referred to as coupling (Figure 2).
The bone remodeling cycle is started by the recruitment of osteoclast precursors to
the site of remodeling. This is believed to involve signaling by apoptotic osteocytes
and bone lining cells initiated by local micro damage (Burr, 2002; Henriksen et al.,
2009b). The signaling is thought to be mediated by decreased transforming growth
factor β (TGF-β) signaling from the osteocytes, which under normal circumstances
inhibits osteoclastogenesis and bone resorption (Henriksen et al., 2009b). Osteocytes
16
were also recently shown to express Receptor Activator of Nuclear Factor κβ Ligand
(RANKL), a key osteoclastogenic cytokine, potentially adding to the recruitment of
osteoclast precursors to the site of remodeling (Xiong et al., 2011; Nakashima et al.,
2011). These mechanisms cause osteocytes and bone lining cells to specifically target
a local bone site for remodeling by attracting osteoclast precursors and stimulate
osteoclastogenesis.
Following activation, bone lining cells remove a thin layer of matrix followed by
retraction to make space for the osteoclasts (Everts et al., 2002; Henriksen et al.,
2009b), and formation of the BRC by the bone lining cells. Osteocyte signaling
recruits vessels to penetrate the BRC wall allowing access of bone cell precursors from
the BM and circulation (Andersen et al., 2009; Eriksen et al., 2007). Osteoclast
precursors summoned from the circulation or BM (Andersen et al., 2009; Parfitt,
2006; Sims and Gooi, 2008) are in the presence of RANKL differentiated into
mature osteoclasts and initiate resorption, lasting 2-4 weeks for the individual
osteoclast (Manolagas, 2000). As the osteoclasts complete resorption of the target site,
they undergo apoptosis (Manolagas, 2000), likely induced by calcium or TGF-β
released from the bone matrix (Houde et al., 2009; Lorget et al., 2000; Nielsen et al.,
2007). The remodeling cycle enters a lag phase, called the reversal phase, where the
osteoclasts are removed from the resorption pit, which is then prepared for bone
formation. This phase involves the formation of a “cement line” separating old bone
from new. The cellular origin of this line is currently not clear, but recent findings
suggest that the presence of so called “reversal cells” of osteoblastic origin might be
involved in the initiation of the formation phase (Andersen et al., 2013). Also
osteoclasts have been implicated in this process, by possibly depositing osteoblast
targeted signals for regulation of bone formation (Everts et al., 2002; Karsdal et al.,
2007; Karsdal et al., 2008). Osteoblast precursors are recruited to the site of bone
remodeling and are differentiated into mature osteoblasts through mechanisms, which
are still being investigated. Recruitment signals possibly consist of a mix of osteoclast
and bone derived signals (Henriksen et al., 2009b; Henriksen et al., 2014; Karsdal et
al., 2007; Karsdal et al., 2008; Kim et al., 2012; Lotinun et al., 2013; Martin et al.,
2009; Martin and Sims, 2005; Pederson et al., 2008; Ryu et al., 2006; Takeshita et
al., 2013). During the formation phase mature osteoblasts deposit osteoid, followed
by layering of collagens and mineralization of the matrix, resulting in newly formed
bone. The formation phase lasts 4-6 months, and after adequate amounts of bone
have been formed, the cycle enters the termination phase, concluding the bone
remodeling cycle. Osteoblasts either become bone embedded osteocytes, develop into
bone lining cells, or undergo apoptosis (Henriksen et al., 2009b). The exact
mechanism for this transition is not yet fully understood, but possibly includes the
secretion of sclerostin by osteocytes, inhibiting further bone formation by the
osteoblasts (Henriksen et al., 2009b; Poole et al., 2005).
17
Microfracture
Mineralization
4‐6 months
Formation
Reversal
Activation
Resorption
Figure 2. An overview of the bone remodeling cycle
Bone remodeling is likely initiated by micro-fractures inducing osteocyte apoptosis, which leads to
activating signals to the osteoclasts and initiation of bone resorption. After ended bone resorption, the
cycle enters reversal phase, with recruitment of osteoblasts followed by bone formation leading to
replenishment of resorbed bone. Adapted from (Segovia-Silvestre et al., 2008)
Coupling in Remodeling and Between Bone Cells
In order for bone mass to remain relatively constant throughout life, the amount of
bone formation must equal the amount of bone resorption during bone remodeling
(Hattner et al., 1965; Takahashi et al., 1964). Since bone remodeling requires the
combined efforts of both bone resorbing osteoclasts and bone forming osteoblasts,
cross talk between the different cell types is necessary to control this process, also
referred to as coupling (Howard et al., 1981; Sims and Walsh, 2012; Karsdal et al.,
2007; Martin et al., 2009; Martin and Sims, 2005). It is important to emphasize that
the strict coupling described here only occurs at the time of peak bone mass in adults.
Earlier in life, the balance is dominated by a positive gain in bone, which is mainly
mediated by modeling. After reaching peak bone mass, the balance shifts towards an
overall negative bone balance for the remaining lifetime (Martin et al., 2009). Cells of
the osteoblast lineage, from progenitor to osteocyte, produce stimulatory and
inhibitory signals and factors that tightly regulate osteoclastogenesis function and
activity; the most prominent probably being the osteoclastogenic factor RANKL and
its soluble decoy receptor osteoprotegerin (Lacey et al., 1998; Simonet et al., 1997;
Boyle et al., 2003). In addition the osteoclasts have also been shown to produce
18
signals affecting the osteoblast recruitment, formation and activity, both through
release of factors from the degraded bone and from the osteoclasts themselves. A large
effort has been aimed at identifying osteoclast derived factors affecting osteoblasts and
bone formation, for obvious therapeutic reasons, but the signaling mechanisms seem
to be more complicated than initially thought, and not limited to a single molecule
acting as master regulator (Henriksen et al., 2014; Karsdal et al., 2007; Karsdal et al.,
2008; Kim et al., 2012; Lotinun et al., 2013; Martin and Seeman, 2008; NegishiKoga et al., 2011; Pederson et al., 2008; Ryu et al., 2006; Takeshita et al., 2013;
Walker et al., 2008; Zhao et al., 2006).
Osteoporosis
Changes in the delicate balance of bone remodeling can result in pathological
conditions, such as for example osteoporosis, where changes in osteoclast and
osteoblast activity lead to a general degradation of the skeleton; an effect often
mediated by reductions in hormone production (Manolagas, 2000).
Epidemiology and pathology
Osteoporosis is a common disease affecting an estimated 12 million people in the US
alone, and the disease accounts for roughly 1.5 million osteoporotic fractures in the
US each year (U.S.Department of Health and Human Services, 2004). With an aging
population, the medical and socioeconomic repercussions will increase over time
(Burge et al., 2007). The disease is characterized by a systemic loss of bone mass,
strength, and micro architecture, which leads to an increased risk of fractures (NIH
Consensus Development Panel on Osteoporosis Prevention, 2001). Bone mineral
density (BMD) is commonly used as a hallmark for osteoporosis, and can be assessed
with dual x-ray absorptiometry (DXA). Osteoporosis is defined by a T score of less
than 2.5, meaning more than 2.5 standard deviations below the average of a young
adult mean BMD (Kanis, 1994). There are a range of significant risk factors for the
development of osteoporosis, the most prominent being loss of sex hormones during
menopause but they also include age, diet, ethnicity, alcohol intake, smoking, genetic
predispositions, immobility, and medication (Seeman, 2003).In most cases,
osteoporosis is caused by a gradual unbalancing of the bone remodeling process,
where bone resorption exceeds bone formation resulting in a net bone loss (Drake and
Khosla, 2013). This change in remodeling is in itself a naturally occurring process
caused by aging. The estrogen deficiency occurring in postmenopausal women,
however, has a profound effect on the balancing of bone resorption and bone
formation, leading to increased osteoclastogenesis, increase in osteoclast activity, and
osteoclast longevity (Drake and Khosla, 2013).
19
Current treatment for osteoporosis
Different treatments modalities are available for osteoporosis, such as anti-resorptiveor anabolic treatments, or dietary supplements such as Vitamin D and calcium. The
anti-resorptive drugs are currently the most widely used treatments for osteoporosis
and, as the name indicates, they focus on removing the osteoclasts or inhibiting their
ability to resorb the bone matrix. Anti-resorptives very efficiently lower bone
resorption and this results in significant reduction of fracture rate in patients.
However, secondary to their anti-resorptive effect is a decrease in bone formation
which limits their overall effect. The anti-resorptives include bisphosphonates (BPs),
oestrogen and selective oestrogen receptor modulators (SERMs), and Denosumab, a
neutralizing RANKL antibody, as well as calcitonin and strontium renalate (McClung
et al., 2006; Papapoulos, 2013; Russell et al., 2008; Watts, 2013; Cummings et al.,
2009; Rogers et al., 2011; Delmas et al., 1997; Ettinger et al., 1999; Chesnut, III et
al., 2000; Meunier et al., 2004). Parathyroid hormone (PTH), or active fragments
here of, are currently the only treatment increasing bone formation, however, besides
the effect on osteoblasts is a general increase in bone turnover actively increasing bone
resorption secondary to bone formation, limiting its overall potential (Cosman and
Greenspan, 2013; Neer et al., 2001). An overview of the different treatments is given
in Table 1. Overall the current treatments suffer from the coupling between
resorption and formation which limits their overall efficacy. New treatments may be
targeted at osteoclast function while preserving bone formation, or by activating
osteoblasts in face of normal bone resorption (Thudium et al., 2012; Eisman et al.,
2011; McClung et al., 2014).
Table 1. Osteoporosis treatments
Treatment
Example
Target
OCL→ OB
coupling
Change in
formation
References
Current treatments
Bisphosphonate
Alendronate
Bone resorption
Yes
Decreased
(Liberman et al.,
1995)
α-RANKL
Denosumab
Bone resorption
Yes
Decreased
(McClung et al.,
2006)
Calcitonin
Miacalcin
Bone resorption
Yes
Decreased
(Sexton et al., 1999;
Chesnut, III et al.,
2000)
SERMs
Raloxifene
Bone resorption
Yes
Strontium
Ranelate
Protelos
Bone resorption/bone
formation
Teriperatide
Bone formation/bone
resorption
Cathepsin K
inhibitor
Odanacatib
α-Sclerostin
Romosozumab
PTH
(or fragments)
(Delmas et al., 1997;
Ettinger et al., 1999)
(Increased)
(Meunier et al.,
2004)
Yes
Increased
(Neer et al., 2001)
Bone resorption
(Yes)
Decreased
(Eisman et al., 2011)
Bone formation
No (modeling)
Increased
(McClung et al.,
2014)
In development
20
Osteoclasts
Osteoclasts are unique cells in that they are the only cells responsible for the
resorption of bone. In light of this fact, the correct functionality and regulation of the
osteoclast is crucial for maintaining bone homeostasis.
Hematopoiesis
Osteoclasts are derived from hematopoietic stem cells (HSCs) which represent a rare
population of cells with the potential of long-term self renewal and a capacity for
multi lineage differentiation (Figure 3). This process leads to the replenishment of
billions of new blood cells for the human body every day (Ema et al., 2005; Ogawa,
1993). HSCs mainly reside in the BM and are primarily present in quiescent state in
human adults. HSCs are capable of either self renewal through symmetric division
giving rise to two identical progeny cells or through asymmetric division giving rise to
two different daughter cells that can either sustain long-term hematopoiesis or act as
progenitor cell for the distinct blood lineages (Morrison and Kimble, 2006). The fate
of the HSC is believed to be directed by a combination of different stochastic events
during division and extracellular signals from the HSC niche (Enver et al., 1998;
Metcalf, 1998; Morrison and Weissman, 1994; Ogawa, 1999). Hematopoietic stem
cells have a very low representation in blood and BM and because no single marker
identifying this population is known, generally a combination of markers/antibodies
is used to identify and describe purified stem- and progenitor cell populations, such as
cluster of differentiation (CD). CD34 is expressed on the surface of human
progenitor and HSCs regardless of cell cycle status, and this marker is often used to
enrich human HSCs (Moore, 2009; Sidney et al., 2014). The hematopoietic nature
of CD34+ cells make them rare in the peripheral blood and they are instead often
isolated from the BM or umbilical cord blood or through mobilization into peripheral
blood (Civin et al., 1984; Gianni et al., 1989).
