rAAVrh74.MCK.GALGT2 Demonstrates Safety and Widespread Muscle Glycosylation after Intravenous Delivery in C57BL/6J Mice

Abstract

rAAVrh74.MCK.GALGT2 is a surrogate gene therapy that inhibits muscular dystrophy in multiple animal models. Here, we report on a dose-response study of functional muscle GALGT2 expression as well as toxicity and biodistribution studies after systemic intravenous (i.v.) delivery of rAAVrh74.MCK.GALGT2. A dose of 4.3 × 1014vg/kg (measured with linear DNA standard) resulted in GALGT2-induced glycosylation in the majority of skeletal myofibers throughout the body and in almost all cardiomyocytes, while several lower doses also showed significant muscle glycosylation. No adverse clinical signs or treatment-dependent changes in tissue or organ pathology were noted at 1 or 3 months post-treatment. Blood cell and serum enzyme chemistry measures in treated mice were all within the normal range except for alkaline phosphatase (ALP) activity, which was elevated in serum but not in tissues. Some anti-rAAVrh74 capsid T cell responses were noted at 4 weeks post-treatment, but all such responses were not present at 12 weeks. Using intramuscular delivery, GALGT2-induced muscle glycosylation was increased in Cmah-deficient mice, which have a humanized sialoglycome, relative to wild-type mice, suggesting that use of mice may underestimate GALGT2 activity in human muscle. These data demonstrate safety and high transduction of muscles throughout the body plan with i.v. delivery of rAAVrh74.MCK.GALGT2.

Short-term treatment of golden retriever muscular dystrophy (GRMD) dogs with rAAVrh74.MHCK7.GALGT2 induces muscle glycosylation and utrophin expression but has no significant effect on muscle strength

“…Non-muscle organs assessed included the brain (cerebellum and frontal cortex), spinal cord, lung, pancreas, liver, spleen, adrenal gland, kidney, and gonads (ovary or testicle) ( Fig 1C ). As expected, based on previous work in mice and rhesus macaques [ 68 , 69 ], the greatest transduction for rAAVrh74 was in the liver and spleen (12 and 8 vg/nucleus, respectively), while the lowest level of transduction was in the brain (both cerebellum and frontal cortex <0.1vg/nucleus). Unexpectedly, pancreas also showed a very low level of transduction (also <0.1vg/nucleus), a finding not seen previously in mice or in macaques [ 68 , 69 ].…”

“…As expected, based on previous work in mice and rhesus macaques [ 68 , 69 ], the greatest transduction for rAAVrh74 was in the liver and spleen (12 and 8 vg/nucleus, respectively), while the lowest level of transduction was in the brain (both cerebellum and frontal cortex <0.1vg/nucleus). Unexpectedly, pancreas also showed a very low level of transduction (also <0.1vg/nucleus), a finding not seen previously in mice or in macaques [ 68 , 69 ]. Transduction of the spinal cord was significantly higher than brain, again as before [ 68 , 69 ].…”

“…Because this study entailed a 3-month treatment period and single-stranded AAV. GALGT2 vectors typically take 3–4 weeks after muscle transduction to achieve full gene expression [ 36 ], we chose to use the stronger MHCK7 [ 67 , 68 ] promoter instead of the MCK (CK7-like [ 69 ]) promoter, which is currently being tested in a phase 1/2a DMD clinical trial using rAAVrh74.MCK. GALGT2 (NCT03333590).…”

High Levels of Frataxin Overexpression Lead to Mitochondrial and Cardiac Toxicity in Mouse Models

“…In addition, previous mouse studies have shown that similar doses of vector, but with different DNA cargo, were well tolerated up to 5 × 10 14 vg/kg, without noticeable cardiotoxicity at long term. 47 , 48 , 49 , 50 , 51 In the current study, we did not investigate the potential involvement of the innate or adaptive immune response against the vector, transgene, and/or transduced cells. 35 , 36 , 52 , 53 It is likely that the treated mice developed a humoral response against the capsid, including neutralizing antibodies, as shown previously in many mouse gene therapy studies.…”

“… 35 , 36 , 52 , 53 It is likely that the treated mice developed a humoral response against the capsid, including neutralizing antibodies, as shown previously in many mouse gene therapy studies. 50 , 51 However, the emergence of antibody against the capsid has not been shown to preclude the long-term expression of transgene or to drive cardiac inflammation but only the re-administration of the same vector. 54 Here, we did not explore the potential presence of antibodies against the transgene and, more importantly, the development of a potential cytotoxic cell response targeting the transduced cardiomyocytes.…”