A Phase I/II Open Label, Dose Escalation, Safety Study in Subjects with Mucopolysaccharidosis type VI (MPS VI) Using Adeno-Associated Viral Vector 8 to Deliver the human ARSB gene to Liver.

Mucopolysaccharidosis VI is an autosomal recessive lysosomal storage disorder
(LSD), belonging to the group of mucopolysaccharidoses (MPS). The MPS are
caused by defects in lysosomal enzymes resulting in widespread intra- and
extra-cellular accumulation of glycosaminoglycans (GAGs).
MPS VI, also known as Maroteaux-Lamy syndrome, is caused by deficiency of the
enzyme arylsulfatase B
Deficiency of ARSB results in the intralysosomal storage and urinary excretion
of these partially degraded GAGs.
The biochemical diagnosis of MPS VI is based on the detection of elevated
urinary dermatan sulfate levels and is confirmed by reduced ARSB activity in
cell extracts. The diagnosis is generally confirmed by ARSB enzyme activity
below 10% of the lower limit of normal range in cultured fibroblasts or
isolated leukocytes.
The rate of clinical progression in MPS VI subjects varies considerably,
generating a spectrum of clinical presentation ranging from rapidly to slowly
progressive disease. Nevertheless, all subjects within this spectrum will
eventually experience significant morbidity and in most cases early mortality.
The classic features of MPS VI include hydrocephalus and spinal cord
compression, coarse facial features, hearing loss, corneal clouding,
macroglossia, heart valve disease, respiratory difficulties,
hepatosplenomegaly, inguinal and abdominal hernias, dwarfism/growth
retardation, skeletal dysplasia, and joint stiffness. Mental development is
usually normal, although physical and visual impairments affect psychomotor
performances .
Communicating hydrocephalus is a typical feature in MPS VI . Increased
intracranial pressure is thought to be caused by dural thickening and
dysfunction of arachnoid villi. Typical signs of obstructive hydrocephalus such
as early morning headache, vomiting, and papilledema are often absent,
although some subjects may present with rapid visual deterioration.
Visual impairment is common and occurs in about 40% of subjects with MPS VI.
Ocular abnormalities include corneal clouding, glaucoma, and abnormal optic
disk.
Aortic and mitral valvular dysfunction, due primarily to thickened calcified
stenotic valves, are the most prominent cardiac involvement.
Disease complications related to the anatomical and pathological changes in the
airways of subjects are obstructive sleep apnea, pneumonia, and hearing loss .
Upper-airway obstruction and decreased lung capacity often lead to obstructive
sleep apnea. Less frequently, daytime somnolence, failure to thrive, pulmonary
hypertension, and cor pulmonale may develop. Behavioral and learning problems
may also occur as a consequence of disrupted sleep. Recurrent pneumonia may be
secondary to decreased pulmonary functions and poor clearance of airway
secretions. Recurrent otitis and conductive hearing loss are common in MPS VI.
Hepatomegaly is always present and enlarged spleen is found in about half the
subjects . Umbilical and/or inguinal hernias are common.
The skeletal changes are striking examples of dysostosis multiplex. An enlarged
head and a deformed chest may be present at birth. Claw-hand deformities are
classic features and nerve entrapment syndromes, particularly of the carpal
tunnel, are common.
Advances in the understanding of the biochemical and molecular bases of LSDs
have led to the development of specific treatment regimens. In addition to
supportive care, specific therapies have been developed to provide the
deficient enzyme by hematopoietic stem cell transplantation (HSCT) and enzyme
replacement therapy (ERT).
Enzyme replacement therapy (ERT), approved by FDA has several limitations
1) rhARSB has a short half-life requiring weekly intravenous infusions that
carry a risk of allergic reaction and often require a central venous access
2) some organs and tissues are not corrected, likely because of limited
biodistribution of rhARSB. For example, in MPS VI subjects, ERT failed to
ameliorate cardiac function, visual impairment
3) the cost of ERT is extremely high, thus representing a significant burden
for the health system

Primary safety objective: To evaluate the safety of systemic intravenous
administration of the Investigational Medicinal Product (IMP) in
pediatric and adult MPS VI patients.

Primary efficacy objective: To investigate the efficacy of the IMP through
measurements of a clinically relevant biochemical endpoint in pediatric and
adult MPS VI patients.

This Phase I/II clinical trial is designed as an open label dose escalation
study and will be carried out as a multi-center clinical trial, involving the
Department of Translational Medicine (DISMET) of *Federico II* University,
Naples, Italy as the primary site and the following secondary sites:

- Center for Lysosomal and Metabolic Diseases, Department of paediatrics,
Erasmus MC University Medical Center, Rotterdam, The Netherlands
- Children*s Hospital, Hacettepe University, Ankara, Turkey.