21
Figure 3. An overview of the hematopoietic hierarchy.
Hematopoiesis comprises the process in which HSCs residing in the bone marrow (BM) give rise to all
types of mature blood cells in the peripheral blood (PB). Osteoclasts are differentiated from progenitors
of the monocytic lineage. LT-HSC (long term HSC), ST-HSC (short-term HSC), MPP/LMPP
(multipotent progenitor/lymphoid-primed multipotent progenitor, CMP (common myeloid
progenitor), CLP (common lymphoid progenitor), MEP (megakaryocyte-erythrocyte progenitor), GMP
(granulocyte-monocyte progenitor), Pre-DC (pre-dendritic cell), Pre-NK (pre-natural killer cell), Pre-B
(pre-B lymphocyte), and Pre-T (pre-T lymphocyte).
Osteoclastogenesis
Mature osteoclasts are large multinucleated cells which originate from the
hematopoietic stem cell (HSC) and which are formed through fusion of mononuclear
precursors in a process known as osteoclastogenesis. During osteoclastogenesis, HSCs
differentiate into common myeloid progenitor cells and then into CD14 expressing
monocytes directed by the presence of a range of specific cytokines and the expression
of transcription factors such as PU.1. (Shalhoub et al., 2000; Way et al., 2009;
Tondravi et al., 1997). PU.1 deficient mice lack both macrophages and osteoclasts
emphasizing its importance in osteoclastogenesis (Tondravi et al., 1997). Two
cytokines are crucial for osteoclast differentiation, namely macrophage colony
stimulating factor (M-CSF) and RANKL. M-CSF acts on proliferation and survival
22
of the monocytes (Felix et al., 1990) and induces the expression of receptor activator
of nuclear factor κB (RANK) on the surface of maturating osteoclasts (Arai et al.,
1999). Binding of RANKL to RANK activates a number of intracellular pathways, of
which nuclear factor κB (NFκB), c-fos, nuclear factor activated T cells c1 (NFATc1),
and TNF receptor associated factor 6 (TRAF6) are the most prominent and required
for osteoclastogenesis (Wang et al., 1992; Lacey et al., 1998; Li et al., 2000; Zhao et
al., 2007). The importance of the RANK/RANKL interaction is underlined by the
complete lack of osteoclasts in RANK knock-out (KO) mice (Dougall et al., 1999),
and development of infantile malignant osteopetrosis in patients with homozygous
mutations in RANK and RANKL (Guerrini et al., 2008; Sobacchi et al., 2007).
Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and acts as a negative
regulator of osteoclastogenesis and the overall osteoclast differentiation is dictated by
the RANKL/OPG ratio (Simonet et al., 1997). Committed osteoclast precursors will
start to fuse, a process which is regulated by dendritic cell-specific transmembrane
protein (DC-STAMP) (Yagi et al., 2005). As pre-osteoclasts fuse, they start expressing
a range of proteins, such as tartrate resistant acid phosphatase (TRAP), cathepsin K,
Chloride channel 7 (ClC-7), and the osteoclastic vacuoloar H+-ATPase (V-ATPase),
including its subunit T-cell immune regulator 1 (TCIRG1)(also referred to as the a3
subunit), which are important for correct osteoclast resorptive function (Figure
4)(BURSTONE, 1959; Kornak et al., 2000; Kornak et al., 2001; Gowen et al.,
1999).
OPG
Hematopoietic
stem cell (CD34+)
OPG
OPG
Lineage commitment
Multipotent progenitor
Monocyte
(CD14+)
PU.1
CSF1R
c‐Fos
c‐Fms
M‐CSF
RANKL
Pre‐osteoclast
RANK
NFκβ
TRAF6
NFATc1
TRAP
DC‐
STAMP
RANKL
Fused
polykarion
c‐Src
αvβ3
MITF
NFATc1
RANKL
Mature
osteoclast
Cathepsin K
V‐ATPase (a3)
ClC‐7
OSTM1
AE2
CAII
CalcitoninR
Figure 4. Osteoclastogenesis.
um>3221</RecNum><IDText>Lowve from HSCs. Commitment to the osteoclast lineage involves
differentiation of monocytes through pre-osteoclasts, fusion of pre-osteoclasts, and polarization of
mature osteoclasts. A number of transcription factors and cytokines direct the expression of multiple
proteins needed for proper differentiation and function of the osteoclasts. Inspired by (Boyle et al., 2003;
Mellis et al., 2011).
23
Bone Resorption
The osteoclast is unique in its capacity to dissolve the inorganic and organic part of
the bone matrix (Blair et al., 1986; Baron et al., 1985), processes which require a
range of osteoclast specific proteins and functions. In addition, the degradation
process depends upon cytoskeletal rearrangements leading to the formation of a
specialized compartment between the osteoclast and the bone crucial for bone
resorption (Zou and Teitelbaum, 2010). A schematic overview of a bone attached
osteoclasts is given in Figure 5.
At osteoclast maturation, cytoskeletal changes in the osteoclast lead to polarization of
the cell and the formation of a sealing zone which defines a closed space between the
osteoclast and the bone called the resorption lacunae (Vaananen and Horton, 1995).
The function of the sealing zone is to create an environment closed off from the
immediate extracellular milieu, which functions like an “extracellular lysosome”. The
sealing zone is formed by the assembly of an intracellular actin ring composed of Factin fibers (Vaananen and Horton, 1995; Lakkakorpi et al., 1989). Upon attachment
to the bone surface, the tight binding of the sealing zone is mediated by the osteoclast
cell surface protein αvβ3-integrin and also requires the signal transducers c-src, Syk,
and proline-rich tyrosine kinase 2 (Boyle et al., 2003; Teitelbaum, 2007; Zou et al.,
2007). In vivo studies in mice lacking the β3 part of the receptor have shown that
osteoclasts are formed but mice become osteopetrotic due to osteoclast dysfunction
(McHugh et al., 2000). Within the sealing zone, the osteoclast generates a highly
folded part of the plasma membrane called the ruffled border (membrane). This
ruffled border is formed by the fusion of intracellular acidic vesicles with the plasma
membrane bounded by the sealing zone (Teitelbaum, 2007). Active bone resorption
is taking place in the resorption lacuna below the ruffled border. The capacity of the
mature osteoclast to resorb bone depends on the secretion of a series of ions and
enzymes. Dissolution of the inorganic matrix is mediated through acidification of the
resorption lacunae (Baron et al., 1985). This acidification process involves proton
transporting V-ATPases, including TCIRG1, brought to the ruffled border via vesicle
fusion, which then transports protons across the ruffled border (Baron et al., 1985;
Frattini et al., 2000; Kornak et al., 2000; Toyomura et al., 2000; Blair et al., 1989).
In order to maintain electroneutrality, the proton secretion is balanced by transport of
chloride ions through the chloride anti-porter ClC-7 (Kornak et al., 2001; Kasper et
al., 2005; Schaller et al., 2005; Schlesinger et al., 1997; Henriksen et al., 2004). The
transport of electrolytes through the V-ATPase and ClC-7 results in secretion of
hydrochloric acid into the resorption lacunae, creating an acidic environment of
approximately pH 4.5. The low pH is crucial for dissolution of the inorganic matrix
and proteolytic enzyme activity (Blair et al., 1989; Baron et al., 1985; Kornak et al.,
2001). Pathological mutations in the V-ATPase or ClC-7 can lead to severe
osteopetrosis in both mice and humans due to lack of bone resorption by the
osteoclasts (Table 2)(Del Fattore et al., 2006; Frattini et al., 2000; Kornak et al.,
24
2000; Kornak et al., 2001; Li et al., 1999; Marks, Jr. et al., 1985; Taranta et al.,
2003; Waguespack et al., 2007).
Protons needed for transport via the V-ATPase are provided by the intracellular
enzyme carbonic anhydrase II (CAII), which catalyses the conversion of CO2 and
H2O into H2CO3, and further hydrolyzed into H+ and HCO3 (Gay and Mueller,
1974; Henriksen et al., 2008). Patients lacking CAII function develop osteopetrosis
from loss of osteoclast function (Sly et al., 1983) . HCO3 is then exchanged with
chloride ions through the anion exchanger 2 (AE2) in the basolateral membrane,
ensuring continued availability of ions for ongoing acidification and bone resorption
(Teti et al., 1989).
The acidification of the resorption lacunae mobilizes the inorganic part of the bone
exposing the collagen rich organic matrix (Nielsen et al., 2007). The exposed collagen
matrix is then degraded by proteases released into the resorption lacunae (Henriksen
et al., 2006; Delaisse et al., 2003; Gelb et al., 1996). The most prominent enzyme in
this regard is the lysosomal cysteine protease cathepsin K, which is secreted from the
ruffled border and activated in the acidic environment (Lecaille et al., 2008). Upon
activation, cathepsin K efficiently cleaves collagen type I. Failure to produce
functional cathepsin K due to mutations in the CTSK gene encoding cathepsin K
results in the bone disease pycnodysostosis (Gelb et al., 1996), characterized by
impaired degradation of the collagen matrix leading to osteosclerosis of the bone,
skull deformities, short stature and weak bones with increased fracture rate (FratzlZelman et al., 2004; Gowen et al., 1999; Gelb et al., 1996).
The role of matrix metallo-proteases (MMPs) in bone degradation is not completely
clear (Delaisse et al., 2003; Everts et al., 1993; Henriksen et al., 2006). Studies have
shown that MMPs might participate in bone degradation. However, under normal
circumstances MMP activity seems to be negligible compared to cathepsin K, and
restricted to specific skeletal sites (Delaisse et al., 2003; Henriksen et al., 2012). The
exact protease profile of osteoclasts appears to be at least partly dependent on the
bone type that is being resorbed, such flat bones or long bones, and context, such as
in pathological disease states. This can also be referred to as osteoclast subtype
(Henriksen et al., 2011).
Products generated through bone resorption are endocytosed by the osteoclast,
transported through the cell and released through the secretory domain opposite the
osteoclasts resorptive domain (Nesbitt and Horton, 1997). After a number of
resorptive cycles, the osteoclast undergoes apoptosis; a process likely induced by the
release of calcium and TGF-β from the resorbed matrix (Houde et al., 2009; Hughes
et al., 1996; Lorget et al., 2000; Nielsen et al., 2007).
25
Figure 5. Schematic drawing of the bone resorbing osteoclasts.
After attachment to the bone surface through integrin binding, the osteoclast secretes acid into the
resorption lacuna through the coordinated efforts of the osteoclastic V-ATPase and the chloride channel
ClC-7. This process ensures breakdown of the inorganic matrix and is followed by the secretion of the
collagen degrading proteinase cathepsin K. Protons and chloride ions needed for proper acidification are
provided by the membrane protein AE2 and the enzyme CAII. ClC-7, chloride channel 7; CAII,
carbonic anhydrase; AE2, anion exchanger 2.
26
Osteoblast Lineage Cells
The other key player in bone remodeling is the osteoblast. The osteoblasts are
mononuclear bone cells responsible for bone formation and derive from
multipotential mesenchymal stem cells (MSCs) located in the BM (Capulli et al.,
2014). Cells of the osteoblast lineage exist as three functiionally different cell types in
the skeleton: osteoblasts, osteocytes, and bone lining cells. The differentiation of the
MSCs into osteoblast lineage cells is controlled by a range of hormones, cytokines and
corresponding receptors which regulate the expression of transcription factors needed
for correct lineage differentiation.
Maybe the most important role of the osteoblast is the formation of new bone. Bone
formation by osteoblasts can be divided into three phases: I) a proliferation phase
involving biosynthesis of the extracellular components, II) development and
organization of the extracellular matrix, and III) extracellular matrix mineralization.
