A multifunctional AAV-CRISPR-Cas9 and its host response

Abstract

CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

Figure 1

Postnatal genome-editing with AAV9-Cas9-gRNAs and transcriptional activation with AAV9-Cas9-VPR-gRNAs. (a) AAV9-Cas9-gRNAs targeting the endogenous Mstn gene or the 3×Stop cassette in neonatal mice. (b) Mutation frequency correlates with AAV9 transduction efficiency (Pearson’s R = 0.73, Spearman’s ρ = 0.74, P < 0.05) (n = 4 mice, 4E12 vg of AAV9-Cas9-gRNAs^M3+M4). Horizontal dashed line, sequencing error rate; vertical dashed line, qPCR false positive rate. Error bars denote s.e.m. for sequencing and qPCR replicates. (c) Fluorescent images of AAV9-Cas9-gRNAs^TdL+TdR-edited tdTomato+ cells in the indicated organs (2 upper rows, n = 3 total mice at 4E12 vg; 2 lower rows, n = 4 total mice at 4E12 vg). Gray, tdTomato. Scale bar, 5 mm. (d) AAV-Cas9-VPR-gRNAs-mediated gene activation of the indicated genes in myotubes (black) and spermatogonial cells (red). Closed dots denote single-gRNA and open circles denote dual-gRNAs (AAV-Cas9^N-gRNA:AAV-Cas9^C-VPR, 1:1). Error bars denote s.e.m. (e) AAV9-Cas9-VPR-gRNAs-mediated gene activation in adult mice (FDR = 0.05). Volcano plot shows total mRNA-sequencing of the same muscle samples used for qRT-PCR in Supplementary Figure 7e (n = 3 mice per condition).

Figure 2

AAV9 and Cas9 evoke host immune responses. (a) Intramuscular Cas9-expression via AAV9-split-Cas9 injection or plasmid-Cas9^FL electroporation. (b) Heat maps depict fold-difference of each immune cell-type fraction compared to that of vehicle-injected muscles (right column) (n = 4 mice per condition). (c) Lymphocyte TCR-β CDR3 repertoires after Cas9-exposure (n = 4 mice per condition; 6 pair-wise comparisons) (Welch’s t-test, Bonferroni corrected). (d) Clonotypic abundance of Vβ16 CDR3 CASSLDRGQDTQYF (Welch’s t-test). Numbers in parentheses denote clonotypic rank within each TCR-β CDR3 repertoire after Cas9 re-stimulation. (e) Epitope mapping by M13 phage display (all Ig subclasses). (f) Cas9 epitopes from Cas9-exposed animals (top, n = 4 DNA-electroporated; bottom, n = 4 AAV9-delivered). P-values from Wald test, Benjamini-Hochberg adjusted for FDR = 0.1. (g) Capsid epitopes from AAV9-exposed animals (n = 8). Counts denote number of animals with capsid-specific antibodies covering each amino acid position (x-axis), and red bars denote positions of immunodominant epitopes. AAV9 capsid expresses as three isoforms (VP1/2/3).

Figure 3

Immunological phenotypes towards AAV-CRISPR-Cas9. (a) Deconvoluted hematopoietic lineage tree from muscle samples. Node sizes scale with fold-differences in gene signatures induced by AAV9-Cas9-VPR-gRNAs^{set 2} treatment (n = 3 mice per condition). Detailed annotations are available in Supplementary Figure 11. (b) Cellular compositions determined by deconvolution reflect fractions quantified by FACS. Two groups of AAV9-Cas9-VPR-gRNAs-injected mice were compared to AAV9-turboRFP-injected negative control mice (n = 3 mice per condition). Error bars denote within-group s.e.m. (c) Tissue sections immunostained for the indicated proteins in muscles injected with AAV9-Cas9-VPR-gRNAs (4E12 vg) (n = 3 mice per condition). All injections included 1E11 vg of AAV9-turboRFP to demarcate transduction. Additional data are presented in Supplementary Figure 12. (d) Quantification of the fraction of centrally nucleated myofibers (n = 3 mice per condition) (one-way ANOVA, followed by Dunnett’s test against mice injected with 1E11 vg of AAV9-turboRFP).