proteintech anti smn2  (Proteintech)

Journal: Nature biomedical engineering

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

doi: 10.1038/s41551-023-01132-z

Figure Lengend Snippet: 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 ABEmax33,38, ABE8.20m35, and ABE8e36 fused to wild-type SpCas9 (panel c) or SpRY37 (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 variants44, 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-SpRY37 or ABE8e-iSpyMac45 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.

Article Snippet: Blots were incubated with anti-GFP (1:2,000; A11122 ThermoFisher Scientific) or Anti-SMN2 human (1:2000; MABE230 Sigma).

Techniques: Sequencing

Journal: Nature biomedical engineering

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

doi: 10.1038/s41551-023-01132-z

Figure Lengend Snippet: 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).

Article Snippet: Blots were incubated with anti-GFP (1:2,000; A11122 ThermoFisher Scientific) or Anti-SMN2 human (1:2000; MABE230 Sigma).

Techniques: Transfection, Sequencing, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Nature biomedical engineering

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

doi: 10.1038/s41551-023-01132-z

Figure Lengend Snippet: 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.

Article Snippet: Blots were incubated with anti-GFP (1:2,000; A11122 ThermoFisher Scientific) or Anti-SMN2 human (1:2000; MABE230 Sigma).

Techniques: Expressing, In Vivo, Sequencing, Injection