The process itself includes the deposition of osteoid layer consisting mainly of
collagen type I and bone specific proteins. Osteoid deposition is followed by
mineralization where hydroxyappatite crystals are deposited in the collagen
framework (Capulli et al., 2014). Morphology and expression of mRNA and proteins
vary greatly in response to the requirements of the different phases of bone formation.
After bone formation is completed, the osteoblasts either become embedded in the
bone as osteocytes, turn into bone lining cells, or die through apoptosis (Manolagas,
2000; Aubin, 2001; Capulli et al., 2014).
In addition to bone formation, a secondary role for the osteoblast lineage cells is the
production of cytokines regulating osteoclastogenesis; the most important being
RANKL and its soluble decoy receptor OPG. The expression is thought to be mainly
isolated to osteoblast precursors and terminally differentiated osteocytes (Nakashima
et al., 2011). The production of cytokines is in turn regulated by hormones such as
PTH, Vitamin D, and estrogen, as well as cytokines such as TGF-β, but the
regulatory mechanisms are complex and still under investigation.
Osteocytes are cells derived from osteoblasts embedded in the bone following bone
formation (Manolagas, 2000). Osteocytes are the most abundant bone cell in the
body, mainly due to its long lifespan, estimated to be around 25 years (Knothe Tate
et al., 2004). The osteocytes are morphologically characterized by having dendritic
cytoplasmic extensions connecting them to each other and the bone surface allowing
them to survive and signal from within the bone matrix. The general function of the
27
osteocyte is thought to include mechanosensory activity actively directing the actions
of osteoblasts and osteoclasts within the BMU, in response to external physiological
stress (Manolagas, 2000; Bonewald, 2011). In addition, osteocytes are believed to
direct the targeted remodeling happening in older bones and in response to
microcracks; a signal which is mediated through the apoptosis of the osteocyte and
sclerostin signaling (Bonewald, 2011; Sims and Vrahnas, 2014).
The thin layer of cells covering all quiescent bone surfaces is called bone lining cells
and also derive from the osteoblast lineage (Manolagas, 2000; Miller et al., 1989).
Here they form a barrier between the BM and the bone matrix. The bone lining cells
are believed to be important for the process of targeted bone remodeling (Everts et al.,
2002; Andersen et al., 2013).
28
Osteopetrosis
Osteopetrosis is a heterogenous group of rare genetic diseases characterized by either
lack of osteoclasts or defects in bone resorption by osteoclasts (Balemans et al., 2005;
Bollerslev et al., 1993; Del Fattore et al., 2006; de Vernejoul and Benichou, 2001;
Del Fattore et al., 2008; Tolar et al., 2004). Its pathological impact aside,
investigation of patients and mice with osteopetrosis have provided invaluable insight
into how osteoclasts function and how bone cells communicate with each other.
Pathogenesis
Radiographic findings in a patient with increased bone density were first described by
Albers-Schönberg (Albers-Schönberg, 1904) and since then a number of different
osteopetrosis forms have been described, ranging from mild or asymptomatic such as
autosomal dominant osteopetrosis (ADO), to very severe or lethal as in autosomal
recessive osteopetrosis (ARO), often referred to as infantile malignant osteopetrosis
(IMO)
The disease presents with a general increase in skeletal bone mass due to increased
density of the bone. Radiological findings in different forms of osteopetrosis can
display as evident sclerostation of the bone, often with a bone-in-bone appearance in
phalanges, long bones and pelvic bones, all varying in severity dependent on type
(Balemans et al., 2005; Shapiro, 1993; Loria-Cortes et al., 1977; Tolar et al., 2004).
The sclerotic bones are brittle and prone to fractures, a phenotype that is probably
attributed to the congenital developmental nature of the disease (Neutzsky-Wulff et
al., 2010).
Osteopetrosis is often associated with compression of the cranial nerves due to bone
thickening at the base of the skull, which can cause visual impairment and hearing
loss (Sobacchi et al., 2013). In addition, patients suffer from disturbed tooth
eruption, as osteoclasts are normally responsible for resorbing a path through the jaw
for the developing tooth (Helfrich, 2005).
29
Table 2. Mutations leading to osteopetrosis
Protein
(Gene)
Disease
Function
Osteoclast/Osteoblast
profile
Reference
TCIRG1/a3
(TCIRG1)
ARO
Acidification
Increased osteoclast number
Normal/increased bone
formation
(Kornak et al., 2000; Frattini
et al., 2000)
CLC-7
(CLCN7)
ARO/ADO
Acidification
Increased osteoclast number
Normal/increased bone
formation
(Kornak et al., 2001; Frattini
et al., 2003; Cleiren et al.,
2001; Campos-Xavier et al.,
2003)
OSTM1/
(OSTM1)
ARO
Acidification
Increased osteoclast number
Normal/increased bone
formation
(Chalhoub et al., 2003;
Pangrazio et al., 2006)
SNX10/
(SNX10)
ARO
Endosomal/vesicular
trafficking
Unknown
(Pangrazio et al., 2013)
RANKL/
(TNFSF11)
ARO
Osteoclastogenesis
No osteoclasts
Decreased bone formation
(Sobacchi et al., 2007)
RANK/
(TNFRSF11A)
ARO
Osteoclastogenesis
No osteoclasts
Decreased bone formation
(Guerrini et al., 2008;
Pangrazio et al., 2012)
cathepsin K/
(CTSK)
Pycnodysostosis
Proteolysis
Increased osteoclast number
osteoblast phenotype unclear
(Gelb et al., 1996)
Current Treatment for Osteopetrosis
While mild forms of osteopetrosis can go undetected for many years, ARO is a fatal
disease and without hematopoietic stem cell transplantation the disease is lethal before
the age of 6 years. Often the lethal outcome is caused by pancytopenia and recurrent
infections which are treated with transfusions and antibiotics to alleviate the
symptoms (Fasth and Porras, 1999; Wilson and Vellodi, 2000). The first BM
transplantations of osteopetrotic children were performed in the seventies (Ballet et
al., 1977; Coccia et al., 1980) after transplantation studies had shown permanent cure
of osteopetrotic gl/gl and microthalamic (mi/mi) mice (Walker, 1975a; Walker,
1975b). Today BM transplantations haves been applied in a number of patients and
the long term clinical outcome evaluated (Driessen et al., 2003; Fasth and Porras,
1999; Gerritsen et al., 1994; Sobacchi et al., 2013).
The outcome of BM transplantation is highly dependent on human leukocyte antigen
(HLA) matching, and the age of the patient at transplantation (Sobacchi et al., 2013).
Studies have shown that transplantations performed before the age of 10 months
increase the chance of full engraftment, while transplantations performed at older age
increase the risk of graft rejection or autologous reconstitution (Sobacchi et al., 2013).
Transplantations performed in patients older than 2 years markedly increase the risk
of hypercalcemia and the risk of neuronal damage from compression of the skull (as
reviewed in (Askmyr et al., 2009a; Sobacchi et al., 2013). This is in line with
transplantation studies in mice showing that the osteopetrotic phenotype in oc/oc
30
mice can be reversed using non-ablative neonatal transplantation of healthy donor
cells (Flores et al., 2010). Overall, BM transplantation is an effective therapy for
treatment of osteopetrosis if initiated early. However, in cases where no HLA
matched donors can be found, there is a clear need for alternative treatment options,
such as gene therapy.
Osteoclast-rich Osteopetrosis
The severe form of osteopetrosis, ARO, is a rare recessive disorder affecting
approximately 1 in 200,000-300,000 (Balemans et al., 2005). ARO is most often
caused by homozygous or compound heterozygous mutations in one of the two
genes, TCIRG1 or CLCN7, resulting in ablated osteoclast function, but not number
and is therefore referred to as an osteoclast-rich osteopetrosis (Figure 6)(Del Fattore et
al., 2006; Frattini et al., 2000; Frattini et al., 2003; Kornak et al., 2000; Kornak et al.,
2001; Segovia-Silvestre et al., 2008; Sobacchi et al., 2001; Sobacchi et al., 2013).
Mutations in CLCN7 can also lead to autosomal dominant osteopetrosis (ADO) and
intermediate autosomal recessive osteopetrosis (IARO), with an osteoclast-rich
cellular phenotype although the disease manifestation in both cases is generally more
benign than the homozygous form. Mutations in the genes encoding osteopetrosisassociated transmembrane protein 1 (OSTM1), sorting nexin 10 (SNX10) and
Pleckstrin homology domain-containing family M member 1 (PLEKHM1), are also
described as being osteoclast-rich forms of osteopetrosis, but will not be mentioned
further in this thesis, as they are relatively poorly understood, and only few cases have
been identified (Aker et al., 2012; Megarbane et al., 2013; Pangrazio et al., 2006;
Pangrazio et al., 2013; Van et al., 2007). An overview of the different mutations is
given in Table 2
Osteoclast-rich osteopetrosis is characterized by an increased number of nonresorbing osteoclasts that may result in osteosclerosis of the bone and abnormal BM
cavity development which limits hematopoiesis. Despite reduced bone resorption,
bone formation is ongoing leading to large increases in bone mass further worsening
the pathology (Bollerslev and Andersen, Jr., 1988; Del Fattore et al., 2006; Frattini et
al., 2000; Kornak et al., 2000; Kornak et al., 2001). Although bone mass is increased,
the bones are brittle and prone to fracture.
31
Figure 6. Osteopetrosis, osteoclast number, and bone turnover
Schematic representations of the correlation between osteoclast number, bone resorption and bone
formation in different forms of osteopetrosis. Adapted from (Karsdal et al., 2007)
TCIRG1 deficiency
TCIRG1 is a subunit of the osteoclast vacuolar type H+-ATPase (V-ATPase) encoded
by the TCIRG1 gene. The a3 subunit is a 7 transmembrane domain protein and is a
member of the V-ATPase a-subunit family. The subunit is primarily expressed in
osteoclasts, where it is crucial for functional bone resorption (Manolson et al., 2003).
V-ATPases in general are important for regulation of pH within many intracellular
compartments, such as lysosomes, endosomes, and secretory vesicles (hence the name
“vacuolar”), and does so by ATP-driven transportation of H+ ions across the plasma
membrane (Sun-Wada and Wada, 2013). The V-ATPase itself is a large
multisubunit/multidomain membrane localized protein consisting of two
subdomains, a structural V0 domain and an enzymatic V1 (Jefferies et al., 2008). A
schematic overview of the V-ATPase is given in Figure 7.
Pathological mutations in the TCIRG1 gene lead to defective acidification and lack of
bone resorption in both human patients and animal models (Blair et al., 2004; Del
Fattore et al., 2006; Frattini et al., 2000; Kornak et al., 2000; Moscatelli et al., 2013;
Taranta et al., 2003). Mutations in TCIRG1 accounts for nearly 50% of all ARO
cases, making this the most frequent cause of osteopetrosis (Del Fattore et al., 2008;
Sobacchi et al., 2001; Sobacchi et al., 2013). Much of the current knowledge about
the V-ATPase and its role in osteoclasts are derived from extensive studies in
osteopetrotic patients and mouse models bearing mutations in TCIRG1.
