Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Abstract

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

Figure 1

In vivo delivery of Cas9 mRNA mediates efficient genome editing in cells. (a) C12-200 lipid nanoparticle delivery of Cas9 mRNA into cells. 293T cells stably expressing both EF1a promoter-GFP and U6 promoter-GFP targeting sgRNA (sgGFP) were ncubated with Cas9 mRNA nanoparticles (nano.Cas9). Cas9-mediated frameshift NHEJ events will result in GFP-negative cells. Red arrowhead indicates the Cas9 cutting site. (b) FACS analysis shows that Cas9 mRNA generates GFP-negative cells. Gate R2 indicates 80% GFP-negative cells after nano.Cas9 treatment (n = 3). (c) GFP locus was deep sequenced in nano.Cas9 treated cells (n = 4). Shown are representative indels. (d) Distribution of indels. (e) Indel phase shows that most indels cause a frameshift. For example, 3N + 1 include 1-, 4- and 7-bp indels, 3N + 2 include 2-, 5- and 8-bp indels, and 3N include 3-, 6- and 9-bp indels. (f,g) Transient Cas9 expression by mRNA delivery can reduce off-target genome editing for a VEGFA sgRNA. 293T cells were co-transfected with Cas9 mRNA and pLKO. sgVEGFA (mRNA). 293T cells infected with lentiviral Cas9 were transfected with pLKO.sgVEGFA alone to represent long-term Cas9 expression (lenti). On-target (TS2) (f) and off-target (OT2-2) (g) indel rate was measured by surveyor assay at 2 d. Arrows denote indel bands. *, nonspecific bands. (h) Relative off-target/on-target ratio. The ratio in lenti.Cas9 was set as 1. *P < 0.01 (n = 3). Error bars, mean ± s.d.

Figure 2

In vivo delivery of Cas9 mRNA and AAV-HDR template cures type I tyrosinemia mice. (a) Design of AAV-HDR template and experiments. G->A point mutation at the last nucleotide of exon 8 in Fah^mut/mut homozygous mice leads to exon skipping of exon 8. A dual function AAV vector harbors U6-sgRNA and a HDR template (1.7 kb) with the “G” nucleotide to repair the “A” mutation. The “TGG” PAM was modified to “TCC” to prevent self-cleavage. Dashed lines denote homologous recombination. ITR stands for inverted terminal repeat. Black arrows indicate PCR primers for deep sequencing analysis. Fah^mut/mut mice were injected with AAV-HDR and nano.Cas9 at indicated time points. Mice were kept off NTBC water at DO. Body weight normalized to pre-injection was monitored over time. (b) Delivery of AAV-HDR and nano.Cas9 fully rescues weight loss upon NTBC withdrawal (n = 3 mice). Error bars, mean ± s.e.m. (c) Liver damage markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin) were measured in serum *P < 0.01 (n = 3 mice) using one-way ANOVA. Error bars, mean ± s.e.m. (d) Fah⁺ cells after 30 d off NTBC. Scale bar, 100 μm.

Figure 3

In vivo delivery of Cas9 mRNA and AAV corrects Fah mutation. (a) Fah^mut/mut mice were kept on NTBC water and euthanized 7 d after nano.Cas9 treatment to estimate initial repair rate. (b) Fah immunohistochemistry (IHC). Scale bars are 200 μm for upper and lower panels, respectively. The lower panel of AAV-HDR + nano.Cas9 is a high-magnification view (box with black dashed line). (c) Fah⁺ positive cells were counted to determine the percentage. (d) Quantitative RT-PCR measurement of wild-type expression of Fah mRNA. (e) Sequence of repaired Fah mRNA in treated mice. QRT-PCR band of exon 8 to exon 9 spliced mRNA in (d) was sequenced. The corrected G nucleotide is circled. (f) Indels and (g) “G-CC” recombination pattern in total DNA from liver by Illumina sequencing. *P < 0.01 (n = 4 mice) using one-way ANOVA. Error bars, mean ± s.e.m.