Cell-Based Therapeutics for Duchenne Muscular Dystrophy

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Ahlke Heydemann	--	1509	2023-03-23 04:01:33	\|
2	format correct	Catherine Yang	Meta information modification	1509	2023-03-24 02:43:45	\| \|
3	format correct	Catherine Yang	-1 word(s)	1508	2023-03-27 08:03:43	\|

This entry is adapted from the peer-reviewed paper 10.3390/biomedicines11030830

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene that cause an almost complete lack of the dystrophin protein in the patient. The dystrophin gene is found on the X-chromosome, causing the disease to be X-linked and almost exclusively affecting young boys and young men. Two novel techniques were recently utilized which identify muscle cell transplantation as a viable treatment for DMD. The first is to generate chimeric cells, fussing the patient's cells with donor cells. The patient's cells identify the fused cells as self, thus avoiding the expected immune response. The donor cells provide dystrophin, the mutated and absent protein. The second novel technique is to inject these fused cells via intraosseous injection. This iliac crest, bone marrow route has proven to be a true systemic delivery route. These two techniques have been successfully utilized in the mdx mouse to decrease pathology. The two techniques are now also being investigated in a clinical trial.

Muscular Dystrophy (MD) dystrophin expressing chimeric cells DEC myoblast transplantation

1. Choosing the Correct Cell Type

Several cell types have been investigated for their abilities to rescue DMD in the mdx mouse model. Initially, scientists used neonatal mouse myoblasts, additional studies utilized satellite cells (muscle resident stem cells), induced pluripotent stem cells (iPSCs), normal adult myoblasts, side population cells, bone marrow cells, pericytes, mesenchymal stem cells (MSC) and mesoangioblasts ^[1]^[2]. Many of these cells (especially the MSCs) have the added benefit of secreting cytokines that are myotrophic and/or myogenic ^[3]. At first glance, satellite cells would appear to be the best candidates to repopulate injured muscles. However, satellite cells have issues; very few can be isolated, propagation in culture reduces their stem-ness, they self-aggregate by blocking small vessels and never achieve muscle localization, even when injected directly into the muscle few cells survive ^[1]^[2].

There is also the decision between autologous or allogenic cells. Autologous procedures utilize the patient’s own cells, usually genetically corrected ex vivo, expanded and then reintroduced into the patient. These cells have the advantage of not requiring immune suppressive pharmaceuticals. Allogenic cells are from a different person, a true donor. These cells have the advantage that they contain normal dystrophin, so they only need to be expanded ex vivo. However, unless the cells are from a perfectly matched donor the patients will require immune suppressive medications for the remainder of their lives.

2. Production of Novel Chimeric Cells Which Avoids Immune Rejection

Many of the MD transplant therapies would require donor matching or continuous immunosuppressants. Donor matching requires identical blood types and identical Human Leukocyte Antigen (HLA) between the healthy, normal donor and the recipient. This test must be conducted to match each patient. Currently more than 1200 HLA variants have been identified, making matching difficult, time-consuming and expensive ^[4]. Close relatives are more likely to be matches, making the statistics better in some situations. The recipient’s blood must also be checked for anti-HLA antibodies possibly formed from a previous transplantation or blood transfusion. Additional tests are performed to identify harmful interactions between donor and host. Despite these many tests, graft-versus-host-disease still occurs in more than 25% of transplant recipients ^[5].

If a perfectly matched donor cannot be found, immunosuppressant pharmaceuticals are an option. These drugs are likely to be needed for the life of the patient and missing a single dose can cause severe reactions. These drugs are also associated with some severe side-effects. The patients will have increased chances of infections and may have coughs and colds that require more time to dissipate. Therefore, additional research is required to successfully bring cell transplants to the clinic.

One method of avoiding the immune rejection dilemma is to mark the transplanted organs, tissues or cells as self. An interesting method to mark donor cells as self is to fuse the donor cells to the recipient’s cells in ex vivo cell culture, to create Dystrophin Expressing Chimeric cells (DEC). To ensure chimerism, the cells are first labelled with red or green dyes, the yellow/orange fused cells are fluorescently sorted and expanded in culture. The sorting step is very important as some donor cells will fuse with other donor cells; if enough of these cells are transplanted an immune reaction would occur. In the desired yellow/orange chimeric cells, the donor cell and nucleus provide the dystrophin while the recipient cell and nucleus provide the cell surface proteins that mark the fused cell as self.

This fusion chimerism has been proven to induce tolerance in vascularized composite allotransplantation and bone marrow transplantation studies ^[6]^[7]^[8].

