Improved transduction of canine X-linked muscular dystrophy with rAAV9-microdystrophin via multipotent MSC pretreatment

Abstract

Duchenne muscular dystrophy (DMD) is a severe congenital disease associated with mutation of the dystrophin gene. Supplementation of dystrophin using recombinant adeno-associated virus (rAAV) has promise as a treatment for DMD, although vector-related general toxicities, such as liver injury, neurotoxicity, and germline transmission, have been suggested in association with the systemic delivery of high doses of rAAV. Here, we treated normal or dystrophic dogs with rAAV9 transduction in conjunction with multipotent mesenchymal stromal cell (MSC) injection to investigate the therapeutic effects of an rAAV expressing microdystrophin (μDys) under conditions of immune modulation. Bone-marrow-derived MSCs, rAAV-CMV-μDys, and a rAAV-CAG-luciferase (Luc) were injected into the jugular vein of a young dystrophic dog to induce systemic expression of μDys. One week after the first injection, the dog received a second intravenous injection of MSCs, and on the following day, rAAV was intravenously injected into the same dog. Systemic injection of rAAV9 with MSCs pretreatment improves gene transfer into normal and dystrophic dogs. Dystrophic phenotypes significantly improved in the rAAV-μDys-injected dystrophic dog, suggesting that an improved rAAV-μDys treatment including immune modulation induces successful long-term transgene expression to improve dystrophic phenotypes.

Keywords: AAV; DMD; DMD dog model; MSC; immune modulation; microdystrophin.

Graphical abstract

Figure 1

Genotypes and gender of the rAAV-treated dogs 112 series dogs (11202FN, 11205MN, and 11206FN) were injected with rAAV at 8 weeks old; 118 series dogs (11806MN, 11803MN and 11805MA) were injected with rAAV at 11 weeks old. For the first administration, MSCs 1.0–2.28 × 10⁶ cells/kg BW with or without rAAV9-CAG-Luc-WPRE and rAAV9-CMV-microdystrophin 5.0 × 10⁵ v.g./cell were injected. For the second administration, MSCs 1.8–2.0 × 10⁶ cells/kg BW were injected. For local injection, rAAV9-CAG-Luc-WPRE 1.0 × 10¹² v.g./muscle were injected. For systemic injection, rAAV9-CAG-Luc-WPRE and rAAV9-CMV-microdystrophin 1.0 × 10¹² v.g./kg BW were injected. F, female; M, male; N, normal; A, affected.

Figure 2

Immunohistochemical detection of the transgene in the skeletal muscle of rAAV-transduced dogs Tibialis anterior muscles of the wild-type dogs were injected with rAAV-luciferase (N1), MSC pretreatment with rAAV-luciferase (N2), and PBS (N3), and analyzed 4 weeks after injection. Luciferase, CD8a, and CD4 were stained immunohistochemically. Scale bar, 100 μm.

Figure 3

T cell responses to the rAAV and transgene product in transduced dogs IFN-γ mRNA expression in PBMCs stimulated with rAAV. PBMCs from transduced dogs at each time point were stimulated overnight with rAAV9-luciferase or rAAV-microdystrophin. IFN-γ mRNA expression was determined by quantitative RT-PCR (ΔΔCt method). Expression levels of IFN-γ in the untreated normal dog are shown (N3). The RNA transcripts were normalized to endogenous 18S rRNA.

Figure 4

Immunohistochemical detection of the transgene in the rAAV-transduced dogs Transgene expression in the skeletal muscle at 134 weeks old (121 weeks after systemic rAAV-microdystrophin delivery). Skeletal muscle (tibialis anterior muscle) from transduced (A1) and untransduced normal dogs (N3) were cryo-sectioned and stained with anti-N-terminal of dystrophin antibody (DysB) and anti-C-terminal of dystrophin antibody (Dys2). $\times$ shows same muscle fiber in adjacent sections. Scale bar, 100 μm.

Figure 5

Clinical manifestations in the rAAV-transduced dogs Clinical grading scale used to assess the transduced dogs. Clinical scores used to measure gait disturbance, mobility disturbance, limb or temporal muscle atrophy, drooling, macroglossia, and dysphagia are shown. N5, normal dog, A1, dystrophic dog, treated with MSCs and rAAV-microdystrophin. Scores from DMD dog 1 and 2 are from non-treated CXMD_J in our dystrophic dog colony from the same age.

Figure 6

Summary of the echocardiography at 1 year old and 24-h Holter ECG recordings for the transduced dogs Echocardiography at 1 year old. (A–C) Summary (A), fractional shortening (%, B), and ejection fraction (%, C) are shown. Dog N5 is a normal beagle administered AAV9-luciferase and MSCs. Dog A0 is CXMD_J without any treatment. Dog A1 is CXMD_J, treated with AAV9-microdystrophin and MSCs.

Figure 7

Macroscopic findings of the transduced dogs (A) Gross morphology of the heart from each dog at the autopsy. The bottom panels show a cross-sectional view. Scale bar, 1 cm. White dotted lines show the position of cross dissection. LV, thickness of left ventricular wall; IVS, thickness of the intraventricular septum; RV, thickness of the right ventricular wall. (B and C) Immunohistochemical detection of the transgene in the heart of the rAAV-transduced dogs. Cardiac muscle (B, IVS; C, LV) from a transduced (A1) DMD dog were cryo-sectioned and stained with DysB. Scale bar, 50 μm (B) and 100 μm (C).