1. Volume 25, Issue 6, Pages 1328-1341 (June 2017)
Gender-Specific Amelioration of SMA Phenotype upon Disruption of a Deep Intronic Structure by an Oligonucleotide 
Matthew D. Howell, Eric W. Ottesen, Natalia N. Singh, Rachel L. Anderson, Ravindra N. Singh 
Molecular Therapy 
Volume 25, Issue 6, Pages (June 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1
Examination of the In Vivo Efficacy of A15/283, an ISS-N2-Targeting ASO
(A) The top image shows the sequence of A15/283 and its binding location to SMN2 intron 7. The sequence of the scrambled control ASO, CON15, is shown. Numbering starts from the beginning of intron 7. The circle with “S” represents a PEG-282 at the 5′ terminus and the circle with “P” represents a propyl spacer at the 3′ terminus. The bottom image presents a general schematic of A15/283 and CON15 chemistry, including a PS backbone with 2OMe modification of sugar moieties, the 5′ PEG-282 and 3′ propyl spacer. (B) Semiquantitative radioactive RT-PCR gel showing SMN2 exon 7 inclusion in SMA patient fibroblasts in the presence of different ASOs. Mock refers to cells not transfected with ASO. ASO and binds to nucleotides 261–278 and 283–297 in SMN2 intron 7, respectively. Anti-N1 binds to the ISS-N1 target (nucleotides 10–24) in SMN2 intron 7.30 CON15 and A15/283 had the terminal modifications described in (A). ASO , , and Anti-N1 had a PS backbone with 2OMe sugar modification but lacked the terminal modifications. The percentage of exon 7 skipping was calculated based on the total value of SMN2 exon-7-included and exon-7-skipped product. (C) Images of radioactive RT-PCR acrylamide gels showing SMN2 exon 7 inclusion in male and female P7 C/C liver, brain, and kidney after SC injection of PBS, CON15, or A15/283 on P1 and P3 (n = 3 for each tissue, treatment, and sex). Gender and treatments are indicated at the top of the images. The graphs present quantification of the gels with the percent of transcripts with exon 7 skipped shown. (D) Western blots showing full-length SMN (derived from SMN2) and α-tubulin proteins in male and female P7 C/C brain, liver, and kidney after SC injection at P1 and P3 (n = 3 for each treatment, tissue, and sex). Gender and treatments are indicated at the top of the images. The graphs present densitometry for each tissue. Error bars on the graphs indicate SEM. For (C) and (D), data for each sex and tissue were compared with a one-way ANOVA, followed by Tukey’s multiple comparison test. *p < 0.05 indicates a significant difference between the means.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2
Early Peripheral A15/283 Treatment Provided Gender-Specific Amelioration of Tail Necrosis in C/C Mice
(A) Tail length over time for WT males injected with PBS (n = 9), CON15 (n = 17), and A15/283 (n = 23) (left) and WT females injected with PBS (n = 15), CON15 (n = 9), and A15/283 (n = 20) (right). (B) Tail length over time for C/C males injected with PBS (n = 9), CON15 (n = 11), and A15/283 (n = 7) (left) and C/C females injected with PBS (n = 11), CON15 (n = 14), and A15/283 (n = 12) (right). (C) Photograph of male and female WT and C/C tails at P60. Treatments are indicated above each column of tails. Scale bar, 25 mm. (D) Tail necrosis onset age for C/C mice. (E) Maximum tail length for C/C mice. (F) Ear necrosis onset age for C/C mice. (G) H&E-stained tail cross sections from male and female P60 mice injected with A15/283 at P1 and P3. The sex and genotype are indicated above each micrograph. Base refers to a cross section taken from the base of the tail near the body and middle refers to a cross section taken at the middle of the remaining tail stub. Micrographs with blood vessels show a higher magnification of the median caudal vasculature at the base of the tail. Black arrowhead indicates blood vessel muscle disorganization, and blue arrowhead indicates blood vessel obstruction. Base and middle micrographs scale bar, 200 μm; blood vessels micrographs scale bar, 50 μm. See also Figure S3 for tail micrographs from PBS-injected and CON15-treated mice. Error bars on graphs indicate SEM. For (A) and (B), data were analyzed with a two-way repeated-measures ANOVA followed by Tukey’s multiple comparison test. + indicates a significant difference between PBS and CON15, * indicates a significant difference between PBS and A15/283, and ˆ indicates a significant difference between CON15 and A15/283. For (D–F), data were analyzed by a two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 indicates a significant difference between the means.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3
