Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by dystrophin gene mutations and mitochondrial dysfunction, leading to progressive muscle weakness and premature death of DMD patients. We developed human Dystrophin Expressing Chimeric (DEC) cells, created by the fusion of myoblasts from normal donors and DMD patients, as a foundation for DT-DEC01 therapy for DMD. Our preclinical studies on mdx mouse models of DMD revealed enhanced dystrophin expression and functional improvements in cardiac, respiratory, and skeletal muscles after systemic intraosseous DEC administration. The current study explored the feasibility of mitochondrial transfer and fusion within the created DEC cells, which is crucial for developing new therapeutic strategies for DMD. Following mitochondrial staining with MitoTracker Deep Red and MitoTracker Green dyes, mitochondrial fusion and transfer was assessed by Flow cytometry (FACS) and confocal microscopy. The PEG-mediated fusion of myoblasts from normal healthy donors (MBN/MBN) and normal and DMD-affected donors (MBN/MBDMD), confirmed the feasibility of myoblast and mitochondrial fusion and transfer. The colocalization of the mitochondrial dyes MitoTracker Deep Red and MitoTracker Green confirmed the mitochondrial chimeric state and the creation of chimeric mitochondria, as well as the transfer of healthy donor mitochondria within the created DEC cells. These findings are unique and significant, introducing the potential of DT-DEC01 therapy to restore mitochondrial function in DMD patients and in other diseases where mitochondrial dysfunction plays a critical role.

Assessment of Motor Unit Potentials Duration as the Biomarker of DT-DEC01 Cell Therapy Efficacy in Duchenne Muscular Dystrophy Patients up to 12 Months After Systemic-Intraosseous Administration.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disease caused by mutations in the dystrophin gene, leading to muscle degeneration and wasting. Electromyography (EMG) is an objective electrophysiological biomarker of muscle fiber function in muscular dystrophies. A novel, DT-DEC01 therapy, consisting of Dystrophin Expressing Chimeric (DEC) cells created by fusing allogeneic myoblasts from normal donors with autologous myoblasts from DMD-affected patients, was assessed for safety and preliminary efficacy in boys of age 6-15 years old (n = 3). Assessments included EMG testing of selected muscles of upper (deltoideus, biceps brachii) and lower (rectus femoris and gastrocnemius) extremities at the screening visit and at 3, 6, and 12 months following systemic-intraosseous administration of a single low dose of DT-DEC01 therapy (Bioethics Committee approval no. 46/2019). No immunosuppression was administered. Safety of DT-DEC01 was confirmed by the lack of therapy-related Adverse Events or Serious Adverse Events up to 22 months following DT-DEC01 administration. EMG of selected muscles of both, ambulatory and non-ambulatory patients confirmed preliminary efficacy of DT-DEC01 therapy by an increase in motor unit potentials (MUP) duration, amplitudes, and polyphasic MUPs at 12 months. This study confirmed EMG as a reliable and objective biomarker of functional assessment in DMD patients after intraosseous administration of the novel DT-DEC01 therapy.

Safety and Efficacy of DT-DEC01 Therapy in Duchenne Muscular Dystrophy Patients: A 12 - Month Follow-Up Study After Systemic Intraosseous Administration.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal muscle-wasting disease with no known cure. We previously reported the preliminary safety and efficacy up to six months after the administration of DT-DEC01, a novel Dystrophin Expressing Chimeric (DEC) cell therapy created by fusion of myoblasts of DMD patient and the normal donor. In this 12-month follow-up study, we report on the safety and functional outcomes of three DMD patients after the systemic intraosseous administration of DT-DEC01. The safety of DT-DEC01 was confirmed by the absence of Adverse Events (AE) and Severe Adverse Events (SAE) up to 21 months after intraosseous DT-DEC01 administration. The lack of presence of anti-HLA antibodies and Donors Specific Antibodies (DSA) further confirmed DT-DEC01 therapy safety. Functional assessments in ambulatory patients revealed improvements in 6-Minute Walk Test (6MWT) and timed functions of North Star Ambulatory Assessment (NSAA). Additionally, improvements in PUL2.0 test and grip strength correlated with increased Motor Unit Potentials (MUP) duration recorded by Electromyography (EMG) in both ambulatory and non-ambulatory patients. DT-DEC01 systemic effect was confirmed by improved cardiac and pulmonary parameters and daily activity recordings. This follow-up study confirmed the safety and preliminary efficacy of DT-DEC01 therapy in DMD-affected patients up to 12 months after intraosseous administration. DT-DEC01 introduces a novel concept of personalized myoblast-based cellular therapy that is irrespective of the mutation type, does not require immunosuppression or the use of viral vectors, and carries no risk of off target mutations. This establishes DT-DEC01 as a promising and universally effective treatment option for all DMD patients.

Dystrophin Expressing Chimeric (DEC) Cell Therapy for Duchenne Muscular Dystrophy: A First-in-Human Study with Minimum 6 Months Follow-up.

Duchenne Muscular Dystrophy (DMD) is a X-linked progressive lethal muscle wasting disease for which there is no cure. We present first-in-human study assessing safety and efficacy of novel Dystrophin Expressing Chimeric (DEC) cell therapy created by fusion of patient myoblasts with myoblasts of normal donor origin. We report here on safety and functional outcomes of the first 3 DMD patients. No study related adverse events (AE) and no serious adverse events (SAE) were observed up to 14 months after systemic-intraosseous administration of DEC01. Ambulatory patients showed improvements in functional tests (6-Minute Walk Test (6MWT), North Star Ambulatory Assessment (NSAA)) and both, ambulatory and non-ambulatory in PUL, strength and fatigue resistance which correlated with improvement of Electromyography (EMG) parameters. DEC01 therapy does not require immunosuppression, involves no risks of off target mutations, is not dependent upon the causative mutation and is therefore a universal therapy that does not use viral vectors and therefore can be readministered, if needed. This study was approved by the Bioethics Committee (approval No. 46/2019). Mechanism of action of the Dystrophin Expressing Chimeric Cell (DEC) cells created via ex vivo fusion of human myoblast from normal and DMD-affected donors. Following systemic-intraosseous administration, DEC engraft and fuse with the myoblasts of DMD patients, deliver dystrophin and improve muscle strength and function. (Created with BioRender.com).

Optimization of human myoblasts culture under different media conditions for application in the in vitro studies.

Human primary in vitro cell cultures are among the most challenging procedures in cellular biology laboratory practice. Myoblasts-progenitor of skeletal muscle origin represent a promising therapeutic cell source since the procedure of their isolation is not technically demanding, and the in vitro culture is relatively straightforward. Myoblasts could be considered as the candidates for clinical applications due to their regenerative potential, and as the carriers of therapeutic proteins introduced through genetic modifications. The main goal of this prospective study was to evaluate different myoblasts isolation strategies based on the pre-plating technique and cells density characteristics. Moreover, testing of different myoblast media formulations-both commercially available and in-house made was performed. Our goal was to establish the in vitro protocol of myoblasts culture allowing for preservation of the proliferative potential and desired phenotype. Our results revealed that in culture of myoblasts of human muscle origin, the pre-plate technique and cell density differences did not correlate with changes in the proliferative potential, however it was observed that low density cells maintained expression of the CD56 marker up to the higher passages. Assessment of different types of culture media confirmed the best performance for DMEM based media without Chicken Embryo Extract (CEE) addition. Cells cultured in DMEM+FBS medium revealed high expression of CD56 and CD90 antigens, absence of the hematopoietic markers and presented stable proliferation profile. This finding is in line with guidelines of regulatory agencies recommending removal of the xeno-derived reagents from the manufacturing process of Advanced Therapy Medicinal Products (ATMP). In this study, human myoblasts culture was optimized in vitro under different media conditions. The next approach in assessment of myoblasts propagation for potential clinical applications will be testing of the clinical grade human platelet lysate (hPL) instead of the FBS.

Attempts to obtain fully xenogeneic fetuses in rat ↔ mouse model†,‡.

The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse ↔ rat chimeras, the interspecific uterine barrier is less hostile to interspecific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse ↔ rat blastocysts by injection of mouse embryonic stem cells (ESCs) into eight-cell rat embryos and rat ESCs into eight-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat ↔ mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.

Mouse↔rat aggregation chimaeras can develop to adulthood.

In order to examine interactions between cells originating from different species during embryonic development we constructed interspecific mouse↔rat chimaeras by aggregation of 8-cell embryos. Embryos of both species expressed different fluorescent markers (eGFP and DsRed), which enabled us to follow the fate of both components from the moment of aggregation until adulthood. We revealed that in majority of embryos the blastocyst cavity appeared inside the group of rat cells, while the mouse component was allocated to the deeper layer of the inner cell mass and to the polar trophectoderm. However, due to rearrangement of all cells and selective elimination of rat cells, shortly before implantation all primary lineages became chimaeric. Moreover, despite the fact that rat cells were always present in the mural trophectoderm, majority of mouse↔rat chimaeric blastocysts implanted in mouse uterus, and out of those 46% developed into foetuses and pups, half of which were chimaeric. In contrast to mural trophectoderm, polar trophectoderm derivatives, i.e. the placentae of all chimaeras were exclusively of mouse origin. This strongly suggests that the successful postimplantation development of chimaeras is enabled by gradual elimination of xenogeneic cells from the nascent placenta. The size of chimaeric newborns was within the limits of control mouse neonates. The rat component located preferentially in the anterior part of the body, where it contributed mainly to the neural tube. Our observations indicate that although chimaeric animals were able to reach adulthood, high contribution of rat cells tended to diminish their viability.

In Memoriam - Prof. Andrzej Krzysztof Tarkowski (1933-2016).

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.