IMP administration will be performed at the primary site, the DISMET of
*Federico II University, Naples, Italy; primary and secondary sites will
perform screening and follow-up visits and evaluations.

The study follows an adaptive design and the starting dose is 6x10^11 genome
copies (gc) of vector per kg of body weight.
Depending on safety parameters collected in the first three subjects at the
starting dose, dose escalation to the higher dose (2x10^12 gc/kg) or
enrollment of additional two subjects at the same dose level or moving to three
subjects at the lower dose (2x10^11 gc/kg) will be performed.
The time interval between each subject within the two lower dose cohort
s will be at least 6 weeks. The time interval between subjects within the high
dose will be at least 14 weeks.
Dose escalation to the higher dose level will be performed after at least 6
weeks from the IMP administration in the last individual of the starting dose
cohort, once safety outcome measures have been collected. .
Every DLT or every phase requiring an escalation or de-escalation will be
reported and discussed with the DSMB
Five protocol phases are foreseen:
1) Recruitment phase, with signature of informed consent.
2) Screening phase, during which the conditions required by the clinical
protocol for patients* inclusion/exclusion will be evaluated.
3) Baseline phase, from the end of the screening phase to the IMP
administration phase.
4) Treatment phase, day 0, i.e. the day of IMP administration.
5) Follow-up phase, from IMP injection until + 3 years from IMP

IMP administration will be performed by peripheral intravenous access using a
commercially available infusion pump and devices that have been designed for
administration of intravenous medications and solutions

Advantages of gene therapy vs. ERT
As described before, ERT with rhARSB (Naglazyme) has been approved by the FDA
and the EMA and is currently recommended for MPS VI (Giugliani et al., 2007 ;
Giugliani et al., 2014). Three clinical studies using Naglazyme ERT have been
reported and the largest study including 56 MPS VI subjects showed long-term
reduction in urinary GAGs and improvements in endurance (Harmatz et al., 2008).
However, ERT has several limitations. First, rhARSB has a short half-life
requiring weekly intravenous infusions that carry a risk of allergic reaction
and often require a central venous access, which carries risks of sepsis.
Second, some organs and tissues are not corrected likely because of limited
biodistribution. For example, in MPS VI subjects, ERT failed to ameliorate
cardiac function, visual impairment, and bone density while inconsistent
results have been reported in lung volumes (FEV1 and FVC), obstructive apnea
parameters, joint range of motion and stiffness (Harmatz et al., 2006; Harmatz
et al., 2008; Harmatz et al., 2005a; Harmatz et al., 2004). Third, ERT costs
are extremely high, thus preventing access to therapy to several subjects
especially in underdeveloped countries.
For these reasons, we believe gene therapy with a single systemic
administration has enormous potential to provide long-term expression and
secretion of ARSB enzyme from liver resulting in biochemical correction and
ultimately in clinical benefit in MPS VI subjects, as observed in animal models
(Cotugno et al., 2011; Cotugno et al., 2010; Ferla et al., 2014; Ferla et al.,
2013; Tessitore et al., 2008).

Our results in non-clinical models suggest that gene therapy:
-may be more effective than ERT. We believe that this is possible, and even
likely, based on the stable and long-term expression and phenotypic rescue we
have obtained using gene transfer in large animal models (Cotugno et al., 2011;
Ferla et al., 2013). In sharp contrast to the gene therapy, during ERT the
levels of ARSB drop to baseline within a few hours after the protein infusion
(Harmatz et al., 2005d). Thus, given the significant difference in ARSB
pharmacokinetics between the two approaches, we hypothesize that the stable
levels of enzyme mediated by gene therapy result in improved enzyme
biodistribution to body districts which are more resistant to ERT, such as bone
or cartilage. The potential for long-term disease correction is indeed
supported by the hemophilia B clinical trial with AAV2/8 expressing the factor
IX (Nathwani et al., 2011.,Nathwani et al., 2014). This trial showed long-term
expression of factor IX at levels, which were sufficient to improve the
bleeding diathesis, with few side effects;

-may avoid the multiple infusions associated with ERT: our data in non-clinical
models of MPS VI show that a single intravascular administration of
AAV2/8.TBG.felineARSB results in expression of therapeutic levels of ARSB for
up to 6 years, the last time point of our observation (Ferla et al., 2014,
Cotugno et al 2011, Ferla unpublished data). Therefore, gene therapy may
overcome the inconvenience and discomforts related to weekly infusions of
recombinant enzyme. In addition, infusions of rhARSB may be associated with
immune-mediated anaphylactoid reactions, presumably due to frequent infusions
of the recombinant protein. The risk of such reactions may be avoided by the
single administration of the gene therapy vector expressing ARSB.