The first connection between a proton pump gene and osteopetrosis was indicated by
studies in the spontaneous osteopetrotic oc/oc mouse model (Marks, Jr. et al., 1985;
Seifert and Marks, Jr., 1985; Frattini et al., 2000; Kornak et al., 2000). Multiple
32
mutational studies led to the identification of the TCIRG1 gene encoding the a
vacuolar proton pump subunit as a cause of osteopetrosis development in both
humans and mice (Frattini et al., 2000; Kornak et al., 2000; Li et al., 1999). In
humans, mutations are variable and include deletions, insertions, missense mutations
and splice site mutations (Sobacchi et al., 2001; Del Fattore et al., 2006; Del Fattore
et al., 2008). The main cellular phenotype in patients with mutations in TCIRG1 is
the higher amount of non-resorbing osteoclasts found in bone biopsies compared to
osteoclasts in healthy individuals (Flanagan et al., 2000; Frattini et al., 2000; Taranta
et al., 2003). Osteoclasts from ARO patients are larger in size and generally display
with more nuclei, but otherwise act phenotypically as normal osteoclasts in terms of
attachment to bone and polarization (Taranta et al., 2003). In vitro studies of
osteoclasts with mutations in the TCIRG1 gene show that despite normal
development, the lack of proton secretion abolish acid secretion (Blair et al., 2004),
limiting the capacity of osteoclasts to resorb bone (Del Fattore et al., 2006;
Moscatelli et al., 2013; Taranta et al., 2003). Despite lack of bone resorption, these
patients show increased bone formation markers and, in alignment with this, a
correlation has been shown between the number of non-resorbing osteoclasts and the
number of osteoblasts in vivo (Del Fattore et al., 2006; Taranta et al., 2003). In
summary, these findings are indicative of ongoing bone formation in osteoclast-rich
osteopetrosis despite the lack of bone resorption. The increased bone formation might
also explain the severity of the phenotype compared to other types of osteopetrosis.
From a theurapeutic perspective recent studies in the TCIRG1 mutated oc/oc mouse
using neonatal transplantation of healthy or gene corrected precursor cells indicate
that the bone phenotype can be corrected using hematopoietic stem cell targeted gene
therapy(Flores et al., 2010; Johansson et al., 2007).
33
ADP + Pi
A
ATP
B
A
B
B
V1
G
E
A
E
G
H
C
D
a3
V0
e
cytoplasm
F
d2
c
c’
c’’
membrane
lumen
H+
Figure 7. Structural overview of the osteoclastic V-ATPase
The V-ATPase transports protons across the ruffled border, driven by the enzymatic conversion of ATP
to ADP. This maintains the low pH in the resorption lacuna essential for proper bone resorption.
Adapted from (Jefferies et al., 2008). a3 – TCIRG1.
ClC-7 deficiency
ClC-7 is a homodimeric membrane protein encoded by the CLCN7 gene. It is
expressed in the lysosomes and at the ruffled border of mature osteoclasts where it
functions as a voltage gated Cl-/H+ exchanger transporting chloride ions into the
resorption lacuna. This transport actively balances the conductance generated by the
proton flow during bone resorption, as described previously described (Henriksen et
al., 2004; Henriksen et al., 2008; Henriksen et al., 2009a; Neutzsky-Wulff et al.,
2008; Schaller et al., 2005). The importance of ClC-7 in bone resorption is
highlighted by the fact that mutations in ClC-7 lead to osteopetrosis in humans, such
as ARO, ADO and IARO, and in mice as seen in the ClC7-/- mouse or the knockin
ADOII mice (Alam et al., 2014; Bollerslev et al., 1989; Frattini et al., 2003; Kornak
et al., 2001; Neutzsky-Wulff et al., 2008; Cleiren et al., 2001). At the membrane, the
ClC-7 homodimer binds to OSTM1, an interaction which is critical for stabilization
of the channel and protection from degradation (Lange et al., 2006; Leisle et al.,
2011) and lack of OSTM1 leads to osteopetrosis in both mice and humans
(Chalhoub et al., 2003).
Patients with mutations in what was most likely ClC-7 were first described by Albers
Schönberg in the early 20th century (Albers-Schönberg, 1904). In contrast to
osteopetrosis originating from TCIRG1 mutations, ClC-7 derived osteopetrosis exists
in two types; the severe recessive form ARO, and the more benign dominant forms
ADO and IARO, as previously mentioned; of which the later will not be discussed
34
further (Benichou et al., 2000; Bollerslev et al., 1993; Chu et al., 2006; Cleiren et al.,
2001; Frattini et al., 2003; Waguespack et al., 2003; Waguespack et al., 2007;
Kornak et al., 2001).
Homozygous or compound heterozygous mutations in ClC-7 give rise to autosomal
recessive osteopetrosis due to acid secretion failure and the phenotype is similar to
osteopetrosis caused by TCIRG1 mutations. Mutations in ClC-7 account for
approximately 15% of ARO cases (Frattini et al., 2003; Kornak et al., 2001). These
patients, in addition to the severe bone phenotype, suffer from primary neuronal
defects leading to neuronal storage disease and retinal athrophy, an effect accounted
to the secondary expression of ClC-7 in brain tissue (Frattini et al., 2003; Kasper et
al., 2005; Kornak et al., 2001). This is in contrast to patients with TCIRG1
mutations where neuronal damage, such as blindness is most often caused by
compression of the cranial nerve (Fasth and Porras, 1999). The cellular phenotype in
patients with ARO due to ClC-7 mutations is very similar to the one seen in patients
with TCIRG1 mutations.
Although not being characterized as ARO, the milder ADO has contributed greatly
to expanding the knowledge on the underlying osteoclast pathology and mechanisms
of osteoclast-rich osteopetrosis. In contrast to ARO, ADO is a heterogenous disease in
terms of severity, and the penetrance has been estimated to be around 66% of
carriers. This heterogeneity can most likely be attributed to the dimeric nature of
ClC-7. Some carriers show no visual signs of disease except for mild increases in
BMD, but the phenotypic penetrance varies greatly between patients (Benichou et al.,
2000; Waguespack et al., 2003; Waguespack et al., 2007; Frattini et al., 2003;
Cleiren et al., 2001), and seem to also correlate with osteoclast resorptive function in
vitro (Chu et al., 2006). ADO is the most common form of osteopetrosis with an
estimated prevalence estimated to be 5.5:100,000, however a more conservative guess
is around 1:100,000 (Benichou et al., 2000).
Some of the clinical manifestations observed in ADO patients include delayed
fracture healing (Bollerslev, 1989; Benichou et al., 2000; Waguespack et al., 2007),
cranial neuropathies, and osteomyelitis (Waguespack et al., 2007).
Studies of the cellular phenotype in bone biopsies from ADO patients show high
numbers of abnormally large TRAP positive osteoclast on the inner bone surface
(Bollerslev et al., 1993), due to increased survival of osteoclasts not resorbing bone
(Nielsen et al., 2007). In correlation with these findings, high levels of TRAP activity
have been found in serum of ADO patients (Alatalo et al., 2004; Bollerslev and
Andersen, Jr., 1988; Waguespack et al., 2002). The osteoclasts show no ruffled
border formation and have a high number of intracellular vesicles, which correlates
well with studies indicating that functional chloride transport is important for
vesicular trafficking (Blair et al., 2004; Del Fattore et al., 2006; Kornak et al., 2001;
Novarino et al., 2010; Sobacchi et al., 2013). ADO subjects have also shown
increased eroded areas but non-significant decreases in resorption rate, indicating
35
reduced resorption capability of these osteoclasts (Bollerslev et al., 1993). In addition,
mineral apposition rate (MAR) was increased in these patients while osteoblast
parameters were unchanged compared to control subjects (Bollerslev et al., 1989).
From a functional perspective, osteoclasts from ADO patients and ClC-7 deficient
mice have reduced or no capability of extracellular acidification in vitro (Henriksen et
al., 2009a; Neutzsky-Wulff et al., 2008).
Osteoclast-poor Osteopetrosis
Recent studies have identified rare cases of osteopetrosis where mutations lead to a
complete absence of osteoclasts in vivo, described in the literature as an osteoclastpoor osteopetrosis (Guerrini et al., 2008; Sobacchi et al., 2007; Pangrazio et al.,
2012). These patients have a milder phenotype development than classical ARO
despite having no bone resorption due to their lack of osteoclasts. The difference in
phenotype acceleration is suspected to be caused by the lack of anabolic signaling
from osteoclasts.
RANKL deficiency
The absence of osteoclasts in a group ARO patients indicated mutations in the cell
differentiation process and not the bone resorptive function itself. Mutations were
identified in the TNFSF11 gene encoding the osteoclast master regulator cytokine
RANKL. These patients displayed with a severe ARO phenotype resembling that of
TCIRG1- or ClC-7 derived osteopetrosis but the phenotype developed at a slower
pace (Sobacchi et al., 2007). The cellular phenotype display with a general lack of
mature osteoclasts. Due to the non-cell autonomous origin of the RANKL mutation,
which is usually expressed by non-hematopoietic stromal cells in the BM or
osteocytes, these patients did not rescue by BM replacement (Sobacchi et al., 2007).
Interestingly, isolated monocytes readily differentiated into multinucleated resorbing
osteoclasts in vitro, in the presence of exogenous RANKL, corroborating the
involvement of a RANKL mutation in these patients (Sobacchi et al., 2007).
RANK deficiency
Another subset of patients displaying with ARO phenotype similar to patients with
RANKL mutations, and without osteoclast presence was found to bear mutations in
TNFRSF11A encoding RANK (Guerrini et al., 2008; Pangrazio et al., 2012). In
addition to the normal ARO symptoms, these patients also suffer from
hypogammaglobulinemia, indicating that RANK also plays a role in the development
of the immune system (Guerrini et al., 2008; Pangrazio et al., 2012). This is also
evident by the fact that lymph node development is hampered in RANK KO mice
(Dougall et al., 1999). The cellular phenotype in patients is very comparable to that
36
of patients with RANKL mutations in that osteoclasts are absent as a result of
disrupted RANK/RANKL signaling. In addition, very little or no osteoblasts, and
consequently bone formation, is observed in biopsies from these patients (Pangrazio
et al., 2012). In contrast to RANKL derived osteopetrosis, osteoclast precursors from
RANK KO mouse are different, in that they do not differentiate into osteoclasts
when RANKL is added in vitro, in line with the cell-autonomous nature of the
mutation (Villa et al., 2009). As with the osteoclast-rich osteopetrosis, the findings in
mouse models of osteoclast-poor osteopetrosis recapitulate the phenotypes observed
in human. Namely that lacking osteoclasts, and, thus, osteoclast-secreted anabolic
factors, leads to a reduction in bone formation and a less severe bone phenotype
compared to its osteoclast-rich counterpart, supporting the view that osteoclasts
themselves are anabolic (Figure 6 and 8).
Figure 8: Coupling of bone resorption to bone formation in healthy and disease states.
In healthy adults bone formation follows bone resorption and complete replenishment of removed bone.
In osteoclast-rich osteopetrosis the osteoclast number is increased, but resorption decreased, leading to
accumulation of old bone (dark grey). Bone formation, however, appears to follow osteoclast number
leading to increased formation of new bone (light grey). In osteoclast-poor osteopetrosis the lack of
osteoclasts cause an attenuation of bone resorption, preserving old bone (dark grey). However, since no
osteoclasts are present, bone formation is also affected leading to smaller and less severe bone phenotype.
Adapted from (Segovia-Silvestre et al., 2008)
37
38
Evidence for Osteoclast Derived
Coupling Signals
Growing evidence supports that osteoclasts themselves secrete anabolic factors not
related to their resorptive function (Figure 8). The first indications that osteoclasts are
anabolic came from findings in bone organ cultures showing that activation of
resorption by osteoclasts led to secretion of anabolic molecules or “coupling factors”
(Howard et al., 1981). Further indications that these coupling factors are secreted by
the osteoclasts and not only released from degraded bone matrix were provided by
studies in osteoclast-rich forms of osteopetrosis. In these forms, bone resorption is
often dramatically reduced due to loss of osteoclast function, while osteoclast
numbers are increased (Bollerslev et al., 1993; Del Fattore et al., 2006). Despite a lack
of bone resorption, bone formation by osteoblasts is often also increased, and the
number of osteoblasts have been found to correlate with the number of non-resorbing
osteoclasts (Del Fattore et al., 2006). In patients with osteopetrosis due osteoclast
differentation defects, such as in RANK or RANKL deficient subjects, the bone
phenotype is less severe, and bone formation is reduced compared to osteoclast-rich
osteopetrotic patients (Pangrazio et al., 2012). These findings indicate that lack of
osteoclasts reduce anabolic signaling leading to reduced bone formation during
remodeling. In alignment with mutational studies in osteopetrotic subjects, in vivo
studies in OVX rats show that inhibition of acidification inhibits bone resorption
while maintaining bone formation; findings which highlight the anabolic nature of
osteoclasts independent of their resorptive capability (Schaller et al., 2004).
