In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing

Abstract

Objective: High-efficiency genome editing to disrupt therapeutic target genes, such as PCSK9 (proprotein convertase subtilisin/kexin type 9), has been demonstrated in preclinical animal models, but there are safety concerns because of the unpredictable nature of cellular repair of double-strand breaks, as well as off-target mutagenesis. Moreover, precise knock-in of specific nucleotide changes-whether to introduce or to correct gene mutations-has proven to be inefficient in nonproliferating cells in vivo. Base editors comprising CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated 9) fused to a cytosine deaminase domain can effect the alteration of cytosine bases to thymine bases in genomic DNA in a sequence-specific fashion, without the need for double-strand DNA breaks. The efficacy of base editing has not been established in vivo. The goal of this study was to assess whether in vivo base editing could be used to modify the mouse Pcsk9 gene in a sequence-specific fashion in the liver in adult mice.

Approach and results: We screened base editors for activity in cultured cells, including human-induced pluripotent stem cells. We then delivered a base editor into the livers of adult mice to assess whether it could introduce site-specific nonsense mutations into the Pcsk9 gene. In adult mice, this resulted in substantially reduced plasma PCSK9 protein levels (>50%), as well as reduced plasma cholesterol levels (≈30%). There was no evidence of off-target mutagenesis, either cytosine-to-thymine edits or indels.

Conclusions: These results demonstrate the ability to precisely introduce therapeutically relevant nucleotide variants into the genome in somatic tissues in adult mammals, as well as highlighting a potentially safer alternative to therapeutic genome editing.

Keywords: PCSK9; gene therapy; lipids and lipoprotein metabolism; molecular biology; nucleotides.

Figure 1

Schematic of the mechanism of action of the BE3 base editor. The N-terminal end of Cas9 is attached to the cytosine deaminase domain from the RNA-editing enzyme APOBEC1. By virtue of being tethered in the vicinity of DNA by CRISPR-Cas9, the cytosine deaminase domain can convert cytosine bases in the DNA molecule into uracil bases. Specifically, BE3 can edit any cytosine base that lies within a “window” 13 to 17 nucleotides upstream of the NGG PAM sequence and is contained in the same strand as the PAM sequence (but not the opposite strand) into uracil. The C-terminal end of the Cas9 portion of BE3 is attached to a domain that inhibits the activity of uracil-DNA glycosylase. By being tethered in the vicinity of the edited cytosine bases, this inhibitor prevents uracil repair. In parallel with the cytosine deaminase activity, the Cas9 portion of BE3 nicks the opposite strand of DNA. The process of nick repair entails the removal of some bases on the DNA strand around the nick, followed by filling in of the gap using the bases on the edited strand as the complementary template. Because the edited strand has uracil bases, the opposite strand will be filled in with adenine bases rather than the guanine bases that were originally in the strand. Thus, C-G basepairs are converted to U-A basepairs. After BE3 releases from the DNA, each uracil base can be removed by uracil-DNA glycosylase, followed by filling in with a thymine base to match the complementary adenine base now on the opposite strand, rendering the base editing permanent.

Figure 2

Base editing of human PCSK9 in vitro. (A) Codons encoding glutamine, arginine, or tryptophan residues that could potentially be changed to stop codons with C-to-T or G-to-A edits. (B) On-target base editing was assessed by Sanger sequencing for various codons in human PCSK9 in HEK 293T cells using BE3, BE3-VQR, or BE3-VRER. The protospacer and PAM sequences for each target site are listed, with the bold letters corresponding to the editing “window” (13 to 17 bases upstream of the PAM) and the underline indicating the target codon (either sense or antisense), in which a C-to-T change would result in a stop codon on the sense strand. “–” indicates no base editing detected, “+” indicates modest base editing, and “++” indicates substantial base editing. (C) Top left panel: CEL-I nuclease assays were performed with genomic DNA from HEK 293T cells transfected with BE3 and a guide RNA targeting the codon encoding Q302. Arrows show the cleavage products resulting from the CEL-I nuclease assays; the intensity of the cleavage product bands relative to the uncleaved product band corresponds to the mutagenesis rate. MW = molecular weight. Top right panel and bottom panels: Sanger sequencing confirmed base editing (C-to-T) of Q302 (codon CAG) in both HEK 293T cells and induced pluripotent stem cells.

Figure 3

Base editing of mouse Pcsk9 in vitro. (A) On-target base editing was assessed by Sanger sequencing for various codons in mouse Pcsk9 in Neuro-2a (N2a) cells using BE3, BE3-VQR, or BE3-VRER. The protospacer and PAM sequences for each target site are listed, with the bold letters corresponding to the editing “window” (13 to 17 bases upstream of the PAM) and the underline indicating the target codon (either sense of antisense), in which a C-to-T change would result in a stop codon on the coding strand. “–” indicates no base editing detected, and “+” indicates modest base editing. (B) Top panels: CEL-I nuclease assays were performed with genomic DNA from N2a cells transfected with BE3 and a guide RNA targeting various codons. Bottom panels: Sanger sequencing confirmed base editing (G-to-A and C-to-T) of two target codons (codon TGG encoding W159 and codon CAG encoding Q347) in N2a cells.

Figure 4

Base editing of mouse Pcsk9 in vivo. (A, B) CEL-I nuclease assays performed with genomic DNA from organ samples taken from mice 5 days after receiving the BE3-Pcsk9 adenoviral vector, the control vector, or no vector. Arrows show the cleavage products resulting from the CEL-I nuclease assays. (C–F) Data from the mice in Figure 4B (n = 5 mice in each group). The fraction of base-edited alleles was determined by deep sequencing of the target site (W159 codon). The Mann–Whitney U test was performed to compare plasma analyte levels in the two groups. The bars indicate median values within each group. (G) Results of Western blot analysis of liver samples taken from the mice in Figure 4B. LDLR indicates low-density lipoprotein receptor. (H) Hematoxylin/eosin staining of liver sections from representative mice, 10× magnification. (I) Sanger sequencing of the on-target site in liver genomic DNA from the mouse with the greatest degree of base editing (the fourth BE3-Pcsk9 mouse in Figure 4B). (J) Consequences and frequencies of base-edited alleles and indel-bearing alleles in liver genomic DNA from the fourth BE3-Pcsk9 mouse in Figure 4B, determined by deep sequencing. The underlines indicate the target codon. (K) Base-editing and indel rates at on-target and off-target sites determined by deep sequencing of liver samples from 3 individual BE3-Pcsk9 mice (the values are separated by slashes) and 1 control mouse. The underlines indicate the base-editing windows within the protospacer sequences. In some cases, no base-editing rates are shown because the base-editing window contains no C bases.

Figure 5

Effects of base editing on plasma PCSK9 and cholesterol levels over time. (A) CEL-I nuclease assays performed with genomic DNA from liver samples from mice taken 4 weeks after receiving the BE3-Pcsk9 adenoviral vector. Arrows show the cleavage products resulting from the CEL-I nuclease assays. (B, C) Plasma analytes measured immediately prior to treatment, 2 weeks after treatment, and 4 weeks after treatment. The data are from 6 male mice treated with the BE3-Pcsk9 adenoviral vector at 5 weeks of age. * indicates P < 0.05 when compared to the pre-treatment levels as calculated by the Wilcoxon signed-rank test.