Risks associated with gene therapy
However, gene transfer is a fairly new area of medicine and the long-term
health effects are not fully known.
The gene therapy vector we propose to use may cause toxic response related to:

-Infusion of the IMP
The infusion itself may cause toxic reactions (fever, hives, skin rashes, low
blood pressure, difficulty breathing, or death).

-Immune response to the IMP
The immune response system may generate antibodies to the vector as it does
with all viruses. This antibody response will preclude any future
administration of the same vector serotype in the subject receiving the vector,
because the neutralizing antibodies (NAB) to AAV will completely prevent liver
transduction (Ferla et al., 2013; Wang et al., 2010). However, participation in
gene transfer studies using a different vector or different AAV vector
serotypes may still be possible.

-Inflammation of the liver
In a clinical trial study with the AAV2/2 vector expressing the human Factor
IX, an unexpected transient elevation in transaminases, beginning several weeks
after vector infusion, was observed in 2 of 7 enrolled subjects. This increase
in liver enzymes was asymptomatic and transient as the transaminases normalised
after 6-8 weeks (Manno et al., 2006). This complication is thought to be
secondary to previous exposure of the participants to the same virus. It is not
known if this liver inflammation, if it occurs, would be transient or would
result in longer-lasting damage. In the most recent clinical trial for
hemophilia B performed using the same AAV vector serotype proposed in our
study, the increase in liver enzymes was observed with the higher dose and was
controlled with a short course of prednisolone (Nathwani et al., 2011; Nathwani
et al., 2014).

-Spread of the vector to other body tissues, including semen
Following IMP infusion, it is possible that the vector may spread into
different tissues of the body. There is a theoretical risk that vector
particles can enter the genetic information of germ cells or semen. In dose
escalation clinical studies with AAV2/2 and AAV2/8 vectors expressing the human
Factor IX, vector DNA was detected in the semen based on PCR analysis, raising
the possibility of germline transmission (Manno et al., 2006; Nathwani et al.,
2011; Nathwani et al., 2014). However, the presence of viral vector DNA in the
semen was proved to be transient in all subjects, with younger subjects
clearing more quickly than older ones. Moreover, animal studies suggested that
AAV2/2 does not transduce spermatogonia directly (Arruda et al., 2001; Couto et
al., 2004). Additionally, vector shedding in rabbit semen is transient and no
late recurrence of AAV serotypes 2, 5, 6 and 8 was found over several
consecutive cycle of spermatogenesis (Favaro et al. 2011; Favaro et al. 2009;
Schuettrumpf et al. 2006; Arruda et al. 2001). We also performed a GLP germline
transmission study in male rabbits (nr. 20140052TLP), which demonstrated that
AAV2/8.TBG.hARSB shedding in semen is transient. This argues against the
possibility of transduction of early spermatogenesis precursor exposed to AAV8
during the blood dissemination to the gonads. Therefore the risk of germline
transmission can be considered minimal. However, in previous clinical trials
based on systemic administration of AAVs, this potential complication was
managed by recommending to all subjects to bank sperm before enrolment and to
use barrier contraception methods until semen was negative for vector
sequences.

-Potential risk of the IMP to cause cancer
The first evidence about the risk of cancer came from a study in MPSVII mice
that were found to develop hepatocellular carcinoma following neonatal AAV
injections (Donsante et al., 2007). These findings were also observed more
recently in methylmalonic acidemia mice which also developed hepatocellular
carcinoma several months after neonatal AAV injections and showed vector
integration and overexpression of microRNA-341 (Mir341) proximal to the Rian
locus, that has no orthologues in humans (Chandler et al., 2015). Similarly, a
higher frequency of hepatocellular carcinoma was observed in molybdenum
cofactor deficient mice injected with AAV as newborns (Reiss and Hahnewald,
2011). In contrast, no evidence of insertional mutagenesis and cancer were
observed in adult mice for 18 months (Li et al., 2011), in dogs for a period of
8 years (Niemeyer et al., 2009), and in nonhuman primates for up to 5 years
(Nathwani et al., 2011).
A recent study found clonal integration of AAV2 in 11 of 193 cases of
hepatocellular carcinoma. These AAV2 integrations occurred in known cancer
driver genes (CCNA2, TERT, CCNE1, TNFSF10, and KMT2B) leading to overexpression
of the target genes. Tumors with viral integration mainly developed in
non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11
cases), suggesting a pathogenic role for AAV2 in these patients (Nault et al.,
2015). However, integration pattern of recombinant AAVs is different than that
of wild type AAVs (Huser et al 2014) and the gap between the high rate of AAV2
infection in human population and the rare occurrence of HCC with AAV2
integration come out in favor of its safety. Additionally, a recent study
showed recombinant AAV5 integration is not associated with hepatic genotoxicity
in non human primates and patients (Gil-Farina et al 2016)