Early Peripheral A15/283 Treatment Improved Testis Development and Normalized Spermatogenesis
(A) Western blot showing full-length SMN (derived from SMN2) and α-tubulin proteins in P7 C/C testis after SC treatment at P1 and P3 (n = 3 mice per treatment). The graph to the right presents densitometry of the blots. (B) Photograph with representative testes from WT and C/C mice treated with PBS, CON15, or A15/283. Scale bar, 500 μm. The graph to the right presents relative testes mass (mg testis per g BW) at P60. For WT mice, n = 9, 17, and 23 mice for the PBS, CON15, and A15/283 groups, respectively. For C/C mice, n = 9, 11, and 7 mice for the PBS, CON15, and A15/283 groups, respectively. (C) Representative H&E-stained P60 testis cross sections showing seminiferous tubules. Scale bar, 50 μm. (D) Percent of tubules with complete spermatogenesis from P60 mice (see Materials and Methods for further details). Numbers of mice were the same as in (B). (E) Sperm count from P60 mice. Sperm were isolated from the epididymis and counted with a hemocytometer. Numbers of mice were the same as in (B). Error bars on graphs indicate SEM. For (A), data were analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 indicates a significant difference between the means. For (B), (D), and (E), data were analyzed by a two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 indicates a significant difference between the means.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
Early Peripheral A15/283 Treatment Decreased TUNEL-Positive Bodies and Normalized Expression of Apoptosis-Related Genes
(A) Representative TUNEL staining from testis cross sections. Treatment and genotype are indicated above and to the left of the micrographs, respectively. The blue signal indicates DAPI (nuclei), and the green signal indicates TUNEL-positive bodies. Seminiferous tubules are outlined with white dashed lines. Scale bar in each micrograph, 50 μm. (B) Percentage of seminiferous tubules with at least one TUNEL-positive object. For WT, n = 5, 5, and 8 mice for PBS, CON15, and A15/283 groups, respectively. For C/C mice, n = 5, 6, and 6 mice for PBS, CON15, and A15/283 groups, respectively. (C) Average number of TUNEL-positive objects per seminiferous tubule. Numbers of mice were the same as in (B). (D–G) Relative expression of pro- and anti-apoptotic genes in testis. Expression for each gene is relative to the WT PBS group (set at 1.0). For WT, n = 6, 7, and 5 mice for PBS, CON15, and A15/283 groups. For C/C, n = 5, 5, and 4 mice for PBS, CON15, and A15/283 groups, respectively. See also Figure S6 for expression of additional pro- and anti-apoptotic genes. Error bars on graphs indicate SEM. For (B–G), data were analyzed with a two-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05 indicates a significant difference between the means.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5
Early Peripheral A15/283 Treatment Partially Normalized Dysregulated Gene Expression in C/C Testis
(A–M) Each panel shows the relative expression of a gene previously shown to be dysregulated in P42 C/C testis.29 Expression for each gene is relative to the WT PBS group (set at 1.0). For WT, n = 6, 7, and 5 mice for PBS, CON15, and A15/283 groups, respectively. For C/C, n = 5, 5, and 4 mice for PBS, CON15, and A15/283 groups, respectively. Error bars on graphs indicate SEM. For each gene, data were analyzed with a two-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05 indicates a significant difference between the means.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions