Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Abstract

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

“…1 AAV1, 8 AAV6, 9 and AAV8 capsids 10 are suitable for gene transfer to skeletal muscle, whereas AAV1, 11 AAV5, 12 and AAV9 13 are efficient for targeting various regions of *Correspondence: Dr. Jacob Giehm Mikkelsen, Department of Biomedicine, Aarhus University, Wilh. Meyers Alle´4, DK-8000 Aarhus C, Denmark.…”

Long-Term Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Golden Retriever Muscular Dystrophy

“…Restoration of dystrophin expression has been accomplished by gene transfer of mini-and microdystrophin constructs in dystrophic mice (Harper et al, 2002;Gregorevic et al, 2004;Liu et al, 2005), dogs (Wang et al, 2007;Kornegay et al, 2010), and humans (Miyagoe-Suzuki and Takeda, 2010), as well as by induction of exon skipping in dystrophic mice (Goyenvalle et al, 2004;Denti et al, 2006Denti et al, , 2008, dogs (Yokota et al, 2009), and humans (Miyagoe-Suzuki and Takeda, 2010;Moulton and Moulton, 2010;Partridge, 2010). Proof-of-principle studies demonstrating successful gene transfer of microdystrophin (Rodino-Klapac et al, 2010) and successful induction of exon skipping (Moulton and Moulton, 2010) have also been performed in healthy nonhuman primates (NHPs).…”

Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice

“…19 Muscles were subjected to physiological analysis using a protocol described previously by our lab 20,21 with some adaptations. Two linear strips of muscle, approximately 2-3 mm in width, were carefully dissected from each diaphragm.…”