Supporting these findings are experiments showing that implantation of calcium
phosphate rods containing V-ATPase inhibitors in rats attract osteoclasts but inhibit
bone resorption while inducing bone formation (Rzeszutek et al., 2003). PTH
administration in vivo normally induces a bone anabolic response. In the osteoclast
deficient c-fos -/- mouse lacking osteoclasts, administration of PTH failed to elicit an
anabolic response (Demiralp et al., 2002; Luiz de Freitas et al., 2009). In contrast, csrc -/- mice which have mature but dysfunctional osteoclasts have impaired bone
resorption, but maintain an anabolic response similar to wt mice in response to PTH
(Koh et al., 2005), suggesting that PTH mediates its anabolic potential through the
actions of osteoclasts, but independent of bone resorption. In line with animal
experiments, the effect of PTH treatment is blunted in osteoporosis patients
following bisphosphonate administration (Black et al., 2003). This reduction could
39
be caused by the ablation of osteoclasts or inhibited coupling signals following the
inhibitory actions of the bisphosphonate. However, the complex nature of these
mechanisms is highlighted by recent findings showing that combined Denosumab
and PTH treatment leads to increased BMD in postmenopausal women (Tsai et al.,
2013), suggesting that anabolic signaling by osteoclasts may be more complex than
initially thought. Although data are not in complete alignment, these findings do
suggest that osteoclasts secrete factors which are important for the ongoing coupling
between bone resorption and bone formation during bone remodeling.
Much effort has been put into identifying coupling factors, and while findings are still
controversial with no clear candidate, a number of molecules have been suggested
(Pederson et al., 2008; Quint et al., 2013; Ryu et al., 2006; Baron and Rawadi, 2007;
Vukicevic and Grgurevic, 2009; Ishii et al., 2009; Ishii et al., 2010; Takeshita et al.,
2013; Kim et al., 2012; Dacquin et al., 2011; Negishi-Koga et al., 2011; Lotinun et
al., 2013). An overview of possible candidate molecules is given in Table 3.
Table 3 Possible osteoclast derived coupling factors
Name
Effect on BF
Function
References
Afamin
+
Pre-OB migration
(Kim et al., 2012)
BMP6
+
OB stimulation
(Pederson et al., 2008)
OB stimulation
(Walker et al., 2008)
(Matsuoka et al., 2014)
Cardiotrophin-1
+
Complement
component 3a
+
OB differentiation
CTHRC1
+
OB stimulation
(Takeshita et al., 2013)
Semaphorin4D
-
OB inhibition
(Negishi-Koga et al., 2011)
Sphingosine-1phosphate
+
OB recruitment (cell stage dependent)
(Pederson et al., 2008; Quint et al.,
2013; Ryu et al., 2006; Lotinun et
al., 2013)
TRAP
+
Osteoblast activation
(Del et al., 2008)
Wnt10b
+
OB stimulation
(Pederson et al., 2008)
40
Gene Therapy
Another aspect of osteopetrosis is the treatment of this disease, and while BM
transplantations has proven efficient in many cases, there is a general need for
alternative treatment options when lacking HLA matching donors. Much progress
has been made within gene therapy in recent years and efforts are focusing on
increasing efficiency and safety of this new form of therapy.
Gene therapy is based on the principle of inserting genetic information into a cell to
restore or correct functionality. This approach could potentially offer a beneficial
alternative to HSC transplantation when treating monogenic hematological disorders
where HLA matching donors are not available.
Targeting Hematopoietic Stem Cells
A range of monogenetic diseases have by now been treated successfully with
hematopoietic stem cell targeted gene therapy, such as X-linked severe combined
immuno deficiency (SCID-X1) (Cavazzana-Calvo et al., 2000; Gaspar et al., 2004),
adenosine deaminase SCID (ADA-SCID)(Aiuti et al., 2002) and chronic
granulomatous disease (CGD)(Ott et al., 2006). Following treatment, however,
several X-SCID patients developed lymphoproliferative disease due to insertional
mutagenesis (Hacein-Bey-Abina et al., 2003a; Hacein-Bey-Abina et al., 2003b). All of
these treatment approaches used gammaretroviral vectors for gene insertion indicating
the need for better and safer vector design and targeting. In this context, lentiviral
vectors are much less liable to insertional mutagenesis in close proximity to protooncogenes, increasing the general safety profile (Cattoglio et al., 2007; Neschadim et
al., 2007). Not considering the serious side effects observed in SCID-X1 patients, a
disadvantage of gammaretroviral vectors is also their need for cell division for
transduction and integration of the pro-virus. In contrast to gammaretroviral vectors,
lentiviral vectors can transduce both dividing and non-dividing cells which minimizes
the need for using stimulatory cytokines to drive cell division (Naldini et al., 1996;
Case et al., 1999; Miyoshi et al., 1999). Lentiviral vectors are currently being
developed for gene therapy treatment in several inherited diseases, and successful gene
transfer to HSCs using lentiviral vectors has been achieved in diseases such as CGD
41
(Naumann et al., 2007), Wiskott-Aldrich syndrome (WASP) (Martin et al., 2005;
Charrier et al., 2007), and X-linked adrenoleukodystrophy (Cartier et al., 2009).
Lentiviral Vector Mediated Gene Transfer
The framework of lentiviral vectors is build around the genetics of lentivirus, a
member of the retrovirus family. These viruses include human immunodeficiency
virus 1 (HIV-1), simian immunodeficiency virus (SIV), bovine immunodeficiency
virus (BIV), feline immunodeficiency virus (FIV), and equine infectious anemia virus
(EIAV). The hallmark of retroviruses is their ability to perform reverse transcription
generating DNA from two single stranded RNA copies that make up the viral
genome, a process performed by the enzyme reverse transcriptase. It does so by
integrating the reverse transcribed DNA into the genome of the host cell, and utilizes
the cells machinery for transcription of viral genes (Durand and Cimarelli, 2011).
Viral vectors take advantage of the natural ability of lentiviruses to integrate into a cell
and transfer genetic material. The most common lentiviral vectors are based on
human immunodeficiency virus 1 (HIV-1). The severe pathology caused by wt HIV
infection in humans have prompted the design of vectors where most of the viral
genes have been removed, including its ability for replication. This allows for its use
as delivery device for permanent incorporation of a gene of interest into a target cell
genome (Schambach et al., 2013). These replication-incompetent vectors are referred
to as self-inactivating (SIN) vectors. Transcription of transgenes are driven by internal
promoters, allowing for careful selection of promoters depending on setting and
minimizing the risk of insertional activation of neighboring genes (Miyoshi et al.,
1998; Zufferey et al., 1998).
The established method for production of lentiviral vectors is by transient transfection
of plasmids into a producer cell line, such as 293T. Although many of the HIV genes
have been removed in todays lentiviral production systems, reducing risk of
recombination events resulting in replication competent vectors is paramount to their
future use. The so-called third generation lentiviral vectors separate various elements
of viral vector assembly and function onto 4 separate plasmids thereby reducing the
risk of recombination(Dull et al., 1998).
42
Aims of the Present Study
The general aim of this thesis was to develop in vitro and in vivo models for studying
osteoclasts, with special focus on the cellular aspects of the role of osteoclasts in bone
remodeling and osteopetrosis.
These experiments involved the induction of osteopetrosis in adult mice by
transplantation of HSCs from osteopetrotic mice. It also involved osteoclast
generation from human hematopoietic progenitors and the validation of lentiviral
mediated rescue of resorptive function in osteoclasts from patients with osteopetrosis.
Specific aims
1. To establish whether osteoclasts have an anabolic role in vivo, by developing
osteoclast-rich and osteoclast-poor adult mouse models of osteopetrosis
(Papers I and II)
2. To investigate whether osteoclasts, independent of bone resorption, secrete
anabolic signals for osteoblasts in vitro (Paper III)
3. To characterize the correction of resorptive function in osteopetrosis patient
CD34+ cells using a lentiviral based gene therapy approach to rescue
TCIRG1 function in vitro (Paper IV)
43
44
Summary of Findings
Paper I
Dissociation of bone resorption and bone formation in adult mice with a nonfunctional V-ATPase in osteoclasts leads to increased bone strength
Plos One, 2011; 6(11): e27482
Bone resorption by osteoclasts and bone formation by osteoblasts are under normal
circumstances tightly balanced, a process referred to as coupling. Studies within the
last decade suggest that the coupling mechanism is more complex than initially
thought and that osteoclasts play a greater part in regulation via secreted anabolic
factors, independent of bone resorption capability.
Patients and mice with mutations reducing the ability of the osteoclasts to secrete acid
have osteopetrosis, characterized by defective bone resorption, increased osteoclast
numbers, and, interestingly, normal or even increased bone formation. However, the
developmental nature of these phenotypes, particularly the large accumulation of
calcified cartilage in the bones and the short life span, limits the general applicability
of these findings. To overcome this we used fetal liver cell (FLC) transplantation from
oc/oc mice with non-resorbing osteoclasts to replace the BM of irradiated fully grown
adult mice and, hereby, inducing an osteopetrotic phenotype. We then observed the
effects on bone volume and particularly strength over a 3 to 6 months period.
In summary, we found that oc/oc transplanted mice had significantly reduced bone
resorption, while the osteoclast numbers were increased compared to control
transplanted animals. Bone formation was elevated at week 6 after transplantation but
normalized after 12 weeks and reduced from here on. μCT analysis showed a
significant increase in both femoral and vertebral bone volume. These increases
manifested in increased bone strength in both compartments. The increases were
apparent already after 3 months in the vertebrae, while femoral changes took longer,
probably due to differences in remodeling rate between the two compartments. In
conclusion, these data suggests that attenuation of acid secretion by osteoclasts in
adult mice leads to an unbalancing phenotype and increased bone strength; findings
which are important for future development of non-catabolic therapies in bone
metabolic diseases.
45
Paper II
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.
Calcified Tissue International, 2014; 95(1):83-93
After having established that transplantation of oc/oc FLCs could induce a mild
unbalancing phenotype where bone formation was maintained despite inhibition of
bone resorption, we wanted to further investigate the role of osteoclasts in controlling
bone remodeling by comparing our current osteoclast-rich model to an osteoclastpoor system with no or very few osteoclasts. Current osteoporosis treatments largely
seek to inhibit the osteoclasts and in doing so generally eliminate them. The great
drawback to these anti-resorptive treatments is the secondary loss of bone formation
happening from the coupling mechanism between osteoblasts and osteoclasts. By
inhibiting osteoclastic acid secretion instead of abolishing osteoclasts, you possibly
maintain bone formation, thus increasing the overall efficacy on turnover.
In this study we investigated the differences between osteoclast-rich oc/oc and
osteoclast-poor RANK KO transplanted mice and two corresponding control groups,
and analyzed changes in osteoclast profiles, bone volume, bone strength and bone
formation parameters. Transplantation of FLCs resulted in a mean engraftment ratio
of approximately 98%. TRAP5b, a marker of osteoclast numbers, and osteoclast
counts were 50% increased in the oc/oc recipient group as expected from previous
studies (paper I). Osteoclast numbers were decreased in RANK KO recipients, but
not completely abolished, despite 98% engraftment ratio. Analysis of in vitro
differentiated splenocyte derived osteoclasts from oc/oc recipients showed a high
number of non-resorbing osteoclasts, while splenocytes from RANK KO recipients
failed to develop into bone resorbing osteoclasts. Serum CTX-I was equally decreased
in both oc/oc and RANK KO recipients compared to respective controls. Bone
volume and bone strength were equally increased in the femur, while in the vertebrae
changes in oc/oc recipients were higher than for RANK KO recipients compared with
respective controls. Finally, dynamic histomorphometry showed an increase in MAR
as well as bone formation rate per bone surface in both oc/oc and RANK KO
recipients compared to controls. However, changes were more prominent in the oc/oc
recipients. In conclusion, these data indicate that unbalancing of bone remodeling via
oc/oc and RANK KO FLC transplantation into adult wild type (wt) mice leads to
equal declines in bone resorption, but increased bone formation when osteoclast are
preserved in vivo. These findings rationalize targeting the acid secretion process when
developing treatments for bone metabolic diseases.
