Base editing as a genetic treatment for spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C•G-to-T•A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2, reverting the exon 7 mutation via an A•T-to-G•C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.

Keywords: CRISPR; SMA; adenine base editors (ABEs); genome editing; neuromuscular diseases.

Figure 1.. Development of adenine base editing to correct SMN2 exon 7 C6T.

a, Schematic of SMN1 and SMN2 in unaffected individuals and spinal muscular atrophy (SMA) patients. Mutations in SMN1 cause SMA due to a depletion of SMN protein, which may be recovered by editing SMN2. b, Schematic of the SMN2 exon 7 C-to-T (C6T) polymorphism compared to SMN1, with base editor gRNA target sites and their estimated edit windows. c-d, A-to-G editing of SMN2 C6T target adenine and other bystander bases when using ABEs comprised of adenine deaminase domains ABEmax^,, ABE8.20m, and ABE8e fused to wild-type SpCas9 (panel c) or SpRY (panel d), assessed by targeted sequencing. e, A-to-G editing of adenines in SMN2 exon 7 when using SpRY or other relaxed SpCas9 PAM variants, assessed by targeted sequencing. Data in panels c-e from experiments in HEK 293T cells; mean, s.e.m., and individual datapoints shown for n = 3 independent biological replicates.

Figure 2.. SMN2 C6T editing and phenotypes in SMA patient-derived fibroblasts.

a, Characteristics of five different SMA donors; all lines harbor a homozygous deletion of exon 7 in SMN1. b, A-to-G editing of the C6T adenine in SMN2 exon 7 across five SMA fibroblast cell lines transfected with ABE8e-SpRY and gRNA A8, assessed by targeted sequencing. Naïve (N) cells were untransfected; Control (C) cells were treated with ABE8e-SpRY and a non-targeting gRNA. c, SMN2 exon 7 mRNA expression across three edited (E) SMA fibroblast lines, measured by ddPCR. Transcript levels normalized by GAPDH mRNA. d, SMN protein levels determined by an SMN-specific enzyme-linked immunosorbent assay (ELISA). e, Representative immunoblot for SMN, PTEN, and GAPDH protein levels across Naïve, Control, or ABE8e-SpRY treated SMA fibroblast lines. f,g, Quantification of SMN and PTEN (panels f and g, respectively) protein levels normalized to GAPDH and the Naïve treatment, determined by immunoblotting. For all assays, GFP-positive fibroblasts were sorted post-transfection and grown in for at least 3 passages; samples from three independent passages were collected for lines 1, 2 and 3 (passages 4–6; see Sup. Fig. 6a), and one passage was collected for lines 4 and 5. For panels b-d, f, and g, mean, s.e.m., and individual datapoints shown for n = 3 independent biological replicates from separate passages (unless otherwise indicated).

Figure 3.. Analysis of SMN2 C6T base editing specificity.

a, Number of putative off-target sites in the human genome with up to 2 mismatches for each gRNA, annotated by CasOFFinder. Predictions for SpCas9 utilized an NGG, NAG, or NGA PAM; with SpRY, a PAMless NNN search. b, Total number of CHANGE-seq detected off-target sites, irrespective of assay sequencing depth. c, Number of CHANGE-seq identified off-target sites that account for greater than 1% of total CHANGE-seq reads and are common across all 5 SMA fibroblast lines. d, Percentage of CHANGE-seq reads detected at the on-target site relative to the total number of reads in each experiment. For panels b, c, and d, mean, s.e.m, and individual datapoints shown for n = 5 independent biological replicate CHANGE-seq experiments (performed using genomic DNA from each of the 5 SMA fibroblast lines). e,f, Summary of targeted sequencing results from ABE-edited SMA fibroblasts or HEK 293T cells at the top 34 CHANGE-seq nominated off-target sites (common sites across all 5 SMA fibroblast lines and treatments with SpRY or SpRY-HF1), analyzing statistically significant editing of any adenine in the target site (panel e) or of all adenines in positions 1–12 of each of the 34 target sites (panel f).

Figure 4.. AAV-mediated delivery of base editors for in vivo SMN2 C6T editing.

a, Schematics of conventional and intein-split plasmids for ABE and gRNA delivery in cells and in vivo. gRNA, guide RNA; NpuN/NpuC, N- and C-terminal intein domains; Cas9(N) and Cas9(C), N- and C-terminal fragments of SpCas9 variants. b,c, A-to-G editing of SMN2 C6T target adenine and other bystander adenines when using ABE8e-SpCas9 with gRNA A10 (panel b) or ABE8e-SpRY with gRNA A8 (panel c), assessed by targeted sequencing. Data in panels b and c from experiments in HEK 293T cells; mean, s.e.m., and individual datapoints shown for n = 3 independent biological replicates. d, Schematic of P1 intracerebroventricular (ICV) injections in SMNΔ7 mice with dual AAV9 vectors that express intein-split ABE8e-SpRY and gRNA-A8. e, A-to-G editing of SMN2 exon 7 adenines following ICV injections of AAV encoding ABE8e-SpRY with gRNA A8 (panel d). Editing across various tissues (without sorting for transduced cells) assessed by targeted sequencing of n = 6 treated and n = 8 untreated (sham injection) SMNΔ7 mice; mean, s.e.m., and individual datapoints shown.