A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Louise R Rodino-Klapac, Paul M L Janssen, Chrystal L Montgomery, Brian D Coley, Louis G Chicoine, K Reed Clark, Jerry R Mendell, Louise R Rodino-Klapac, Paul M L Janssen, Chrystal L Montgomery, Brian D Coley, Louis G Chicoine, K Reed Clark, Jerry R Mendell

Abstract

Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.

Methods: The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD.

Results: Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 +/- 0.9 (1 month), 91.3 +/- 3.1 (2 months), and 89.6 +/- 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 +/- 6.8 (1 month), 78.9 +/- 7.4 (2 months), and 81.2 +/- 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 +/- 0.3 (1 month), 2.1 +/- 0.8 (2 months), and 2.1 +/- 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 x 1012 vg/kg in mouse vs 2 x 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius - 63.8 +/- 4.9%, lateral gastrocnemius - 66.0 +/- 4.5%, EDL - 80.2 +/- 3.1%, soleus - 86.4 +/- 1.9%, TA - 72.2 +/- 4.0%.

Conclusion: These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD.

Figures

Figure 1
Figure 1
(A) Schematic of murine micro-dystrophin construct. A truncated MCK promoter/enhancer (563 bp) is used to drive muscle specific gene expression. Also labeled is a chimeric SV40 intron (97 bp) and synthetic polyadenylation site (53 bp). The 3,590 bp murine micro-dystrophin construction is depicted in detail. ABD is the complete actin binding domain, hinges 1, 2 and 4 are shown (green boxes), as are the flanking spectrin rod domains (SR blue boxes). The cysteine-rich dystroglycan binding domain is denoted by an orange box. AAV2 ITR are shown as arrowheads. (B) Immunofluorescence detection of micro-dystrophin expression in mdx mouse TA muscle. rAAV1, 6, or 8.micro-dystrophin (1011 vg) was delivered by intramuscular injection (IM Control) or ILP through the femoral artery of 3–4 week old mdx mice. Representative TA muscle sections (12 um) are shown from 4 week post-injected animals (8 and 12 weeks looked similar). Sections were immuno-stained with the N-terminal dystrophin antibody Manex1a. Scale Bar, 50 μm.
Figure 2
Figure 2
Quantification of micro-dystrophin protein expression. Micro-dystrophin distribution in muscle sections (4, 8, 12) weeks was quantified by visual fiber counts (images from 4 random fields) of the number of positive fibers/total fibers and represented as a percent mean ± SEM (n = 4–8 per group).
Figure 3
Figure 3
Micro-dystrophin expression levels result in reduced mdx pathology. Hematoxylin and Eosin (H&E) analysis revealed a reduction in centralized nuclei, a hallmark of DMD pathology. (A, C) H&E staining of rAAV6 and rAAV8 micro-dystrophin muscle sections, respectively (8 week time point). (B,D) Micro-dystrophin immunofluorescence detection in serial sections of rAAV6 and rAAV8 cohorts, respectively. Scale Bar, 25 μm. (E) Micro-dystrophin expression results in a 25% decrease in centralized nuclei. (F) Frequency distribution demonstrating micro-dystrophin expression results in a significant increase in type I, IIa, and IIb fiber diameter for rAAV6 and rAAV8.micro-dystrophin injected mdx mice compared to mdx controls. (n = 5–6 per group) (p < 0.0001 ANOVA analysis).
Figure 4
Figure 4
Components of the dystrophin-associated protein complex are restored in mdx mice treated with rAAV.micro-dystrophin gene transfer. mdx mice treated with rAAV8.micro-dystrophin delivered by ILP (8 week time point) were serial sectioned and stained with manex1a antibody for dystrophin, α-sarcoglycan, β-sarcoglycan, and H &E. Muscle fibers transduced with micro-dystrophin also exhibited restoration of expression of α-sarcoglycan and β-sarcoglycan (no staining in untreated mdx mice – bottom row). Muscle fibers from serial sections exhibited identical expression localization for each protein (arrow).
Figure 5
Figure 5
rAAV.micro-dystrophin gene transfer improves force generation measurements significantly. (A) rAAV8.micro-dystrophin and rAAV6.micro-dystrophin treated mdx extensor digitorum longus (EDL) muscles (8 week time point) demonstrated significant maximum specific force improvement (mN/mm2) over mdx-untreated muscles (p < 0.05) but did not reach levels of age-matched C57BL/10 wild type (WT) controls (P < 0.05). Values are presented as the means ± SEM, (B) EDL muscles were subjected to 10 cycles of isometric stimulation at 150 Hz with a 10% lengthening to induce damage during the last 100 ms of each contraction. Force values are represented as fractions of the first contraction. rAAV8.micro-dystrophin and rAAV6.micro-dystrophin treated EDL muscles exhibited significant protection from damage compared to mdx-untreated during the first two cycles (p < 0.05), while thereafter all 4 groups showed such robust injury that differences could no longer be determined. There was no significant difference between rAAV8- or rAAV6-treated and WT. (C) The ratio of force (ECC2/ECC1) after one lengthening contraction of rAAV-treated versus the initial contraction of WT is not different, but is significantly improved compared to mdx-untreated (n = 6–8 per group; P < 0.05).
Figure 6
Figure 6
Isolated limb perfusion of rAAV8.CMV.eGFP in cynomologous macaques (4–5 kg) resulted in widespread gene expression in lower limb muscles. Using a custom (PE 50 Tubing) made catheter and the aid of a 0.018 m guide wire and fluoroscopy, 2 × 1012 vg/kg (2 ml volume) was administered via the femoral artery. A tourniquet occluded flow during a 10 minute dwell time to allow vector to bind. Three weeks post transfer, robust transduction was observed in muscles perfused by peroneal vessels (TA and EDL) and tibial vessels (Med and Lat. Gas. – medial and lateral gastrocnemius and soleus) (n = 2). Scale Bar, 100 μm.

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