46
Paper III
A specific subtype of osteoclasts secretes factors inducing nodule formation by
osteoblasts.
Bone, 2012; 51(3):353-61
Osteoclasts are important for the coupling process between bone resorption and bone
formation during bone remodeling. The aim of this study was to investigate at what
stage osteoclasts are anabolically active and whether matrix or resorptive function
affects this signaling.
Human monocytes were differentiated into osteoclasts driven by the presence of MCSF and RANKL. Conditioned media (CM) was collected from cultures of
macrophages, pre-osteoclasts or mature functional or osteopetrotic osteoclasts. The
cells were either differentiated on plastic, bone or dentin and with or without the
inhibitors diphyllin, GM6001, or E64. The osteoblastic cell line 2T3 was then treated
with conditioned media or non-conditioned control media for 12 days and bone
formation analyzed with Alizarin Red.
Osteoblasts incubated with CM from mature osteoclasts induced bone formation
while conditioned media from macrophages did not. Media from non-resorbing ARO
osteoclasts still induced bone formation by osteoclasts despite significantly lowered
bone resorptive activity. In alignment, conditioned media from osteoclasts treated
with the V-ATPase inhibitor diphyllin or the cysteine protease also induced bone
formation, although the effect was slightly reduced, compared to vehicle treated
osteoclasts, while conditioned media from osteoclasts treated with MMP-inhibitor
GM6001 were comparable to vehicle conditioned media. Finally, osteoclasts seeded
on either decalcified bone or dentine generated conditioned media that had strongly
impaired anabolic output.
The data suggest that osteoclasts, dependent and independent of their resorptive
capabilities can secrete anabolic signals which induce bone formation. Importantly
the data also show that the anabolic capability is very much dependent on the type of
matrix that cells are present on, since both dentine and decalcified bone impaired the
anabolic response. These findings are in line with studies in patients with
osteopetrosis, where mutations in the osteoclast resorption machinery abolish
resorptive function but maintain osteoclast viability, and importantly bone formation.
47
Paper IV
Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells restores
osteoclast function in infantile malignant osteopetrosis.
Bone, 2013; 57(1):1-9.
ARO (IMO) is a rare genetic disease, and is lethal in infancy in the absence of
matching BM donors. The aim of this study was to rescue to the resorptive function
of osteoclasts derived from ARO patients using lentiviral mediated gene transfer of
TCIRG1 cDNA to osteoclast precursor cells. CD34+ progenitor cells were isolated
from peripheral blood samples from 5 IMO patients and from cord blood samples of
healthy controls. They were then transduced with a self-inactivating (SIN) lentiviral
vector containing TCIRG1 cDNA and a green fluorescent protein (GFP) marker
gene. The cells were expanded (500-fold) for 2 weeks in the presence of myeloid
acting cytokines, and gradually changed from CD34 expressing to CD14 expressing
cells. Following the expansion, cells were seeded on bovine bone slices and
differentiated into mature osteoclasts for 10 days, in the presence of M-CSF and
RANKL, and osteoclastogenesis, osteoclast activity, and expression of TCIRG1 were
investigated.
The transduction efficiency after 2 weeks of proliferation reached approximately
40%. During maturation ARO CD34+ cells as well as transduced cells developed into
large multinucleated osteoclasts in a similar fashion to cord blood CD34+ cells,
expressing the osteoclast marker TRAP. The osteoclasts also maintained a similar
morphology visualized by the presence of the characteristic actin ring. However, ARO
osteoclasts completely failed to resorb bone, as shown by a decreased calcium
secretion and CTX-I release. Transduction of CD34+ IMO cells resulted in increased
TCIRG1 mRNA and protein levels, as shown by qPCR and western blot; an
expression which was comparable to controls. However, TCIRG1 protein was only
present at the late stages of maturation similarly to wt osteoclasts. Vector corrected
ARO osteoclasts generated increased Ca2+ and CTX-I into the media and formed
clearly visible resorption pits. The average resorption was approximately 70 to 80% of
that of osteoclasts generated from normal CD34+ cord blood cells. Furthermore,
transduced CD34+ cells engrafted successfully in NSG-mice, and no signs of toxicity
were observed. Finally osteoclasts generated from isolated splenocytes expressed GFP
in vitro, confirming engraftment.
Overall, these findings justify further development of gene therapy targeting
osteoclasts in the treatment of osteopetrosis.
48
General Discussion
Diseases affecting osteoclast development and function as well as cell culture models
of osteoclasts have been invaluable tools in the understanding of osteoclast regulation
and function, and have greatly assisted in the development of treatments for diseases
caused by osteoclast dysfunction.
The work in this thesis has been divided into two parts; 1) a series of in vitro and in
vivo studies investigating the role of osteoclasts in regulating bone remodeling and,
more specifically, bone formation. The rationale for this line of work is based on the
hypothesis that osteoclasts independent of their resorptive capability secrete anabolic
factors important for ongoing bone formation, as indicated in patients with
osteoclast-rich osteopetrosis. Hence, abolishing resorption through inhibition of
acidification, and maintaining the presence of non-resorbing osteoclasts might be an
advantageous mode of action for treating bone turnover diseases, compared to current
available therapies. 2) An in vitro based study developing an osteoclast model for
validation and development of correction of osteopetrosis in patient cells. The long
term aim of this line of experiments is to develop hematopoietic stem cell targeted
gene therapy for ARO patients with mutations in TCIRG1.
Investigating Osteopetrosis in Adult Mice
The work in paper I and II has largely focused on the development of an in vivo
model that could be used to investigate the mode of action by which bone formation
is maintained in osteopetrotic subjects, independent of bone resorption but not
osteoclast presence. The obvious comparison would be a model which has no
osteoclasts, and, hence, potentially a lowered bone formation as an effect of this, such
as in osteoclast poor osteopetrosis (Guerrini et al., 2008; Pangrazio et al., 2012).
The induction of osteopetrosis was performed in adult mice for several reasons. The
endogenous mouse models of ARO, the oc/oc and the RANK KO mouse, suffer from
severe developmental phenotypes in their bones. The lack of bone resorption due to
lack of acidification or lack of osteoclasts in general, results in the inability to dissolve
the inorganic part of the calcified cartilage which is used to mold the bones during
bone growth (Dougall et al., 1999; Neutzsky-Wulff et al., 2010). This leads to the
development of bones which largely consist of calcified cartilage instead of normal
49
bone and renders them weak and brittle. The undegraded calcified cartilage occupies
marrow space, effectively blocking the cavity for proper hematopoiesis (Dougall et al.,
1999; Neutzsky-Wulff et al., 2010). These traits of the endogenous models lead to
early death, limiting their applicability in the investigation of adult mice. To
overcome this, we induced osteopetrosis in adult mice by transplanting FLCs from
osteopetrotic mice. Using an adult chimera model allowed us to properly investigate
the unbalancing phenotype between osteoclasts and osteoblasts independent of the
developmental phenotype seen in endogenous models, and to isolate remodeling
mechanisms from early modeling and endochondral ossification processes related to
growth. In addition, a transplantation model gave us the opportunity to observe the
effect of inhibiting acidification in old mice instead of young. A treatment targeting
acidification would mainly be developed for the treatment of osteoporosis, a disease
primarily manifesting in the older population. Hence, from a translational perspective
using adult mice is favorable compared to using actively growing mice for these
purposes.
Phenotype Penetrance in Adult Osteopetrotic Mice
As discussed in paper I and II, we were surprised by the mild phenotype in the
transplantation models. While realizing that there are differences in terms of
magnitude of phenotype in the oc/oc model described in the two papers, we speculate
that the fairly mild phenotype in the experiment in paper II is the explanation for the
lack of significant increase in vertebral bone volume in the RANK KO, an effect
which was expected. In terms of why the bone histomorphometry in paper I had a
different outcome, this might be related to the differences observed in phenotype. In
paper I it is clearly seen that a peak bone volume effect is obtained in vertebrae
already after 12 weeks, and this is unchanged at 28 weeks. These data indicate that
coupling has been reestablished in these animals, which fits with the biomarker and
histomorphometry data. In paper II we observed a smaller increase at 15 weeks, which
is best explained by the fact that the phenotype is still accumulating, and, while very
speculative, we might see corresponding increases in bone formation rates in the
previous studies at an earlier time point, before a plateau is reached. We do not feel
that the time frame differences between these two experiments cause the differences,
but rather the milder manifestation of the phenotype; although, this cannot be
excluded.
Engraftment levels reached 95-98% leaving a minor percentage of host cells still
remaining in recipient animals. It is possible that HSCs in this remaining pool can
divide and give rise to a number of functional osteoclasts and, thereby, limiting the
effect of the osteopetrosis induced in these studies.
50
Another possible explanation for the mild phenotype can be the existence of quiescent
osteoclast precursors (QOPs). QOPs are believed to be post mitotic osteoclast
precursors, residing both in circulation and on bone surfaces in proximity to
osteoblasts (Mizoguchi et al., 2009; Muto et al., 2011). This specific pool of
precursors expresses high levels of RANK and c-fms and supposedly exists in a
quiescent state and has a longer halflife than normal osteoclasts (4 weeks longer). The
post mitotic characteristic is a key feature of QOPs, and renders this population
resistant to 5-FU, a strong inducer of apoptosis in cells with high proliferative
potential, as well as the inhibitor of DNA replication hydroxyurea (Mizoguchi et al.,
2009). These findings, while not experimentally confirmed, also imply resistance of
QOPs to conditioning regimens targeting high-proliferation cells such as radiation
conditioning. In papers I and II we use lethal radiation doses to ablate the BM
including any proliferating myeloid precursors to condition mice for stem cell
transplantation. Despite this conditioning we still observe osteoclast in vivo, a finding
not related to the RANK KO model itself, as the endogenous model as well as
patients are completely free of osteoclasts (Guerrini et al., 2008; Pangrazio et al.,
2012; Dougall et al., 1999).
Thus it is possible that QOPs remain in vivo in circulation or on bone surfaces, and
can be recruited to sites of bone remodeling, even after radiation therapy. This would
account for the amount of osteoclasts in RANK KO recipients, and possibly explain
the fairly mild phenotype observed. Whether oc/oc recipients have the same amount of
QOPs would require further analysis, such as analysis of donor and host specific
surface proteins in the osteoclast population. A way to remove all osteoclasts in a
transplantation model might be to transplant stem cells from mice with inducible
expressing of diphtheria toxin driven by the cathepsin K promoter (ctskCre;DTA), as
recently published by Oury and collegues (Oury et al., 2013). The fusion of engrafted
osteoclast precursors and QOPs during osteoclastogenesis lead to entry of diphtheria
toxin in the QOPs, causing a complete ablation of mature osteoclasts. Diphteria toxin
driven by the cathepsin K promoter might solve the issue with osteoclast generation
from QOPs, as the mechanism affects maturating osteoclasts and not proliferating
cells. However, this would of course not be applicable in oc/oc recipients, as we require
the osteoclast to be present in this model, overall limiting the potential of the
diphtheria toxin model in a comparative study.
The ideal adult models of osteoclast-rich and osteoclast-poor osteopetrosis for
studying how osteoclasts affect bone remodeling and particularly bone formation in
vivo would be to generate inducible knock-out models targeting the genes endoding
TCIRG1 and RANK.
51
Secretion of Coupling Factors is Dependent on Osteoclast
Subtype
The in vivo studies clearly support the hypothesis that non-resorbing osteoclasts are
anabolic; however, the understanding of when and how they produce these factors is
limited. Therefore, in paper III, we undertook an extensive analysis of the anabolic
signals as a function of cell phenotype and matrix composition.
