Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy

Abstract

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 (MB^N/MB^N) and normal and DMD-affected donors (MB^N/MB^DMD), 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.

Keywords: Chimeric mitochondria; DMD therapy; Dystrophin expressing chimeric (DEC) cells; Mitochondria in DMD; Mitochondrial fusion; Mitochondrial transfer.

Fig. 1

Mitochondrial Transfer and Creation of Chimeric Mitochondria via PEG-mediated Fusion of Human Myoblast Derived from Normal and DMD-affected Donors for Enhancement of Therapeutic Effect of Dystrophin Expressing Chimeric (DEC) Cells. Human myoblasts isolated from tissue biopsies obtained from a normal donor (MB^N) and a DMD patient (MB^DMD) were propagated in in vitro cell culture. To assess the fate of mitochondria after cell fusion, normal donor (MB^N) cells were stained with the mitochondrial dye MitoTracker Deep Red and DMD patient (MB^DMD) cells with MitoTracker Green. The PEG-mediated fusion resulted in transfer of normal, healthy donor mitochondria and the formation of MB^N/MB^DMD chimeric mitochondria within the Dystrophin Expressing Chimeric (DEC) cells, confirming the enhanced therapeutic potential for DT-DEC01 therapy. The DEC cells were propagated and the DT-DEC01 product was prepared for the systemic intraosseous administration to DMD patient

Fig. 2

Confirmation of Mitochondrial Transfer and Creation of Chimeric Mitochondria via ex-vivo PEG-mediated Fusion of Myoblasts Derived from Normal Human Donors. (a) Representative flow cytometry dot plots confirming fusion of the MB^N/MB^N cells stained with the MTDeepRed or MTGreen mitochondrial dyes assessed by FACS. The upper row shows the identification of single myoblasts populations (singlets) among cell debris and aggregates. Lower row shows fluorescence analysis of the single-stained MTDeepRed (Q1) or MTGreen (Q4) mitochondria, the MIX of single-stained MTDeepRed and MTGreen mitochondria (Q1 and Q4) and the overlapping fluorescence of MTDeepRed/MTGreen (Q2) confirming the chimeric state of mitochondria. (b) Analysis of the single myoblasts after fusion using the FlowSight^® Amnis Imaging flow cytometer. The upper row shows the identification of single cell populations among cell debris and aggregates. The lower row shows (from left to right): the double-positive MTDeepRed/MTGreen signal indicating chimeric mitochondria, the cellular morphology of DEC cells in the brightfield channel (Ch01), the fluorescence image of MTDeepRed (Ch11) and MTGreen (Ch02), and colocalization of the MTDeepRed/MTGreen signal (Ch11/Ch02). (c) Representative confocal microscopy images of the myoblasts before and after fusion. The upper row shows the control, MIX of the single-stained MTDeepRed, MTGreen myoblasts and the merged channels (Merge). The lower row shows the fluorescence image of the chimeric myoblast after fusion in the single channels of the FarRed and Green, and the Merge showing overlapping signals of MTDeepRed/MTGreen indicating the chimeric mitochondria. The red arrows denote the mitochondria of normal donor (MB^N) origin (MTDeepRed), the green arrows indicate the mitochondria of normal donor (MB^N) origin (MTGreen), and the yellow arrows indicate the chimeric mitochondria of normal donors origin (MB^N/MB^N) as shown by the overlapping MTDeepRed/MTGreen fluorescence dyes (magnification 100x/1.49, scale bar 20 μm)

Fig. 3

Confirmation of Mitochondrial Transfer and Creation of Chimeric Mitochondria via ex-vivo PEG-mediated Fusion of Myoblasts Derived from Normal and DMD-affected Human Donors. (a) Representative flow cytometry dot plots confirming fusion of the MB^N/MB^DMD cells stained with the mitochondrial dyes of MTDeepRed or MTGreen assessed by FACS. The first two dot plots show the identification of single myoblasts populations (singlets) among cell debris and aggregates. Next dot plots show fluorescence analysis of MTDeepRed (Q1) or MTGreen (Q4) mitochondria, the MIX of single-stained MTDeepRed and MTGreen mitochondria (Q1 and Q4) and the overlapping fluorescence of MTDeepRed/MTGreen (Q2) confirming the chimeric state of mitochondria. (b) Representative confocal microscopy images of myoblasts after fusion. From left to right, the images show the fluorescence of the stained myoblasts after fusion in a wide field of view in the split channels of the FarRed and Green, and in the merged channels (Merge). Objects marked with (-) are the single-stained normal donor myoblasts (MB^N) with the MTDeepRed mitochondrial dye or the single-stained DMD patient myoblasts (MB^DMD) with the MTGreen mitochondrial dye. Objects marked with (+) represent the double-positive chimeric myoblasts (MB^N/MB^DMD) with the overlapping MTDeepRed/MTGreen mitochondrial dyes. Yellow arrows indicate the chimeric cells (No. 1 and No. 2), selected for the mitochondrial chimerism analysis shown in the panel below. Nuclei were counterstained with DAPI (blue) (magnification 60x/1.4, scale bar 25 μm). (c) Representative confocal microscopy images of the myoblasts before and after fusion. The upper row shows the images of the fluorescence control: the MIX of the single-stained normal donor myoblasts (MB^N) with the MTDeepRed mitochondrial dye and the single-stained DMD patient myoblasts (MB^DMD) with the MTGreen mitochondrial dye in the split channels of the FarRed, the Green and the Merge, confirming the absence of fusion by the lack of fluorescence dyes overlap. The rows below represent confocal microscopy images after fusion (MB^N/MB^DMD) and show images of the chimeric myoblasts: No. 1 and No. 2 (the second and the third row, respectively). The cells were captured in the single channels of the FarRed where the red arrows denote the mitochondria of normal donor (MB^N) origin (MTDeepRed) and in the Green channel where the green arrows indicate mitochondria of DMD patient (MB^DMD) origin (MTGreen). After the merge of the MTDeepRed/MTGreen fluorescence dyes, the chimeric mitochondria are indicated by the yellow arrows. The images on the right, include the Pearson correlation coefficient values for the co-localization of two mitochondrial dyes, confirming the presence of the chimeric mitochondria as shown by the overlapping MTDeepRed/MTGreen fluorescence dyes and determining the degree of mitochondrial chimerism (magnification 60x/1.4, scale bar 5 μm). Nuclei were counterstained with DAPI (blue)