In vivo prime editing of a metabolic liver disease in mice

Abstract

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2^ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2^ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah^enu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10¹⁴ vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Figure 1. Establishment of the size-reduced PE^ΔRnH variant.

(A) Schematic representation of the full-length PE2 and our size-reduced variant PE2^ΔRnH lacking the RNaseH (RnH) domain (A486-677) of the RT. (B) rSTOP reporter: conversion of a TAG stop codon results in GFP expression. (C) TLR reporter: correction of a 2 bp frameshift results in tagRFP expression. (D, E) Performance of PE2 and PE2^ΔRnH in rSTOP (D) and TLR (E) reporter cells using 2 different protospacers (labelled as A and B). Editing efficiency was scored by flow cytometry. (F) Comparative analyses of on-target editing efficiency and indel formation of PE2 and PE2^ΔRnH at seven genomic sites. pegRNA plasmids were transfected as negative controls. (G) In vitro analysis of IL-6, TNFα, and IFNα production upon expression of PE2 and PE2^ΔRnH in supernatants of RAW264.7 macrophages. A plasmid expressing GFP was used as an additional control (two-way ANOVA with Tukey’s multiple comparisons test; ****P<0.0001). Data from all experiments are represented as mean ± s.d. (three independent experiments; D, E, G) or range (two independent experiments; F). PE, prime editor; M-MLV, Moloney murine leukemia virus; RT, reverse transcriptase; NLS, nuclear localization signal; bGH, bovine growth hormone polyadenylation signal; EF-1α, eukaryotic translation elongation factor 1α; rSTOP, remove stop codon; ADRB1, β₁-adrenergic receptor; APP, amyloid β-precursor protein; EIF2B, eukaryotic translation initiation factor 2B; OTC, ornithine carbamoyltransferase; GABAR1α, gamma-aminobutyric acid receptor subunit α-1.

Figure 2. AAV8- and AdV-mediated prime editing at the Dnmt1 locus in the mouse liver.

(A) Schematic outline of the experimental setup with AAV8- or AdV-mediated prime editing in newborn and adult mice. Constructs used for in vivo prime editing at the Dnmt1 locus in the mouse liver are not depicted to scale. (B) Correction and indel rates in newborn and adult animals after AAV8-mediated delivery (injected dose per neonate and adult: 1 × 10¹² and 2× 10¹² vector genomes; vg). Untreated mice were used as negative controls. Percentage of sequencing reads with indels around the protospacer region were determined by deep sequencing. (C) Editing rates in neonates relative to AAV doses per animal (left panel) and per kg bodyweight (right panel). The respective values for adult mice were added (dark green) for comparison. (D) Correction and indel rates in newborn and adult mice at 4 weeks after AdV-mediated delivery (injected dose per neonate and adult: 3×10¹⁰ and 1×10¹¹ viral particles, vp). (E) Editing rates in neonates relative to AdV doses per animal (left panel) and per kg bodyweight (right panel). The respective values for adult mice were added (dark green) for comparison. Untreated mice were used as negative controls. Data are represented as mean ± s.d. (n=3-6 mice per group) and were analyzed using a two-way ANOVA with Tukey’s multiple comparisons test (ns, not significant, P>0.05; *P<0.05; **P<0.005).

Figure 3. In vitro correction of the Pah^enu2 allele using PE-SpCas and -SpRY variants.

(A, B). In vitro editing rates (A) and indel formation (B) of pegRNAs designed for SpCas-(light and dark purple) and SpRY-PEs (light and dark green) to target the disease-causing Pah^enu2 mutation (c.835T>C; p.F263S) on exon 7. pegRNAs for the SpCas-PE were also combined with an additional PE3 nicking sgRNA (pegRNA+sgRNA, dark purple). Two nicking sgRNAs were designed for two PE3b approaches using the SpRY variant (dark green). Experiments were performed in reporter HEK293T cells in which the mutated exon 7 of the Pah^enu2 gene was stably integrated. Data are represented as mean ± range of two independent experiments.

Figure 4. Correction of the Pah^enu2 allele in vivo in mice using prime editing.

(A) Schematic outline of the experimental setup for AAV8- and AdV-mediated treatment in newborn and adult PKU mice (vg, viral genomes; vp, viral particles). (B) In vivo correction and indel rates in newborn and adult animals after AAV8- or AdV-mediated delivery of SpCas-PEs (injected dose of AAV8 in neonates and adults: 1×10¹² and 2×10¹² vg; injected dose of AdV in neonates and adults: 3.0×10¹⁰ and 1.0×10¹¹ vp). Untreated mice were used as negative controls. Indels are calculated as percentage of sequencing reads with indels at the protospacer region. (C) Blood L-Phe concentrations after in vivo prime editing compared to untreated, heterozygous, and homozygous control animals. L-Phe concentrations below 600 μmol/L (U.S.) and 360 μmol/L (Europe) are considered therapeutically satisfactory (61, 62). (D) Enzymatic activity of PAH at experimental endpoints (4 weeks, AdV). (E) Blood L-Phe concentrations in newborn AdV-treated mice over time. Editing rates of the corresponding mice are color-coded and indicated at 9 and 18 weeks. (F) Deep amplicon sequencing of the top 5 experimentally determined off-target (OT) sites for the protospacer of the pegRNA mPKU-2.1 and of the top 5 computationally predicted off-target sites in untreated and AdV-treated mice (>20’000 reads per site). Data are represented as mean ± s.d. (n=3-8 mice per group) and were analyzed using a two-way ANOVA with Tukey’s multiple comparisons test (ns, not significant, P>0.05; *P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001).

Figure 5. Innate and adaptive immunity to AAV8 and AdV vectors.

(A) ALT concentrations in PBS-, AAV8- and AdV-treated mice at 6h p.i., 10d p.i., and experimental endpoints. (B) Cas9 transcript abundance in AAV8- and AdV-treated newborns (light green) and adults (dark green) at 6h p.i., 10d p.i., and endpoints. Transcript counts were normalized to the housekeeping gene Rplp0. (C-E) TNFα (C), IL-6 (D), and IFNα (E) concentrations in the serum of PBS-, AAV8- and AdV-treated adults and neonates at 6h, 10d p.i. and experimental endpoints. (F, G) Presence of neutrophils and monocyte-derived (md) macrophages (F) and pro-inflammatory chemokines ccl2, cxcl1, and cxcl10 (G) in the livers of adult mice at 6h p.i. and 10d p.i. (H) Cas9-specific adaptive immune response in control and treated mice 6h, 10d p.i. and endpoints. Data are represented as mean ± s.d (n=2-4 animals per group). Experimental endpoints for AdV, 4 weeks (newborns and adults); for AAV8, 4 weeks (newborns), 8 weeks (adults).