3. The DEC Fusion Techniques for Muscular Dystrophy Have Been Perfected In Vitro and Preclinically in the mdx Mouse

The proof-of-principle chimeric cell experiments demonstrated that myoblast cells from two different mice could be successfully fused without losing muscle stemness and without losing the ability to proliferate. Furthermore, when myoblasts from an mdx mouse were fused with myoblasts from a wildtype mouse: MB^N/MB^mdx the chimeric cells expressed dystrophin in vitro. After sorting for and expansion of the fused cells, the MB^N/MB^mdx cells were injected directly into the gastrocnemius muscle (GM) of mdx mice. Upon analysis at 90-days post-transplant, dystrophin was found to be expressed and localized correctly to the sarcolemma. The injected GM samples also demonstrated increased strength by in vivo and ex vivo muscle tests and increased resistance to fatigue ^[9]^[10].

The next step was to analyze the possibility that normal human myoblast cells could be fused to DMD patient myoblasts and still retain their ability to differentiate into muscle cells and proliferate ^[9]. After in vitro confirmation of fusion, differentiation and proliferation, these human fused cells were injected into the GM of mdx/scid mice and followed for 90 days. Again, the DEC cells repopulated the GM and expressed dystrophin and restored the expression of other dystrophin glycoprotein complex members. Furthermore, after 90 days the DEC-injected GM produced significantly more force ^[9].

An additional—often raised—cell transplant question is which cells will proliferate best and repopulate the muscle best. Based upon many previous publications, the authors also tested the fusibility of murine myoblasts with mesenchymal stem cells (MSC). The fused MB^N/MSC^N DEC cells performed well in the in vivo battery of tests. They fused well, maintained their muscle stem-ness, expressed dystrophin, and proliferated well. These cells also passed an in vitro Comet Assay, which investigates possible genotoxicity. None of the MB^N, MSC^N or MB^N/MSC^N DEC cells displayed any genotoxicity. Furthermore, to evaluate any possible immune reactions elicited by the transplanted chimeric cells the Mixed Lymphocyte Reaction was utilized. Again, the three chimeric cell types passed by displaying reduced alloreactivity. These DEC cells also passed the localized GM transplantation in vivo tests. At 90 days post-transplantation, dystrophin expression was present and functional tests demonstrated improvement over vehicle-injected controls ^[11].

4. Choosing the Correct Delivery Route

The chimeric cell technique overcomes many of the issues that surrounded cell transplantation experiments. The chimeras evade the immune response thus allowing efficient engraftment and proliferation when locally injected. As important as ensuring that the cells are right is the use of the correct delivery route. Scientists have attempted intravenous in the tail vein ^[12] or intraarterial transplantation ^[13]. None of these produced adequate engraftment or dystrophin expression. Very few of the precious cells implanted into the muscles. A newly tested delivery route is through intraosseous delivery (IO). This route has historically been used in an emergency setting when a vein cannot be accessed. This route was now to be tested for systemic DEC cell delivery ^[14]^[15]. These experiments could then also analyze the global effectiveness of the DEC cells in alleviating cardiac and respiratory pathologies.

Intraosseous delivery has now been effectively utilized preclinically to treat the mdx mouse model of DMD. Human DEC cells (MB^N/MB^DMD) were identified by immunofluorescence to have localized to the gastrocnemius, diaphragm, and cardiac muscles at least to 90 days post transplant ^[14]. The expression patterns correlated with improved, quantitative histopathology and function in all three muscle tissues. The quantitative histology measurements included the analysis of inflammatory foci. As inflammation is ubiquitous with DMD, the found decrease in this characteristic also indicates benefits from the DEC transplantation. Quantitative histology measurements also included fibrosis analysis, and again, the DEC-treated animals displayed reduced fibrosis. The functional improvements include force generation capabilities of the GM, improved ejection fraction and fractional shortening for the heart, and improved tidal volume and relaxation times for the diaphragm (improving respiratory function). These same benefits (in GM, diaphragm and cardiac muscles) were observed in mice that had received DEC therapy via the intraosseous route after 180 days ^[16]^[17]; thus, demonstrating that the DEC cells also have longevity after their engraftment.

Importantly, the mice were assessed for additional safety measures at 180 days after intraosseous transplantation of human DEC cells (MB^N/MB^DMD). By flow cytometry, there was negligible presence of DEC cells in the off-target organs (blood, bone marrow, lung, liver, and spleen) and a dose-dependent increase in DEC cells in the target organs (heart, diaphragm, and gastrocnemius). Furthermore, magnetic resonance imagining was utilized to demonstrate lack of tumors. The lack of tumors was verified by a trained veterinarian conducting a complete necropsy at the study’s endpoint ^[16].

References

Biressi, S.; Filareto, A.; Rando, T.A. Stem cell therapy for muscular dystrophies. J. Clin. Investig. 2020, 130, 5652–5664.
Sun, C.; Serra, C.; Lee, G.; Wagner, K.R. Stem cell-based therapies for Duchenne muscular dystrophy. Exp. Neurol. 2020, 323, 113086.
Cudkowicz, M.E.; Lindborg, S.R.; Goyal, N.A.; Miller, R.G.; Burford, M.J.; Berry, J.D.; Nicholson, K.A.; Mozaffar, T.; Katz, J.S.; Jenkins, L.J.; et al. A randomized placebo-controlled phase 3 study of mesenchymal stem cells induced to secrete high levels of neurotrophic factors in amyotrophic lateral sclerosis. Muscle Nerve 2022, 65, 291–302.
Chee, R.E. Histocompatibility testing. Southeast Asian J. Trop Med. Public Health 2002, 33 (Suppl. S2), 112–114.
Partanen, J.; Hyvarinen, K.; Bickeboller, H.; Bogunia-Kubik, K.; Crossland, R.E.; Ivanova, M.; Perutelli, F.; Dressel, R. Review of Genetic Variation as a Predictive Biomarker for Chronic Graft-Versus-Host-Disease After Allogeneic Stem Cell Transplantation. Front. Immunol. 2020, 11, 575492.
Siemionow, M.; Demir, Y.; Mukherjee, A.; Klimczak, A. Development and maintenance of donor-specific chimerism in semi-allogenic and fully major histocompatibility complex mismatched facial allograft transplants. Transplantation 2005, 79, 558–567.
Siemionow, M.; Klimczak, A.; Unal, S.; Agaoglu, G.; Carnevale, K. Hematopoietic stem cell engraftment and seeding permits multi-lymphoid chimerism in vascularized bone marrow transplants. Am. J. Transpl. 2008, 8, 1163–1176.
Hivelin, M.; Klimczak, A.; Cwykiel, J.; Sonmez, E.; Nasir, S.; Gatherwright, J.; Siemionow, M. Immunomodulatory Effects of Different Cellular Therapies of Bone Marrow Origin on Chimerism Induction and Maintenance Across MHC Barriers in a Face Allotransplantation Model. Arch. Immunol. Ther. Exp. 2016, 64, 299–310.
Siemionow, M.; Cwykiel, J.; Heydemann, A.; Garcia, J.; Marchese, E.; Siemionow, K.; Szilagyi, E. Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy. Stem Cell Rev. Rep. 2018, 14, 370–384.
Siemionow, M.; Cwykiel, J.; Heydemann, A.; Garcia-Martinez, J.; Siemionow, K.; Szilagyi, E. Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy. Stem Cell Rev. Rep. 2018, 14, 189–199.
Siemionow, M.; Szilagyi, E.; Cwykiel, J.; Domaszewska-Szostek, A.; Heydemann, A.; Garcia-Martinez, J.; Siemionow, K. Transplantation of Dystrophin Expressing Chimeric Human Cells of Myoblast/Mesenchymal Stem Cell Origin Improves Function in Duchenne Muscular Dystrophy Model. Stem Cells Dev. 2021, 30, 190–202.
Muskiewicz, K.R.; Frank, N.Y.; Flint, A.F.; Gussoni, E. Myogenic potential of muscle side and main population cells after intravenous injection into sub-lethally irradiated mdx mice. J. Histochem. Cytochem. 2005, 53, 861–873.
Bachrach, E.; Perez, A.L.; Choi, Y.H.; Illigens, B.M.; Jun, S.J.; del Nido, P.; McGowan, F.X.; Li, S.; Flint, A.; Chamberlain, J.; et al. Muscle engraftment of myogenic progenitor cells following intraarterial transplantation. Muscle Nerve 2006, 34, 44–52.
Siemionow, M.; Langa, P.; Harasymczuk, M.; Cwykiel, J.; Sielewicz, M.; Smieszek, J.; Heydemann, A. Human dystrophin expressing chimeric (DEC) cell therapy ameliorates cardiac, respiratory, and skeletal muscle’s function in Duchenne muscular dystrophy. Stem Cells Transl. Med. 2021, 10, 1406–1418.
Siemionow, M.; Malik, M.; Langa, P.; Cwykiel, J.; Brodowska, S.; Heydemann, A. Cardiac Protection after Systemic Transplant of Dystrophin Expressing Chimeric (DEC) Cells to the mdx Mouse Model of Duchenne Muscular Dystrophy. Stem Cell Rev. Rep. 2019, 15, 827–841.
Siemionow, M.; Brodowska, S.; Langa, P.; Zalants, K.; Kozlowska, K.; Grau-Kazmierczak, W.; Heydemann, A. Long-Term Biodistribution and Safety of Human Dystrophin Expressing Chimeric Cell Therapy After Systemic-Intraosseous Administration to Duchenne Muscular Dystrophy Model. Arch. Immunol. Ther. Exp. 2022, 70, 20.
Siemionow, M.; Langa, P.; Brodowska, S.; Kozlowska, K.; Zalants, K.; Budzynska, K.; Heydemann, A. Long-Term Protective Effect of Human Dystrophin Expressing Chimeric (DEC) Cell Therapy on Amelioration of Function of Cardiac, Respiratory and Skeletal Muscles in Duchenne Muscular Dystrophy. Stem Cell Rev. Rep. 2022, 18, 2872–2892.

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.

Upload a video for this entry

Information

Subjects: Biology

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Ahlke Heydemann

Maria Siemionow

View Times: 255

Update Date: 27 Mar 2023

Table of Contents

Video Upload Options

Confirm