Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.

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. f, A-to-G editing in exon 7 of SMN2 when using ABE8e-SpG and gRNAs A9 or A10. g, A-to-G editing in exon 7 of SMN2 when using ABE8e-SpRY or ABE8e-iSpyMac with gRNAs A7 and A8, or wild-type ABE8e-SpCas9 with gRNA A10. Data in panels c-g from experiments in HEK 293T cells; mean, s.e.m., and individual datapoints shown for n = 3 or 4 independent biological replicates.

Figure 2.. SMN2 C6T editing 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, with data presented as normalized fold-change from naïve cells. 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 expression plasmids (left panel) and AAV ITR-containing intein-split plasmids (right panel) 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 (cohort 1). 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 different tissues (without sorting for transduced cells) assessed by targeted sequencing. n = 8 treated and n = 6 untreated (sham injection) SMNΔ7 mice; mean, s.e.m., and individual datapoints shown. f, SMN2 exon 7 mRNA expression in select tissues from mice in cohort 1. Exon 7 transcript levels were measured by ddPCR and normalized by SMN2 exon 1/2 expression. Data presented as fold change from untreated mice. n = 8 treated and n = 6 untreated SMNΔ7 mice; mean, s.e.m., and individual datapoints shown. SC, spinal cord. g, Schematic of P1 ICV injections with longer-term 12-week follow-up in Smn+/Smn+/SMN2 and Smn+/Smn−/SMN2 mice for cohort 2, using the same injection scheme as cohort 1. h, A-to-G editing of SMN2 C6T in tissues of mice from cohort 2. i, Schematic of cohort 3 featuring combined P1 ICV and retroorbital (IV) injections in SMNΔ7 mice. n = 9 treated and n = 3 untreated SMNΔ7 mice; mean, s.e.m., and individual datapoints shown. B, brain; SC, spinal cord; L, liver; H, heart, SM, skeletal muscle. j, A-to-G editing of SMN2 C6T in tissues of mice from cohort 3. n = 8 treated and n = 3 untreated SMNΔ7 mice; mean, s.e.m and individual datapoints. B, brain; SC, spinal cord; L, liver; H, heart, SM, skeletal muscle. k-m, Phenotypic characterization in SMA mice from cohort 3, including body mass (panel k), motor function (panel l) and survival rate (panel m) assessments. Lon-rank test revealed a significant difference between treated and untreated mice in the survival rate (P = 0.01). n = 8 treated and n = 3 untreated SMNΔ7 mice; mean and s.e.m. (panels k-i), and survival rate (panel m) shown.