Figure 1.

Biopotency of the AAVrh74.tMCK.CAPN3 vector, short-term efficacy, and cardiac safety 4 weeks after gene delivery

(A) Schematic of the AAVrh74.tMCK.CAPN3 vector showing 5′ and 3′ AAV2 inverted terminal repeats (ITRs), the tMCK promoter, the chimeric intron (In), the human CAPN3 transgene, and the SV40 polyadenylation signal (poly(A)). (B) Representative western blot images of CAPN3 protein in muscle samples following intramuscular (IM) and systemic gene delivery in CAPN3 KO mice with normal human muscle lysate (gel load of 60% total protein compared with mouse lysates from tibialis anterior [TA], quadriceps, and gastrocnemius), and UT CAPN3 KO mice for comparison. 94-kDa, full-length human CAPN3 was detected in the TA 4 weeks after IM injection of the vector at 1 × 10¹¹ vg. Following systemic delivery, note the prominent increase of full-length CAPN3 protein from the 6 × 10¹² vg treatment (high dose [HD]) compared with the 3 × 10¹² vg (low dose [LD]) cohort. 12.5 μg and 2.5 μg (25% and 5%, respectively) total protein from the corresponding UT WT C57BL/6 muscles were used as reference points for semiquantitative analysis of the AAV-mediated CAPN3 levels in the corresponding treated CAPN3 KO mouse muscles. CAPN3 levels were detected using the CALP-12A2 antibody, which recognizes epitopes in muscle extracts from human and mouse. (C and D) CAPN3 protein levels in the quadriceps (C) and gastrocnemius (D) are quantified as percent of the WT, and the levels of all HD samples (n = 3) are given as mean ± SEM. (C) Representative images of succinic dehydrogenase (SDH)-stained tissue sections from TA muscle in wild-type (WT) and CAPN3 KO mice following IM delivery of AAV.hCAPN3 or Ringer’s lactate (untreated [UT]). (D) A fiber size increase toward normalization was seen with CAPN3 gene therapy. (E) Following systemic AAV.hCAPN3 gene therapy, LD- and HD-treated CAPN3 KO mice performed better on the treadmill test compared with UT counterparts. (F) The mean fiber diameter of TA muscle was increased toward WT values following systemic delivery in LD and HD cohorts. (G) Representative images from H&E-stained fresh-frozen sections through left ventricles inCAPN3 KO mice 4 weeks after systemic injection of the vector at LD and HD with UT control. Necrosis, regeneration, or inflammation was not seen. (H) Western blot analysis of cardiac tissue from the HD cohort showed no detectable CAPN3 protein. A full-length, 94-kDa hCAPN3 band is present in human skeletal muscle as a positive control. (I) Bar graph representing cardiac mRNA levels of individual mice corresponding to samples shown in western blot. Each bar represents the mean ± SEM of 3–4 mice. ∗∗p < 0.01, one-way ANOVA, Tukey’s multiple comparisons test. Scale bars 30 μm in the WT image (applies to all).