CM from mature human osteoclasts grown on bone and plastic induced nodule
formation when added to osteoblast cultures. In contrast, CM from neither preosteoclasts nor macrophages grown on bone were able to induce this nodule
formation by osteoblasts. This is in line with previous findings showing that
osteoclasts precursors are not sources of anabolic signals (Pederson et al., 2008), as
well as findings of reduced bone formation in osteoclast-poor osteopetrosis patients
(Pangrazio et al., 2012).
An anabolic signal have been speculated to be a mix of both osteoclast derived factors
as well as signals released from the matrix during bone resorption as has been
extensively reviewed (Henriksen et al., 2009b; Henriksen et al., 2011; Henriksen et
al., 2014; Karsdal et al., 2007; Martin et al., 2009; Martin, 1993; Martin and Sims,
2005; Martin and Seeman, 2008; Sims and Walsh, 2012; Sims and Vrahnas, 2014).
We found that both the acidification inhibitor diphyllin and the cathepsin K
inhibitor E64 inhibited the release of anabolic factors when grown on bone; an effect
which was absent in osteoclasts grown on plastic. These findings suggest that anabolic
factors are also released from the degraded bone and that this release can be inhibited
using inhibitors of bone resorption. In further support of this was the observation that
acidification deficient ARO osteoclasts showed similar blunting of anabolic signaling
when grown on cortical bone. These findings strengthen the idea that an anabolic
signal from osteoclasts during bone remodeling is a mix of molecules released from
the resorbed matrix in addition to factors released from mature osteoclasts
independent of resorption.
A peculiar finding in this study was the complete absence of anabolic signaling when
osteoclasts were grown on alternative substrates such as dentine or decalcified matrix.
Dentine is a highly calcified matrix which under normal conditions is non-remodeled.
It is likely that dentine contains inhibiting signals which limit the release of anabolic
signals by osteoclasts, thus abrogating bone remodeling. In line with this, osteoclasts
grown on decalcified matrix showed a similar lack of anabolic signals, again indicating
an active role for matrix type. The effect of matrix composition, while speculative,
could indicate that various osteoclast subtypes have different resorption function and
anabolic action directly induced by signals on matrix level.
52
Level of Correction for Gene Therapy of ARO
For future development of gene therapy treatment of osteopetrosis an important
question is what level of osteoclast function is needed for correction of disease
phenotype.
Recent mouse experiments have indicated that only minor reconstitution (5 to 10%)
of the BM is necessary to incur rescue of resorptive function in vivo (Flores et al.,
2010). In line with this, human studies in individuals with mutations in only one
allele of TCIRG1 show no signs of osteopetrosis indicating haplosufficiency.
The low number of rescued cells needed for functional correction might be explained
by the fusing nature of osteoclasts formation. It is possible that wt or corrected
monocytes can fuse with, and correct, dysfunctional monocytes during osteoclast
differentiation, resulting in an overall higher number of rescued osteoclasts (Figure 9).
In alignment, transducing CD34+ cord blood with a GFP containing vector gave a
30% transduction efficiency; an efficiency which after differentiation resulted in GFP
expression in almost 100% of mature osteoclasts.
To see how the fusion mechanism relates to function, we setup a series of experiments
where CD34+ ARO cells, TCIRG1 transduced ARO CD34+ cells, and cord blood
CD34+ cells were mixed at different ratios, differentiated into osteoclasts on bovine
bone slices and analyzed for bone resorption capacity. Findings indicate that small
amounts of cord blood CD34+ cells are enough to restore resorptive function in ARO
co-culture. These data are in line with the GFP expression pattern seen in fusing GFP
transduced osteoclasts, in vivo rescue transplantations, as well as the haplo sufficient
nature of ARO related to TCIRG1 mutations. The increase in function does not
appear to be caused by selective expansion or differentiation of cord blood cells, as we
saw similar results when mixing ARO CD34+ cells with TCIRG1 transduced ARO
CD 34+ cells. Together, these findings indicate that even low transduction and
engraftment efficiencies might be sufficient for functional reconstitution of osteoclasts
when treating ARO.
An interesting parameter to determine would be the maximum rescue achievable in
vitro; however, transduction of CD34+ cells with current vectors rarely exceeds 30%
transduction efficiency, which limits the “dose range”. Sorting cells using fluorescence
activated cell sorting (FACS) might be possible to enrich the number of transduced
cells, but pilot experiments indicate that cell viability is heavily affected, limiting the
use of the method in this setup.
53
BONE
CD34+
TCIRG1
Tranduction
BONE
CD34+
Figure 9. Osteoclast fusion might increase rescue percentage
Schematic figure of how osteoclast fusion might impact overall rescue percentage in mature osteoclasts. If
we assume that osteoclasts fuse randomly, transduced and non-transduced pre-osteoclasts will fuse in
ratios correlating to the transduction efficiency, hereby increasing the overall amount of cells with a
corrected TCIRG1 gene.
Post-transcriptional Regulation of Vector Driven TCIRG1
Expression
Osteoclastogenesis is a tightly regulated process which is highly dependent on the
osteoclastogenic cytokines M-CSF and RANKL. The differentiation into osteoclasts
involves the up-regulation of a number of osteoclast specific genes and proteins, the
most prominent being cathepsin K, ClC-7, MMP9, TRAP, and TCIRG1.
It is well-established in the literature that TCIRG1 is regulated at the transcriptional
level by a number of transcription factors. In pre-osteoclasts, the TCIRG1 gene is
repressed by the poly (ADP-ribose) polymerase-1 (PARP-1)(Beranger et al., 2006).
During osteoclastogenesis, the presence of RANKL causes degradation of PARP-1
and upregulation of TCIRG1 through JunD proto-oncogene (junD) and Fos related
antigen (Fra-2)(Beranger et al., 2007).
Driving a transgene by strong viral promoter such as the spleen focus forming virus
(SFFV) promoter most often causes it to be expressed ectopically, in cell lineages and
at differentiation stages not usually relevant to the normal endogenous gene. In paper
IV the expression of TCIRG1 protein was therefore expected to be present at both
54
the pre-osteoclast stages as well as in mature osteoclasts, when cells were transduced
with a TCIRG1-containing vector. Counter intuitively, we observed no TCIRG
protein in pre-osteoclasts. TCIRG1 protein became detectable at day 5 to 7 of
osteoclast maturation, resembling the endogenous transcriptional regulation
previously described. Co-expressed GFP protein as well as exogenous TCIRG1
mRNA was present in pre-osteoclasts and throughout differentiation, consistent with
exogenous expression. Furthermore, transduced cells cultured without RANKL
differentiated into macrophages but failed to express TCIRG1 protein indicating that
the expression is mediated through RANKL or osteoclast maturation. These
observations indicate that the expression of TCIRG1, in addition to transcriptional
control, is regulated through a post-transcriptional mechanism. Interestingly,
HT1080 cells transduced with the TCIRG1-vector expressed TCIRG1 protein early
on, indicating that the mechanism was absent in non-hematopoietic cells. The
mechanism of this post-transcriptional regulation is unclear, but could involve
proteasomal degradation caused by regulated intracellular processes such as folding or
compartment sorting, or regulation through RNA binding proteins ensuring
expression at the correct cell stage (Glisovic et al., 2008).
If vector mediated expression of TCIRG1 is regulated post-transcriptionally in a
manner resembling the endogenous regulation, this might in the gene therapy setting
eliminate the need for cell specific promotors in the vectors, since TCIRG1 protein
would be expressed only in mature osteoclasts.
55
56
Future Perspectives
Identifying Osteoclast Derived Anabolic Factors
Future experiments will aim at identifying factors expressed by the osteoclasts which
direct the anabolic actions of the osteoblasts. We showed in paper III, that the
anabolic actions of osteoclasts affecting the nodule formation by osteoblasts are highly
dependent on osteoclasts stage and matrix composition. The secretion of anabolic
factors from osteoclasts grown on cortical bone, and the absence of these factors on
dentine, as well as in macrophages provides a unique opportunity to investigate
differences in expression patterns. Briefly, RNA and protein from osteoclasts grown
on matrices differentially affecting their anabolic potential will be isolated, and gene
and protein expression analyzed using affymetrix array and proteomic analysis. Once
a gene or molecule is identified, the role of such a molecule can investigated and
possibly validated by constructing gene specific knockout mice, as was recently done
for the potential coupling factor Cthrc1 (Kimura et al., 2008; Takeshita et al., 2013),
or combining lentiviral knockdown of potential factors with the adult osteopetrotic
mouse model.
The identification of a coupling factor holds great potential in both therapeutic
development and biomarker assessment of bone related diseases.
Targeting Acidification When Treating Bone Turnover
Diseases
Mutations in osteopetrotic patients and animal models with acidification deficiency
have highlighted the importance of having osteoclasts present to maintain ongoing
bone formation (Bollerslev et al., 1989; Bollerslev et al., 1993; Frattini et al., 2000;
Henriksen et al., 2004; Henriksen et al., 2009a; Kornak et al., 2000; Kornak et al.,
2001; Segovia-Silvestre et al., 2008). Most importantly, the finding that osteopetrotic
subjects, despite having a high number of non-resorbing osteoclasts, maintain normal
or even increased bone formation, suggest that osteoclasts, but not necessarily their
resorptive function, are a driving factor in maintaining bone formation locally at sites
57
where bone formation is needed (Henriksen et al., 2004; Henriksen et al., 2014;
Karsdal et al., 2007; Sims and Walsh, 2012).
These findings clearly distinguish inhibition of acid secretion through inhibition of
for example ClC-7 or the osteoclastic V-ATPase in terms of treatment mode of action
in bone turnover diseases, from traditional anti-resorptives such as bisphosphonates or
the recently approved RANKL antibody, Denosumab (Figure 10). These current
anti-resorptive treatments, despite being very potent inhibitors of bone resorption,
suffer from secondary reduction in bone formation. This reduction in bone formation
limits long-term efficacy, and at the same time it is not completely clear how this
“freeze” of the remodeling process affects general bone health.
Currently the only approved anabolic treatment is PTH or fragments hereof.
However, secondary to the anabolic effect of PTH, are catabolic increases in
osteoclast mediated bone resorption limiting the overall efficacy of the drug. To
overcome this limitation, combinational treatments with PTH and bisphosphonates
seeking to limit the secondary catabolic response from the osteoclasts have been tested
in clinical trials, but data indicate a blunting of the anabolic response, which have
been proposed to be mediated by a reduction in osteoclast numbers (Black et al.,
2003). However, recent studies showing osteogenic effects of PTH in combination
with Denosumab question these findings and suggest that mode of action of PTH
might be more complex (Tsai et al., 2013).
Several molecules with proposed superior modes of action are currently in clinical
development. The cathepsin K inhibitor Odanacatib inhibits collagenolysis while
maintaining osteoclasts, and is thus proposed to maintain ongoing osteoblastic bone
formation (Gauthier et al., 2008). However, thus far clinical trials have failed to
provide compelling evidence that bone formation is ongoing in face of reduced bone
resorption, although the reduction in biomarkers of formation is less than seen for
bisphosphonates and Denosumab (Bone et al., 2010; Eisman et al., 2011). The
reason for the observed decrease in bone formation is currently unclear, but the
mechanism might involve modified signaling at osteoclast attachment sites
(Henriksen et al., 2009b; Karsdal et al., 2007).
Where Odanacatib is proposed to work through mechanisms of maintaining bone
formation similar to inhibition of acid secretion, the antibody anti-sclerostin exerts its
effect through the osteoblastic lineage. Its mode of action is mediated through the
binding of sclerostin; an osteocyte secreted inhibitor of the Wnt/beta-cathenin
pathway (Poole et al., 2005). This binding results in osteoblast activation leading to
bone formation (Li et al., 2009; McClung et al., 2014). It should be noted that
despite its anabolic profile, anti-sclerostin also decreases bone resorption, but the
reason for this is currently not understood (McClung et al., 2014). The effect of not
having the osteoclasts directly involved in guiding the bone forming actions of the
osteoblasts as seen in normal bone remodeling have not yet been fully investigated in
long term clinical trials. It is possible that bone formation is instead activated in a
58
systemic manner similar to bone modeling; a differing mode of action which could
potentially lead to increases in bone at unwanted sites such as joints or nerve endings.
