Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Abstract

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34⁺ cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

Keywords: adeno-associated virus; genome editing; hematopoietic stem cells; homologous recombination; in vivo genome editing.

Fig. 1.

Genome editing of a promoterless GFP into intron 1 of the human PPP1R12C gene. (A) Schema for genome editing assay. (Upper) Map of the PPP1R12C-GFP editing vector genome. In line 1, the insert cassette consists of a GFP ORF (green) preceded by SA/T2A (SA/2A) sequences and followed by a pA (PA) signal (cyan), and it is flanked on either side by 800-bp HAs (blue) complementary to chromosomal sequences in PPP1R12C. HAL, left HA; HAR, right HA. The entire editing construct is bounded by AAV2 ITRs (purple). In line 2, the PPP1R12C gene is depicted. Promoter (P) is shown in yellow, exons (Ex) are shown in dark green, and introns are shown in pink. Line 3 depicts a hypothetical HR schema. Line 4 depicts the edited PPP1R12C gene showing the insertion of the GFP cassette into intron 1. (B) Dose–response curves correlating genome editing with the MOI of the PPP1R12C-GFP editing vector. The editing vector genome was packaged in the capsids noted within each box. (Left) Editing of primary human CD34⁺ cells. (Right) Editing of the human hepatocellular carcinoma cell line HepG2. (C) Heat map showing genome editing efficiency of targeted insertion of GFP into PPP1R12C by AAV serotype (columns) in different human cell types (rows) as follows: (1) transformed cell lines, (2) primary human cells, and (3) immortalized LCLs carrying genetic mutations. Values represent specific GFP expression as assessed by flow cytometry. GFP expression was assessed only in viable cells, as determined by DAPI exclusion. Data shown are aggregates of >906 individual measurements. Cell type details are shown in SI Appendix, Fig. S1. (D) Comparison of median genome editing efficiency across AAV clade A (AAV6), clade B (AAV2), clade E (AAV8), and clade F (AAV9) (AAVHSCs). Editing efficiencies were measured by GFP expression by flow cytometry. Cells were transduced at a MOI of 1.5E5 and evaluated by flow cytometry at 48 h. Sample sizes for the experimental groups are as follows: untreated (Untd), n = 25; AAV6, n = 33; AAV2, n = 17; AAV8, n = 19; AAV9, n = 30; and AAVHSCs combined, n = 390. AAVHSC represents data compiled from AAVHSC1, AAVHSC4, AAVHSC5, AAVHSC7, AAVHSC9, AAVHSV12, AAVHSC13, AAVHSV15, AAVHSV16, and AAVHSC17. Outliers are represented by individual circles. Significance was determined by a paired two-tailed t test using AAVHSCs as the comparison reference. The vector genomes (VG) were quantitated in nuclei purified from AAV-treated CD34⁺ cells 48 h posttreatment. The number of VG per nucleus was determined by real-time PCR for GFP and the housekeeping gene hApoB. Values shown are averages of three replicates per transduction and three transductions with each AAV vector.

Fig. 2.

AAVHSCs mediate precise and efficient genome editing in primary human cells. (A) Flow cytometric analysis of GFP expression in cord blood CD34⁺ cells following editing of the promoterless GFP ORF into intron 1 of PPP1R12C. Cells were transduced at a MOI of 1.5E5 and assayed for GFP expression after 48 h. The fraction of specific GFP expression is noted in each flow profile. Untd, untreated. (B) Schema depicting the edited PPP1R12C locus showing targeted insertion of GFP, the expected 1.7-kb TI amplicon, and the location of primers for the TI assay. Gels show TI amplicons (red arrow) specific for GFP insertion into intron 1 of PPP1R12C in HepG2 and CD34⁺ cells at 24 h and 39 d posttransduction in vitro. FWD, forward; HAL, left HA; MW, molecular weight marker; P, promoter; REV, reverse. (C) Correlation of GFP expression in edited cells with molecular ddPCR-based quantitation of edited alleles. CD34⁺ cells were treated with AAVHSC17 PPP1R12C-GFP editing vector at a MOI of 1.5E5. Flow cytometry-based GFP expression and ddPCR-based allele quantitation were measured from the same samples after 48 h. (D) Enrichment of edited alleles in flow-sorted K562 cells. K562 cells were treated with the AAVHSC9 PPP1R12C-GFP editing vector at a MOI of 150,000. Cells were assessed for the frequency of edited alleles before and 3 wk after enrichment of GFP-expressing cells. (E) NGS analysis of the edited PPP1R12C genomic region from edited CD34⁺ cells. Shown is the NGS read depth (y axis) and the covered chromosomal region (x axis), including HAs and the GFP insert. The fidelity of editing and errors per allele is noted, showing seamless editing with no inserted viral sequences being detected. The locations of errors are denoted by red arrows under the map. Each arrow signifies a single error. (F) Targeted editing of the promoterless GFP cassette into two locations within the human IL2RG gene in primary human CD34⁺ cells. The map depicts the IL2RG gene. Insertion locations are at either the ATG or in intron 7 (HA; depicted in blue). Flow analyses of GFP expression following insertion of the GFP ORF at either the initiation codon or in intron 7 are shown. Expression is driven by the chromosomal IL2RG promoter. Percent-specific GFP is noted within each flow profile.

Fig. 3.

