Assess safety of intra-arterial autologous myogenic stem cell therapy for m.3243A>G mutation carriers

Mitochondrial disorders are progressive, often fatal multisystem disorders, in
20-25% of the cases caused by heteroplasmic mutations in the mitochondrial DNA
(mtDNA). Epidemiological studies have shown that mtDNA disorders affect about 1
in 10,000 of the general population, inducing significant morbidity and
mortality and high health and societal costs. Clinical manifestations are most
prominent in organs with a high energy demand, like muscle and brain. At this
moment, there is no effective treatment known to influence the disease process
or manifestation. Myogenic stem cell-based therapies complementing defective
muscle cells and fibres, are highly promising to combat the myopathy and
exercise intolerance which affect >50% of heteroplasmic mtDNA mutation
carriers. Myogenic stem cells called mesoangioblasts (MABs), are currently the
only myogenic precursors that fulfill all criteria to be used as advanced
therapy medicinal product (ATMP) for systemic treatment, namely good ex vivo
proliferation capacity, high myogenic capacity and a capability to cross blood
vessels, allowing intra-arterial (systemic) delivery towards affected muscle.
The only experience today is using allogeneic MABs transplantation in mice and
dog models and in patients with Duchene muscular dystrophy (DMD). Treatment
with ex-vivo expanded MABs resulted in significant regeneration of DMD positive
muscle fibers in both mice and dog models. Intra-arterial delivery of
allogeneic MABs in DMD boys (phase I/IIa clinical study) demonstrated that the
treatment was relatively safe and that some dystrophin was produced by the new
muscle fibers, although not sufficient for functional improvement. Our approach
has key advantages as we use autologous MABs, which do not require an
immunosuppressive regime. Also, mitochondrial function is partly preserved in
mtDNA mutation carriers and partial supplementation by healthy fibres should
suffice to ameliorate mitochondrial function. We have demonstrated that MABs of
most m.3243A>G carriers contain no or only a low amount (<10%) of the mtDNA
mutation, allowing direct ex vivo expansion of patient-derived MABs. The
overall aim is to induce muscle regeneration using these autologous MABs with a
mutation load of <10%, as advanced therapy medicinal product (ATMP).

The phase I/IIa trial will consist of an intra-arterial injection (via catheter
in femoral artery) of autologous MABs in the left lower leg of 5 m.3243A>G
patients. The primary objective is assessing safety of administration of
autologous MABs, which have not been used as treatment before in humans.
Secondary objectives are:
(1) to assess homing of the labelled autologous MABs to the tibialis anterior
muscle after i.a. delivery
(2) assess effectiveness at the tissue level by measuring myogenesis and mtDNA
mutation load

Mono-center prospective open label intra-subject controlled phase I/IIa
clinical study.

Intra-arterial administration of 5*10E7 MABs/kg in left lower leg (total
dosage: 1,9*10E8 MABs for treatment of one lower leg of a 75kg individual).

15 participants will be included, of which 5 will be selected for the clinical
study.
- At the first visit (inclusion visit), a routine physical and neurological
examination will be performed and a vastus lateralis skeletal muscle biopsy
(~30mg) will be collected for quantification of the m.3243A>G mutation load in
MABs and muscle.

The 5 participants, who have the lowest m.3243A>G mutation load in MABs (max
10%) and highest mutation load in muscle (min. 40%) will be selected for
participation in the clinical study and will visit the Maastricht UMC four
additional times. For the other 10 participants, the study ends after the first
visit.

- At the second visit (5 patients), a routine clinical physical examination and
will be performed, and a vastus lateralis skeletal muscle biopsy (~100mg) and a
venous blood sample (~10 ml) will be collected and they will try-out the bout
of eccentric exercise on the Biodex machine.
- At visit 3: routine physical examination, a bout of maximal eccentric
exercise will be performed and a venous blood sample (10 ml) will be collected.
- At visit 4: routine physical examination, intra-arterial administration of
5*10E7 MABs in one lower leg, followed by 24hrs observation in the hospital
including regular blood sampling (4x ~10ml) and a ~30mg tibialis anterior
muscle biopsy of both leg 24 hours after infusion of the asCTMP.
- Visit 5 consists of a neurological and routine clinical physical examination,
a venous blood sample (~10ml) and a ~30mg muscle biopsy in the tibialis
anterior muscles of both legs.

The burden and risk associated with participation in the clinical study (5
patients) will consist of the collection of in total 6 skeletal muscle
samples, 7 times venous blood sampling (10ml) and the intra-arterial
administration of 1,9*10E8 labelled MABs (based on a 75kg person). Muscle
biopsies can be painful in some cases. Infections and bleeding afterwards are
possible, but rare. To minimize patient burden, the five small (~30mg) muscle
biopsies collected at visit 1, 4 and 5 will be collected using the Pro-Mag I
automatic biopsy instrument, which is a fast and routinely used procedure to
harvest a small muscle fragment with patient burden being limited to the time
of the procedure (anecdotic information of >500 patients). Intra-arterial
injection can cause bleeding and an allergic reaction to the contrast agent is
possible. Intra-arterial injection of autologous cells are not expected to
trigger an immune response, especially, because no missing protein is
introduced as in DMD. Migration of MABs is possible when injected
intra-arterially, and allogeneic i.a. delivery of 1.6-9 x10E8 allogeneic MABs
in femoral artery was shown to be safe in children with DMD. Intra-arterial
infusion of 1,9x10E8 MABs may cause obstruction of a blood vessel. To prevent
formation of cell aggregates, 5 IU/ml heparin is added to the medicinal product
and a bolus of 3,000 IU heparin is given at insertion of the catheter. The
total dose of heparin is <3,200 IU, which is far below the maximum of 40,000
IU/day. After i.a. infusion, patients are monitored for 24 hrs and medication
to react in case of an adverse event (thrombosis, anaphylactic shock, allergic
reaction) is available.
The burden and risk associated with the first visit, for the 10 persons that
are excluded from participation in the clinical study, is one visit the MUMC at
which a small (~30mg) muscle biopsy will be collected using the Pro-Mag I
automatic biopsy instrument.
The expected benefit of the current study for mtDNA patients is the
characterization of the safety of autologous MABs as treatment for myopathy and
secondly, an indication of the homing and effectiveness at the tissue level of
the labelled asCTMP (myogenesis and m.3243A>G mutation load). This cannot be
studied in animal models, as no model is available for the specific mtDNA
mutation studied.