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. 2020 Feb;31(3-4):241-252.
doi: 10.1089/hum.2019.224. Epub 2020 Jan 10.

Restoration of Functional Full-Length Dystrophin After Intramuscular Transplantation of Foamy Virus-Transduced Myoblasts

Jinhong Meng  1   2 , Nathan Paul Sweeney  3 , Bruno Doreste  1   2 , Francesco Muntoni  1   2 , Myra McClure  3 , Jennifer Morgan  1   2
Affiliations

Restoration of Functional Full-Length Dystrophin After Intramuscular Transplantation of Foamy Virus-Transduced Myoblasts

Jinhong Meng et al. Hum Gene Ther. 2020 Feb.

Abstract

Stem cell therapy is a promising strategy to treat muscle diseases such as Duchenne muscular dystrophy (DMD). To avoid immune rejection of donor cells or donor-derived muscle, autologous cells, which have been genetically modified to express dystrophin, are preferable to cells derived from healthy donors. Restoration of full-length dystrophin (FL-dys) using viral vectors is extremely challenging, due to the limited packaging capacity of the vectors, but we have recently shown that either a foamy viral or lentiviral vector is able to package FL-dys open-reading frame and transduce myoblasts derived from a DMD patient. Differentiated myotubes derived from these transduced cells produced FL-dys. Here, we transplanted the foamy viral dystrophin-corrected DMD myoblasts intramuscularly into mdx nude mice, and showed that the transduced cells contributed to muscle regeneration, expressing FL-dys in nearly all the muscle fibers of donor origin. Furthermore, we showed that the restored FL-dys recruited members of the dystrophin-associated protein complex and neuronal nitric oxide synthase within donor-derived muscle fibers, evidence that the restored dystrophin protein is functional. Dystrophin-expressing donor-derived muscle fibers expressed lower levels of utrophin than host muscle fibers, providing additional evidence of functional improvement of donor-derived myofibers. This is the first in vivo evidence that foamy virus vector-transduced DMD myoblasts can contribute to muscle regeneration and mediate functional dystrophin restoration following their intramuscular transplantation, representing a promising therapeutic strategy for individual small muscles in DMD.

Keywords: Duchenne muscular dystrophy; codon-optimized full-length dystrophin; foamy virus; intramuscular transplantation; mdx nude mice.

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Conflict of interest statement

No competing financial interests exist.

Figures

Figure 1.
Figure 1.
Human skeletal muscle CD133+ cells retain their in vivo myogenic capacity post-transduction with FV. The percentage of GFP+ cells (a) and the mean fluorescence intensity (b) of cells transduced with different MOIs of FV coding for GFP, 6 days after transduction. (c) The contribution of transduced cells (MOI 10) to muscle regeneration after their intramuscular transplantation into immunodeficient mice. Donor-derived nuclei expressed human lamin AC+ (red), and donor muscle fibers were identified by their GFP expression. Scale bar = 25 μm. FV, foamy virus; GFP, green fluorescent protein; MOI, multiplicity of infection. Color images are available online.
Figure 2.
Figure 2.
In vitro transduction of human DMD myoblasts with FV-coFLDys. (a) In vitro proliferation curve of cells transduced with different amounts of FVs, for 30 days after their transduction. (b) The expression of dystrophin in cells with FV of MOI 2. (c) Western blot indicating the correct molecular weight of the transgene in differentiated myotubes derived from the transduced cells. (d–f) Immunostaining of myosin heavy chain (d, green), dystrophin (e, red), and merged images (f). Nuclei were counterstained with DAPI. Scale bar = 25 μm. coFLDys, codon-optimized full-length dystrophin; DMD, Duchenne muscular dystrophy. Color images are available online.
Figure 3.
Figure 3.
FV-coFLDys-transduced DMD myoblasts contribute to muscle regeneration after intramuscular transplantation into immunodeficient mdx nude mice. (a–c) Immunostaining of human lamin AC/human spectrin (a, both red), human dystrophin (green, b), and (c) merged image of (a, b). (d, e) Quantification of the number of human spectrin+ fibers, which contained at least one human lamin A/C+ nucleus (S+L); and dystrophin+ fibers in each transplanted muscle. Color images are available online.
Figure 4.
Figure 4.
Restored full-length dystrophin recruits members of the DAPC at the sarcolemma of donor-derived myofibers in vivo. (a–c) Immunostaining of dystrophin (green, a) and α-sarcoglycan (red, b) on donor fibers. (c) Merged image of (a, b). (d–f) Immunostaining of dystrophin (green, d) and γ-sarcoglycan (red, e). (f) Merged image of (d, e). (g) Intensity of α-sarcoglycan on dystrophin+ fibers and dystrophin− fibers within the transplanted muscle. Data were compared using paired t-test. (h) Linear regression of the intensity of α-sarcoglycan and dystrophin in a transverse section of a representative muscle. (i) Intensity of γ-sarcoglycan on dystrophin+ fibers and dystrophin− fibers within transplanted muscles; data were compared using paired t-test. (j) Linear regression of the intensity of γ-sarcoglycan and dystrophin in a transverse section of a representative muscle. Scale bar = 50 μm. DAPC, dystrophin-associated protein complex. Color images are available online.
Figure 5.
Figure 5.
Upregulation of nNOS on dystrophin+ donor-derived myofibers within transplanted muscles. (a–c) Immunostaining of dystrophin (green, a) and nNOS (red, b). (c) Merged image of (a, b). Scale bar = 50 μm. (d) Quantification of the intensity of nNOS on dystrophin+ and dystrophin− fibers; data were compared using paired t-test. (e) Linear regression analysis of the intensity of nNOS and dystrophin in a transverse cryosection of a representative muscle showed positive correlation between the two proteins in donor-derived muscle fibers. nNOS, neuronal nitric oxide synthase. Color images are available online.
Figure 6.
Figure 6.
Restoration of dystrophin decreases the utrophin expression on donor-derived fibers. (a–f) Immunostaining of dystrophin (green, a, d) and utrophin (red, b, e) in transplanted muscles. (c, f) Merged image of (a, b) or (d, e), respectively. (d–f) Enlarged images of the square in (a–c). Scale bar = 50 μm. (g) Paired t-test of the intensity of utrophin in dystrophin+ and dystrophin− fibers. (h) Linear regression analysis of the intensity of the utrophin and dystrophin in dystrophin+ donor fibers from one of the representative muscles suggested no correlation of the expression of the two proteins. Color images are available online.
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