Effect and Safety MABs Administration m.3243A>G Mutation Carriers

Assess Effect and Safety of Intra-arterial Autologous Mesoangioblast Administration to the Upper Arm of m.3243A>G Mutation Carriers

The first primary objective is to assess the effect of three intra-arterial administrations of autologous mesoangioblasts (MABs) with respect to improving muscle strength and reduce fatigue of the treated biceps brachii (BB) compared to the untreated BB. The second primary objective is safety of three intra-arterial administrations of autologous MABs, which the investigators will assess by monitoring (serious) adverse events ((S)AEs), blood flow in left arm pre- and post-intervention, and neurological vital signs during 8h post-intervention observation in the hospital. Secondary objectives are to assess changes in muscle mass of the treated and untreated BB muscle, and microscopic changes and m.3243A>G mutation load at tissue level in treated biceps brachii (BB) muscle at baseline and after treatment. Up to 20 adult m.3243A>G patients will undergo a ~30mg m. biceps brachii muscle biopsy at visit 1. The first six eligible patients will enroll the clinical study based on their m.3243A>G mutation load in skeletal muscle (50-90%) and mesoangioblasts (<10%), and on a decreased BB muscle strength and increased fatigue. These 6 selected patients will visit the Maastricht University Medical Center for 8 additional times. From each patient, during visit 2 till 9: BB muscle biopsies of the left arm will be collected (1x ~130 mg at visit 2 and 1x ~30mg at visit 9) MRI of the BB muscles in both arms will be performed (visit 2 and 9). Autologous MABs will be injected into the left arm via axillary artery delivery. Angiography will be performed before and after infusion to assess vascular obstructions, and the participant will be monitored in the hospital for 8 hours (visit 4,6,8). Tc99m macroaggregated albumin (MAA) is infused to quantify blood flow to the BB muscle (visit 4). A bout of maximal eccentric exercise of BB muscles on both sides will be executed at visit 3, 5 and 7. BB muscle strength will be assessed using a Biodex dynamometer (visit 3-9) venous blood samples will be taken for assessing muscle damage and inflammation markers (visit 3-9), kidney functioning, coagulation and viral screening (visit 1 and 2).

Intra-subjected controlled clinical study. Treatment is performed in left arm of the subjects and right arm (no intervention), measurements are performed before the first and after the last administration.

three times intra-arterial administration of autologous mesoangioblasts in biceps brachii of the left arm at 4-6 week interval

Assess blood flow in left arm using digital subtraction angiography (DSA) before and directly after MABs administration in week 1, 5 and 10.

Using Medical Research Council (MRC) scale for muscle strength, muscle strength of left arm will be assessed 0,1,2,3,4,6 and 8 hours after administration in week 1, 5 and 10. MRC scale ranges from 0 (no visible contraction) to 5 (normal)).

Breathing frequency will be checked 0,1,2,3,4,6 and 8 hours after administration in week 1, 5 and 10.

Systolic and Diastolic blood pressure will be checked 0,1,2,3,4,6 and 8 hours after administration in week 1, 5 and 10.

Glasgow Coma Scale (GCS) score will be determined 0,1,2,3,4,6 and 8 hours after administration in week 1, 5 and 10. GCS score ranges from 3 to 15, 3 being the worst and 15 being the best score.

Pupil size of both eyes will be assessed 0,1,2,3,4,6 and 8 hours after administration in week 1, 5 and 10. If pupil size is not equal, this can indicate disease or trauma.

To assess brain functioning, reaction of pupils to light will be determined 0,1,2,3,4,6 and 8 hours after administration. Upon light, both pupils should become smaller in week 1, 5 and 10. No reaction or only reaction in one eye can indicate nerve damage.

Assess changes in muscle strength of biceps brachii muscle in both arms at baseline and 4-6 weeks after third i.a. delivery of autologous MABs in left arm, which is 12-16 weeks after baseline measurements.

Determine maximum muscle force generating capacity (peak torque N/m) of the biceps brachii muscle in both arms using Biodex dynamometer measurements. Measurements will be performed in left (intervention) and right (no intervention) biceps brachii muscle using Biodex dynamometer measurements at baseline and 5 weeks after the third autologous MABs administration, which is 15 weeks after baseline measurements.

Assess changes in muscle fatigue of biceps brachii muscle in both arms at baseline and 4-6 weeks after third i.a. delivery of autologous MABs in left arm, which is 12-16 weeks after baseline measurements.

Asses changes in muscle fatigue by measuring percentage decrease in maximum muscle force generating capacity (peak torque N/m) between first and sixth repetition. Measurements will be performed in left (intervention) and right (no intervention) biceps brachii muscle using Biodex dynamometer measurements at baseline and 5 weeks after the third autologous MABs administration, which is 15 weeks after baseline measurements.

Assess changes in muscle volume biceps brachii muscles of both arms following 3 i.a. deliveries of autologous MABs in left arm.

MRI T3 analysis to assess changes in muscle volume in both arms at baseline and 5 weeks after the third autologous MABs administration, which is 15 weeks after baseline measurements.

Perform embryonic myosin heavy chain (MHC)+ immunostaining to assess percentage of new / regenerating muscle fibers in muscle biopsies collected at baseline and 5 weeks after the third autologous MABs administration, which is 15 weeks after baseline measurements.

Assess changes in m.3243A>G mutation load in new/regenerating muscle fibers compared to existing muscle fibers isolated via laser microdissection from muscle biopsies left arm collected at baseline and 5 weeks after the third autologous MABs administration, which is 15 weeks after baseline measurements.

Assess changes in mitochondrial functioning by performing Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) staining in muscle biopsies from left arm collected at baseline and 5 weeks after the third autologous MABs administration, which is 15 weeks after baseline measurements.

Assess creatine kinase level in blood plasma as marker for muscle damage following 3 i.a. deliveries of autologous MABs

Assess creatine kinase (CK) in blood plasma following eccentric exercise prior and 8 hours after administration in week 1,5 and 10.

Inclusion Criteria: Written informed consent Age: 18-64 Sex: male/female Patients with the m.3243A>G mutation load of 50%-90% determined in skeletal muscle or derived from age-corrected calculation of blood m.3243A>G mutation load Exclusion Criteria: A potential subject who meets any of the following criteria will be excluded from participation in this study: Use of dabigatran, apixaban, edoxaban or rivaroxaban (DOACs) as anti-coagulants Have a weekly alcohol intake of ≥ 35 units (men) or ≥ 24 units (women) Current history of drug abuse Deficient immune system or autoimmune disease Significant concurrent illness Ongoing participation in other clinical trials with intervention Pregnant or lactating women Psychiatric or other disorders likely to impact on informed consent Patients unable and/or unwilling to comply with treatment and study instructions A history of strokes with signs of extra-pyramidal or pyramidal syndrome Allergy for contrast fluid Peripheral signs of ischemia or vasculopathy Claustrophobia Metal implants Any other factor that in the opinion of the investigator excludes the patient from the study

van Tienen F, Zelissen R, Timmer E, van Gisbergen M, Lindsey P, Quattrocelli M, Sampaolesi M, Mulder-den Hartog E, de Coo I, Smeets H. Healthy, mtDNA-mutation free mesoangioblasts from mtDNA patients qualify for autologous therapy. Stem Cell Res Ther. 2019 Dec 21;10(1):405. doi: 10.1186/s13287-019-1510-8.