Osman EY , Bolding MR , Villalón E , Kaifer KA , Lorson ZC , Tisdale S , Hao Y , Conant GC , Pires JC , Pellizzoni L , Lorson CL .

NS102451 U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS), R01 NS102451 NINDS NIH HHS

	Figure 1. SMN conservation, functional domains, and homology across species. (a) The 50% majority-rule consensus tree was generated from Bayesian analysis of 21 protein sequences encoded by SMN orthologues (see Methods). The value at each branch is the Bayesian posterior probability for the split. Schizosaccharomyces pombe was assumed to be an out-group for rooting purposes. (b) The SMN protein map is divided into exon regions labeled with exon number and corresponding amino acid (AA) above. Domains of SMN that have been identified by their sequence characteristics and/or binding partners are labeled and defined by colored triangles. (c) Nomenclature of the species examined in the study. Scientific names, corresponding viral vectors and the appropriate abbreviations used. (d) Percent identity and evolutionary divergence of the species examined. Conservation relationships among tested SMN homologs. The percent identity is calculated from amino acid sequence differences. Divergence values represent information established from the phylogenetic relationship between the species.
	Figure 2. Delivery of SMN homologs results in variable lifespan extension in a severe mouse model of SMA. Viral vector injections for each tested SMN homolog were administered by ICV on P2 and life span was recorded. (a) Comparison of average survival time by Log-rank Mantel-Cox test. Kaplan-Meier survival curve depicts life span of healthy (unaffected), SMA (untreated) mice in comparison to SMN homologs-treated SMA mice. (b) Representative images of SMN∆7 mice (Smn−/−;SMN2+/+;SMN∆7+/+) injected with xSmn (left panel) and zSmn (right panel) homologs, respectively, past the age of P40. The treated mice display noticeable distal necrosis, which is exaggerated on the tail, eyelids and/or ears. (c) Representative image of SMN∆7 mice injected with xSmn, cSmn, dSmn homologs compared to control littermates at P12. The xSmn injected mouse has the appearance and gait of the unaffected littermate, while the dSmn and cSmn injected animals show slower growth and development delay similar to the untreated SMA mouse.
	Figure 3. SMN homologs induce healthy weight gain in SMNΔ7 animals. (a) Weight gain curves in grams of SMNΔ7 mice treated with various SMN homologs and control littermates. Error bars indicate SEM. Statistical analyses were performed by Student’s t-test (of each treatment), where significance was shown with p < 0.0001 for untreated vs. hSMN, zSMN and xSMN cohorts; where no significant weight gain difference was observed between untreated and dSMN (p = 0.0681), untreated and cSMN (p = 0.2813), untreated and ySMN (p = 0.2874). (b) Individual body weights from all cohorts on P7. The scatter plot shows two divergent groups of SMN homologs that are distinct at earlier stages of life.
	Figure 4. Assessment of motor function of treated SMNΔ7 mice after treatment with various SMN homologs. (a) Line graph representing raw data of the average time to right from P7 to P17. Animals injected with hSMN, zSmn and xSmn displayed significant improvement in motor function through their life span. (b) Scatter plot of time-to-right ability of treated cohorts highlights the difference in performance for each individual mouse (values are shown for P12). Statistical analysis was carried out by one-way ANOVA and significance is represented by “*”p ≤ 0.05; “**”p ≤ 0.01; “***”p ≤ 0.001; and “n.s.”p > 0.05. (c) Bar graph showing the average time-to-right in seconds of each treatment group from ages of P7 through P17, where error bars represent significance compared to the control groups. Data expressed as S.E.M.
	Figure 5. Neuromuscular junction pathology in SMN∆7 mice after delivery of SMN homologs. (a) Representative immunohistochemistry of NMJs of individual cohorts injected with xSmn, dSmn, cSmn and compared to the age-matched controls. Neurofilament and synaptic vesicle are shown in green. Acetylcholine receptors are stained with α-Bungarotoxin (red). NMJs were analyzed from the longissimus capitis (shown) and the splenius muscles, harvested from cohorts at P12. (b) Quantification of percent NMJ innervation in the splenius muscle. (c) Quantification of percent NMJ innervation in the longissimus muscle. Data was analyzed using by one-way ANOVA and statistical significance is represented by “*”p ≤ 0.05; “**”p ≤ 0.01; “***”p ≤ 0.001; “****”p ≤ 0.001; “n.s.”p > 0.05 and expressed as S.E.M.
	Figure 6. Functional analysis of SMN homologs in RNA processing. Total mRNA levels from mouse spinal cord tissues (n = 3) were assayed by RT-qPCR. Relative mRNA levels are normalized and compared to unaffected wild type animals. (a) Levels of total endogenous mouse Smn, full-length (SMN2 FL) and total human SMN transcripts from the SMN2 gene. (b) Analysis of representative SMN-dependent RNA processing events that are dysregulated in SMA mice, which include aberrant U12 splicing of Stasimon mRNA, 3′-end processing of H1c histone mRNA, accumulation of Cdkn1a mRNA, and decreased Chodl mRNA expression. cSmn is not able to correct mRNA processing defects induced by SMN deficiency in SMA mice (untreated, red), whereas xSmn rescue of these defects was as effective as treatment with full-length human SMN (hSMN). Data represent mean and S.E.M from independent biological replicates. Statistical analysis was carried out by one-way ANOVA and significance is represented by “*”p ≤ 0.05; “**”p ≤ 0.01; “***”p ≤ 0.001.
	Figure 7. AAV delivery of SMN236 – an artificial protein containing only the most highly evolutionarily conserved domains of human SMN – improves survival and weight gain in SMA mice. SMN homologs demonstrate regions of high conservation in the amino acid sequences encoded by human SMN exons 2a, 2b, 3 and 6. Therefore, the SMN236 construct was designed to contain these regions and incorporated into a viral vector for gene delivery in mouse models of SMA. (a,b) Analysis of SMN236 injected in the severe SMNΔ7 mice on survival (a) and weight gain (b) compared to the age-matched control littermates. The difference in survival between treated (median 15 days) and untreated SMNΔ7 (median 13 days) mice was calculated by the log-rank Mantel-Cox test (p = 0.015). (c,d) SMN236 delivered into the intermediate Smn2B/− mouse model of SMA delayed early deaths with median survival 25 days for untreated vs. 36 days for the treated animals (Mantel-Cox test; p = 0.00023) (c) and has the greatest weight gain effect in the long-lived animals (d). Data expressed as mean and S.E.M.

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