Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse

Abstract

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.

Figure 1

Morphology, human minidystrophin expression and ex vivo muscle function analysis of diaphragm muscle. (A) Sections of diaphragm from experimental and control mice were stained with hematoxylin and eosin (H&E) and immunolabeled for minidystrophin (minidys) transgene expression detected with green fluorescence and stained with DAPI to detect nuclei (image bar = 200 μm). (B) Human minidystrophin-expressing muscle fibers were quantified and expressed as an average percentage of the total number of muscle fibers per section. Necrotic and regenerating muscle fibers were identified by binding to mouse immunoglobulin G (IgG) and expression of embryonic myosin heavy chain (eMyHC), respectively, and expressed as the average percentage of the total number of muscle fibers per section. Diaphragms from C57BL/10 healthy control mice lack human minidystrophin expression, necrosis and regeneration (data not shown in B). Ex vivo functional analysis of the diaphragm is shown by specific force production (N/cm²) (C) and as the percent force production in response to 10 isometric lengthening activations (D). All quantitative data are shown as mean ± standard error of the mean (SEM); *significant difference from untreated mdx mice (P < 0.05); n = number of animals analyzed. —♦—, C57BL/10; , untreated mdx; —□—, mdx, AAV + NBD peptide; —●—, mdx, AAV + saline.

Figure 2

Morphology and human minidystrophin expression analysis of quadriceps muscle. (A) Sections of quadriceps muscle were studied with hematoxylin and eosin (H&E) staining and immunohistochemical detection of human minidystrophin expression (image bar = 200 μm). (B) Quantitative analysis of transgene expression (minidys), necrosis (IgG) and regeneration (eMyHC) is shown as the average percentage of positive fibers per total number of muscle fibers per section. Quadriceps from C57BL/10 healthy control mice lack human minidystrophin expression, necrosis and regeneration (data not shown in B). All quantitative data are shown as mean ± SEM; *significant difference from untreated mdx mice (P < 0.05); **significant difference from AAV + saline–treated mdx mice (P < 0.05); n = number of animals analyzed.

Figure 3

Quantification of human minidystrophin expression in quadriceps, heart and diaphragm muscles. Minidystrophin transgene protein expression was detected from total protein extracts by Western blot analysis. (A) Three representative bands reflecting the range of results are shown for quadriceps, heart and diaphragm muscles. There is no human minidystrophin expression in quadriceps, heart and diaphragm in mdx and C57BL/10 controls (data not shown). (B) Densitometric quantification of bands from all mice in each group was normalized to GAPDH expression. All quantitative data are shown as mean ± SEM.; *significant difference from AAV + saline–treated mdx mice (P < 0.05); n = number of animals analyzed.

Figure 4

Activation of NF-κB. (A) NF-κB activation was analyzed by electrophoretic mobility shift assay of nuclear protein extracts of diaphragm and quadriceps muscle tissue in age-matched, untreated C57BL/10 and mdx mice and AAV + NBD peptide– and AAV + saline–treated mdx mice. Three representative bands reflecting the range of results are shown. (B) Densitometric quantification of bands from all treated and control mice were averaged and are presented as mean ± SEM. *Significant difference from untreated mdx mice (P < 0.05); n = number of animals analyzed.

Figure 5

Biodistribution of minidystrophin transgene expression. Sections of muscle tissues (diaphragm, quadriceps, tibialis anterior, gastrocnemius, forelimb triceps, heart and tongue) were analyzed for human minidystrophin expression by immunohistochemistry to assess levels and distribution of transgene expression from the AAV minidystrophin vector with (AAV + NBD peptide) or without (AAV + saline) treatment with the 8K-NBD peptide. One representative mouse from each treatment group is shown. Representative images are from individual muscles of AAV + NBD peptide–treated (A–G) and AAV + saline–treated (H–N) groups. Image bar = 200 μm. L, Left; R, right.

Figure 6

Quantification of vector DNA and mRNA transcripts in quadriceps. DNA and RNA were isolated from quadriceps muscle tissues from AAV + NBD peptide–and AAV + saline–treated mdx mice and analyzed by real-time qPCR. Vector DNA levels were quantified by primers/probes specific for AAV9 minidystrophin DNA, using isolated AAV9 minidystrophin vector DNA to generate a standard curve. The numbers of nuclei per sample were quantified by real-time qPCR using primers/ probes specific for the ApoB gene. (A) Vector copy numbers were normalized to nuclei copy number to yield vector genomes/nucleus. Isolated muscle mRNA was reverse-transcribed, and cDNA samples were analyzed by real-time qPCR with primers/probe specific for minidystrophin cDNA to assess levels of vector mRNA and also with primers/probe specific for GAPDH. (B) Vector mRNA expression was normalized to GAPDH. Data are shown as mean ± SEM; *significant difference from AAV + saline–treated mdx mice (P < 0.05); n = number of animals analyzed.

Figure 7

Muscle damage analysis. Serum from untreated C57BL/10, untreated mdx, AAV + NBD peptide–treated and AAV + saline–treated mdx mice was analyzed for the muscle protein α-sarcomeric actin, a marker of muscle degeneration. Serum levels (μg/mL) of α-sarcomeric actin were quantified using a standard curve of purified α-actin protein. (A) Representative bands from each of the treatment groups are shown. (B) Quantitative densitometry of samples from each control and treatment group is shown as mean ± SEM. *Significant difference from both untreated mdx and AAV + saline–treated mdx mice (P < 0.05); n = number of animals analyzed.