Replacement of a specific nucleotide to model correction of point mutations. (A) Schema of dinucleotide replacement editing assay. (Right) In line 1, the nucleotide (NT) replacement vector was designed to replace the wild-type genomic “TA” in intron 1 of PPP1R12C with “AT”, replacing the wild-type NheI site with a SphI site. The nucleotide replacement vector was also designed to insert a 12-bp linker 245 bp downstream (depicted in orange). (Left) In line 1, control vector was designed to only insert the 12-bp linker, without replacing the TA dinucleotide. HAL, left HA; HAR, right HA. Line 2 shows the wild-type chromosome, highlighting the region to be edited. Line 3 shows the expected configuration of the edited chromosome. Green boxes represent exons, and introns are shown as pink lines. FWD, forward; REV, reverse. (B) Sequence alignment showing dinucleotide editing in intron 1 of PPP1R12C in HepG2 cells. Both 500-bp and 80-bp windows are shown. Alignment across sequenced regions is displayed as a stack plot by position (green). Precise dinucleotide editing is displayed as a cumulative position-weighted matrix (PWM) of each edited sequence aligned to a reference trace.

Fig. 4.

Role of DNA repair genes in AAVHSC- and AAV9-mediated genome editing. (A) Heat map of genome editing efficiencies in LCLs harboring mutations in DNA repair genes. LCLs were treated with AAVHSC PPP1R12C-GFP vectors at a MOI of 1.5E5. Cells were analyzed by flow cytometry after 48 h. Data represent percent GFP expression in live cells following editing of the GFP ORF into intron 1 of PPP1R12C (Fig. 1A). Row 1 depicts primary human CD34⁺ cells for comparison. Subsequent rows depict immortalized cell lines bearing DNA repair mutations as follows: row 2, BLM; row 3, ERCC4/XPF; row 4, NBS1; row 5, RAG1; row 6, ATM; row 7, FANCF; row 8, FANCB; row 9, FANCC; row 10, FANCA; row 11, FANCD2; row 12, BRCA2^+/−; and row 13, BRCA2^−/−. Columns depict different AAV serotypes. (B) Stack plot of average cumulative editing efficiencies in LCLs harboring loss-of-function mutations in selected DNA damage response genes. Genes in the x axis are as follows: 1, GM04408 (BLM); 2, GM08437 (ERCC4); 3, GM15818 (NBS1); 4, ID00078 (RAG1); 5, GM03332 (ATM); 6, GM13023 (BRCA2^−/−); 7, GM13071 (FANCB); 8, GM14622 (BRCA2^+/−); 9, GM12794 (FANCC); 10, GM16749 (FANCA); 11, GM16756 (FANCD2); and 12, GM16757 (FANCF). Each AAV serotype is denoted by a specific color. (C) AAVHSCs successfully mediate gene transfer into BRCA2^−/− fibroblasts but do not edit. Flow cytometric analysis of the EUFA423 BRCA2^−/− or BRCA2^+/+ fibroblast line transduced with AAVHSC17 PPP1R12C-GFP editing vector or a CBA-GFP gene transfer vector is shown, where GFP expression is driven by the CBA promoter. Robust GFP expression is observed after gene transfer, but no GFP is observed following treatment with the editing vector in BRCA2^−/− fibroblasts. The absence of editing in BRCA2^−/− cells is denoted by lack of GFP expression. Untd, untreated.

Fig. 5.

AAVHSCs mediate efficient in vivo genome editing after i.v. injection. (A) Schema showing the vector and genome maps for the Rosa26-Luc editing vector that inserts the promoterless luciferase ORF into intron 1 of the murine Rosa26 locus. The luciferase ORF is preceded by SA/T2A (SA/2A) and followed by pA (PA). The luciferase cassette is flanked by 800-bp HAs targeting intron 1 of Rosa26. HAL, left HA; HAR, right HA. Also depicted is the map of the edited Rosa26 locus. (B) Serial in vivo imaging of luciferase expression in mice injected with AAVHSC15 Rosa26-Luc vector (5e11 vg per mouse). Luciferase expression is under the control of the chromosomal Rosa26 promoter. Also shown are AAVHSC15 noHA, a negative control containing the luciferase cassette but noHA, and an AAV8 Rosa26-Luc editing vector. Days postinjection (D) are depicted below each image. The ventral (Upper) and dorsal (Lower) images of each mouse are shown. Sample sizes for the experimental groups are as follows: AAVHSC15 Rosa26-Luc group, n = 5; AAVHSC15 noHA group, n = 3; and AAV8 Rosa26-Luc group, n = 3. (C) Sanger sequence analysis of the junction sequences between the HA and genomic DNA (gDNA) confirmed precise editing of the luciferase ORF into the Rosa26 locus. (D) Southern blot analysis of the edited murine Rosa26 locus. Genomic DNA from the liver was digested with SpeI, gel-electrophoresed, transferred to a nylon membrane, and hybridized with a luciferase-specific probe. The expected 7-kb band is observed with the luciferase probe in the edited liver DNA (lane 5), but not from untreated liver DNA (lane 4). Also shown is a titration of the vector plasmid as a positive control for the probe (lanes 1–3). MW, molecular weight marker. Maps show the location of the 7-kb band in the edited Rosa26 locus and the 3,049-bp band in the Rosa26-Luc vector plasmid (pRosa26-Luc) cut with DrdI and SpeI. Potential sizes are also depicted on a map of the vector genome. Red double-headed arrows depict the luciferase-binding site.