The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy

Abstract

Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.

Keywords: AAV; CRISPR; Duchenne muscular dystrophy; gRNA.

Figure 1.

Quantification of viral genome copy number confirmed preferential depletion of the gRNA AAV vector. (A) Quantification of the AAV genome copy number. The ration in X-axis refers to the gRNA to Cas9 ratio at the time of injection. (B) Quantification of the gRNA:Cas9 vector ratio in tissues harvested from AAV-injected mice. Asterisk, p < 0.05. AAV, adeno-associated virus; gRNA, guide RNA; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein 9.

Figure 2.

Evaluation of dystrophin restoration at 3 months post-treatment. (A) Quantitative western blots revealed a significantly higher dystrophin level in skeletal muscle in mice that received the gRNA and Cas9 vectors at the 3:1 ratio. Note, the loading in the WT lane is one-tenth of other lanes. (B) Quantitative western blots revealed similar dystrophin levels in the heart for both the 1:1 and 3:1 groups. (C) Evaluation of dystrophin expression by immunofluorescence staining in skeletal muscle. Left panels, representative dystrophin (red) and laminin (green) immunostaining photomicrographs; right panel, percentage of dystrophin-positive myofibers. (D) Evaluation of dystrophin expression by immunofluorescence staining in the heart. Left panels, representative dystrophin (red) and laminin (green) immunostaining photomicrographs; right panel, percentage of dystrophin-positive myofibers. (E) ddPCR quantification of the total dystrophin transcripts in skeletal muscle of WT, mdx, and CRISPR-treated mdx mice. For CRISPR-treated mdx mice (1:1 group and 3:1 group), we also determined the percentage of edited and unedited dystrophin transcripts. (F) ddPCR quantification of the total dystrophin transcripts in the heart of WT, mdx, and CRISPR-treated mdx mice. For CRISPR-treated mdx mice (1:1 group and 3:1 group), we also determined the percentage of edited and unedited dystrophin transcripts. (G) Cartoon illustration of the method used to evaluate DNA-level editing efficiency. Primers b and c detect the unedited genome, and primers a and c detect the edited genome. The editing efficiency is calculated by dividing the copy number of the edited genome with the copy number of the edited and unedited genome. (H) ddPCR quantification of DNA-level editing efficiency in skeletal muscle. (I) ddPCR quantification of DNA-level editing efficiency in the heart. Asterisk, p < 0.05. ddPCR, droplet digital PCR; WT, wild type.

Figure 3.

Western blot showed similar levels of Cas9 protein expression in the 1:1 and 3:1 groups. (A) Quantitative Cas9 western blots from skeletal muscle. (B) Quantitative Cas9 western blots from the heart.

Figure 4.

The 3:1 group showed better histological and physiological rescue in skeletal muscle. (A) Myofiber size distribution in the quadriceps muscle. (B) Minimal Feret diameter of the quadriceps muscle. (C) Percentage of centrally nucleated muscle fibers in the quadriceps muscle. (D) Specific tetanic force in EDL muscle. (E) Overall profiles of 10 cycles of eccentric contraction. (F) Scatter plot evaluation of individual cycle of eccentric contraction. Asterisk, p < 0.05. EDL, extensor digitorum longus.

Figure 5.

The 3:1 group showed better ECG improvement. ECG evaluation of the heart rate, PR interval, QRS duration, QTc interval, Q amplitude, and the cardiomyopathy index. Asterisk, p < 0.05. ECG, electrocardiography.