CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle

Abstract

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.

Progress and prospects: gene therapy for genetic diseases with helper-dependent adenoviral vectors

“…For example, intravenous injection of HDAd expressing the canine coagulation factor IX in hemophilia B dogs resulted in sustained phenotypic improvement of the bleeding diathesis for the duration of the experiment of over 1 year after vector administration. 3 These, as well as other recent studies, [4][5][6][7][8][9][10][11][12][13] provide compelling evidence for using HDAd to treat genetic disorders.…”

“…A similar outcome may occur in humans as well, as many DMD patients have large dystrophin gene deletions. To overcome this problem, several strategies have been successfully utilized including co-delivery of immunomodulatory molecules such as CTL4Aig, 10,50 and use of an alternative transgene such as utrophin, a functional homolog of dystrophin. 51 HDAds have also been explored for in utero gene therapy of DMD.…”

Comparison of high-capacity and first-generation adenoviral vector gene delivery to murine muscle in utero

“…Helper virus level was less than 1% of the vector preparation. 13 The structure of the HC-Ad vectors was confirmed by restriction digest of vector DNA isolated from purified virions. There was no detectable recombination.…”

“…An HC-Ad vector carrying the full-length murine dystrophin cDNA driven by the muscle creatine kinase (MCK) promoter (AdmDys) 13 was delivered intramuscularly to one hind limb of E-16 fetal mdx mice. At 9 weeks after vector delivery, muscles from both hind limbs were collected for dystrophin, a-sarcoglycan and b-sarcoglycan immunostaining.…”

Full-length dystrophin gene transfer to the mdx mouse in utero

“…12 The HC-Ad vector can carry a large expression cassette (up to approximately 30 kb of insert capacity) and lacks all viral genes. Therefore, the vector can deliver a full-length dystrophin cDNA and confers lower induction of immunity compared to first-generation Ad vectors.…”