All in all, although bone turnover diseases are well treated, there is still room for new
treatments. Acidification inhibitors, acting locally on osteoclasts, and maintaining
bone remodeling in the BRU including bone formation, similar to the normal
physiological state, could be superior to current modalities.
Figure 10. Treatment modalities for osteoporosis
Schematic representations of the correlation between osteoclast number, bone resorption and bone
formation in different treatments. BP, bisphosphonate. Adapted from (Karsdal et al., 2007)
Critical Issues for Further Development of Clinical Gene
Therapy
Safety concerns
When developing gene therapy history calls for careful consideration of the safety of
treatment. Clinical gene therapy trials targeting hematopoietic stem cells have now
been successfully performed in a range of diseases, including ADA-SCID, SCID-X1
and Wiskott Aldrich syndrome (Braun et al., 2014). However, a number of clinical
gene therapy trials have resulted in serious side effects due to genotoxicity, in many
cases leading to leukemia. Among these are chronic granulomatous disease, SCID-X1,
and Wiskott-Aldrich (Stein et al., 2010; Braun et al., 2014; Hacein-Bey-Abina et al.,
2003a; Hacein-Bey-Abina et al., 2003b). These trials have generally made use of
gamma-retroviruses for viral transduction, and the link between vector origin and
insertional mutagenesis has naturally led to a great effort being invested in developing
safer gene delivery methods (Naldini et al., 1996; Case et al., 1999; Miyoshi et al.,
59
1999; Cattoglio et al., 2007; Neschadim et al., 2007). For further development of
clinical gene therapy the vectors will be third generation SIN lentiviral vectors.
Similar vectors are currently being implemented in clinical trials for treatment of
WASP, ADA-SCID, SCID-X1, and CGD (Aiuti et al., 2013; www.Clinicaltrials.gov,
2014). The vectors themselves will be analyzed for integration sites and clonal
proliferation in a setting involving transplantation of rescued cells into NSG mice and
long term follow-up.
Vector adaption to clinical settings
One of the primary objectives of this thesis has been to develop and validate a system
for transduction of osteoclast precursors to be used in the development of gene
therapy for osteopetrosis. After having shown that osteoclast function can be
corrected through rescue of the TCIRG1 gene in CD34+ cells from osteopetrotic
patients we are now aiming at gene therapy development for clinical application. In
this regard, safety is a prime aspect and the SFFV promoter, used up to this point as a
proof of concept vector, has to be exchanged with cellular promoters derived from
human genes. We are currently testing vectors making use of the mammalian
promoters elongation factor 1 alpha (EF1-alpha) or ChimP. Preliminary data indicate
that transduction efficiencies and correction are lower than SFFV-promoter driven
TCIRG1 expression in vitro, but sufficient for functional rescue. Further studies will
be aimed at optimizing transduction protocols to be in line with further clinical
development.
In vivo models of correction using human CD34+ cells
It is clear that clinical development of gene therapy not only requires preclinical
optimization of therapeutic vectors but also the use of relevant mouse models.
Efforts are currently put into the development and optimization of a xenograft model,
which can be used to study disease correcting treatment strategies of human patient
cells in vivo. The immunodeficient NSG mouse has proved valuable in analyzing the
transplanted cells ability to graft, and assessment of vector mediated toxicity. The
NSG mouse is, however, limited in terms of studying functional rescue due to lack of
cross reactive cytokines, especially M-CSF, which causes lack of human myeloid
differentiation in vivo. Therefore, it might be necessary to isolate the CD34+
population from transplanted NSG mice, which can then be differentiated ex vivo
and analyzed for osteoclastogenic potential after in vivo transplantation. Finally, the
NSG mouse is not an osteopetrotic model and, as such, is not suitable for functional
analysis of rescue in vivo. Such an analysis may require a combination of the
immunodeficient properties of the NSG mouse, with the osteopetrotic features of the
oc/oc model.
60
Selection of patients and clinical treatment scenario
Clinically, a gene therapy trial will be offered and aimed at children diagnosed with
ARO, which are without a suitable stem cell donor. Due to the high prevalence of
CD34+ cells in peripheral blood of IMO patients, cells will be harvested through
exchange transfusions without the use of mobilization regimens (Steward, Blair et al
2005). CD34+ cells will be transduced with the optimal therapeutic vector based on
preclinical validation. Depending on the need for multiple exchange transfusions,
transduced cells will be either kept in culture or frozen before being re-infused into
the patient. It is unclear whether myeloablation will be needed for stable engraftment
of cells, but in order to ensure adequate space in the niche, patients will be treated
with low-dose busulphan, similar to ADA-Scid and CGD trials, as well as the oc/oc
model (Askmyr et al., 2009b; Aiuti et al., 2002; Ott et al., 2006). Follow up will be
performed regularly analyzing the patients for clonal composition of hematopoiesis
(Schmidt et al., 2007).
Exchange transfusion
Viral transduction
Culture
Exchange transfusion
Viral transduction
Freesing
Reinfusion of cells
Conditioning
Figure 11. Schematic outline of proposed clinical gene therapy setup.
61
62
Popular Scientific Summary
My PhD thesis has focused on increasing the understanding of the osteoclasts, the
cells responsible for degradation of bone, by studying the rare hereditary disease
autosomal recessive osteopetrosis (ARO). ARO is present already at birth and is
caused by mutations affecting the osteoclasts ability to degrade bone. Non-functional
osteoclasts lead to dramatic increases in bone thickness affecting bone marrow, blood
vessels, and nerves due to having smaller cavities in the skeleton. Despite the increase
in bone mass, the bones are weak and brittle because of how the bones are molded
during growth, and fractures often occur. In almost half the cases, the disease is
caused by mutations in the gene called TCIRG1, which encodes a protein important
in the degradation of calcium in bone. As osteoclasts come from blood stem cells, the
disease can be corrected by bone marrow transplantation, replacing sick stem cells
with healthy cells from a donor. However, this treatment is risky and is highly
dependent on a matching donor. Without bone marrow transplantation the disease is
fatal within five years of birth.
Under normal circumstances the osteoclast mediated bone degradation process leads
to release of signals that direct new bone formation by specialized cells called
osteoblasts. In ARO patients these osteoclast derived signals are maintained despite
the lack of bone degradation, suggesting that simply the osteoclasts being present may
be enough for bone formation. A great effort has been put into identifying these
osteoclast derived signals as they may lead to new treatments for patients with too
little bone, such as osteoporosis.
In the first two papers we developed an adult mouse model with osteopetrosis to
investigate how signals from non-functional osteoclasts affected bone formation. We
induced osteopetrosis by transplanting stem cells from osteopetrotic mice, with
defects in the mouse version of TCIRG1, into normal adult mice. The healthy
osteoclasts are thereby replaced by non-functional osteoclasts and the mice develop a
mild form of osteopetrosis. In paper I we found that these “osteoclast-rich”
osteopetrotic mice have increased bone mass and surprisingly increased bone strength
compared to control mice.
In Paper II we compared the osteoclast-rich model to an osteoclast-poor model. For
this we instead used stem cells from a mouse model which has a mutational defect in
a gene crucial for osteoclast development. Comparing these two types of osteopetrosis
in mice, we found that bone formation was increased in osteoclast-rich osteopetrotic
63
mice compared to their osteoclast-poor counterparts. These findings suggest that
blocking bone degradation, while keeping osteoclasts, maintains the signals directing
bone formation. A molecule blocking the osteoclasts ability to degrade bone but
without removing the cell itself, may be interesting as potential treatment for low
bone mass diseases such as osteoporosis.
In paper III we used different ways of modifying osteoclast function to investigate
how the release of signals from osteoclasts, directing bone formation is affected by
these changes in various osteoclast related functions. We also used osteoclasts from
ARO patients to show that signals are still being released directing bone formation
despite their lack of bone degradation. The studies indicate that the signals are both
dependent on how old the osteoclast is, and what type of bone the osteoclast is
degrading.
Blood stem cells can renew themselves and produce all the different kinds of blood
cells in the blood throughout a lifetime. By correcting these cells one can change the
function of all osteoclasts that are to be made. The principle of gene therapy is to
insert the normal functional gene into a cell having dysfunctional gene. This may
result in the patient’s ability to produce the correct functional protein themselves.
In Paper IV we used a modified HIV-virus as a tool to insert the correct functioning
TCIRG1 gene into human ARO blood stem cells. We found that the corrected cells
expressed the functional protein and that function was almost fully restored in these
osteoclasts, as could be measured by the release of degradation products such as
calcium and collagen fragments.
In summary these studies have increased the understanding of ARO and the
osteoclast related defects causing this disease as well as taken a step towards gene
therapy as a treatment for this ARO. The experiments have also shed light on the
important regulation undertaken by the osteoclasts in the maintenance of bone, and
findings suggest that targeting the osteoclasts ability to degrade bone without
removing the osteoclasts may be a novel target for treatment of bone turnover
diseases.
64
Articles not Included in this Thesis
Disruption of the V-ATPase functionality as a way to uncouple bone formation and
resorption – a novel target for treatment of osteoporosis
Christian S. Thudium, Vicki K. Jensen, Morten A. Karsdal, Kim Henriksen.
Curr Protein Pept Science, 2013; 13(2):141-51; (Review)
Osteoclasts are not crucial for hematopoietic stem cell maintenance in adult mice
Carmen Flores, Ilana Moscatelli, Christian S. Thudium, Natasja S. Gudmann, Jesper S.
Thomsen, Anne-Marie Brüel, Morten A. Karsdal, Kim Henriksen, Johan Richter.
Haematologica, 2013; 98(12):1848-55
65
66
Acknowledgements
First and foremost I would like to thank my external supervisor, Kim Henriksen, for
all the help and dedication you have put into the project throughout my years
working in your lab. Your commitment to science and insight into the many different
areas of bone research have truly been an inspiration.
Johan Richter, my internal supervisor, for your dedication to both the clinical and
basic research and for always being available for helpful scientific as well as practical
discussions.
Ilana Moscatelli and Carmen Flores for being wonderful “long-distance” colleagues
and for all the interesting discussions and exciting experiments we have done together.
Carmen Montano, Zsuzsanna Kertész and Henrik Löfvall for all your help with mice
and cells.
Morten Karsdal for your scientific enthusiasm and guidance in an evolving biotech
industry.
Christina Hansen and Mia Jørgensen for your invaluable help with animal care and
experimentation.
Marianne Ladefoged, Inge Kolding, Mie Andersen and Tülay Dastan for all your help
in the lab and for always being able to have the answer I need. Thanks to all the rest
of my colleagues at Nordic Bioscience, past en present, for providing a friendly, fun
and scientific atmosphere.
To my colleagues at the division of molecular medicine and gene therapy for always
being friendly and helpful despite my limited time physically in Lund.
Jesper Thomsen and Mie Brüel for enormous help with the bone analyses and
valuable scientific input.
Ellen Hauge for expert scientific advice and introducing me to bone
histomorphometry. Natalie Sims and Vincent Everts for providing technical expertise
and bone analyses. Ansgar Schulz, Anders Fasth, Anna Villa and Oscar Porras for
providing patient samples and expertise.
Nordforsk and Nordic Bioscience for generously providing financial support for my
PhD.
67
Thanks to my friends and family for your help and support and for always showing
interest in my work even though it is not always completely clear to you what I do. A
special thanks my mom, Dorte for endless support in everything I do, and my brother
Emil for always reminding me that not everyone does natural science.
Finally, to my better half and best friend, Rebekka, for your love and support, for
always believing in me, and for making me happy.
68
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