Gene Therapy Restores Auditory and Vestibular Function in a Mouse Model of Usher Syndrome Type 1c

Because there are currently no biological treatments for deafness, we sought to advance gene therapy approaches to treat genetic deafness. We reasoned that gene delivery systems that target auditory and vestibular sensory cells with high efficiency would be required to restore complex auditory and balance function. We focused on Usher Syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and used a knock-in mouse model, Ush1c c.216G>A, which carries a cryptic splice site mutation found in French-Acadian patients with Usher Syndrome type IC (USH1C). Following delivery of wild-type Ush1c into the inner ears of neonatal Ush1c c.216G>A mice, we find recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders

Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

<div><p>Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (<i>SMN1</i>) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, <i>SMN2</i>, which is nearly identical to <i>SMN1.</i> However, <i>SMN2</i> cannot compensate for the loss of <i>SMN1</i> because <i>SMN2</i> has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from <i>SMN2</i> lacks exon 7. <i>SMN2</i> mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving <i>SMN2</i> exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in <i>SMN2</i> exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased <i>SMN2</i> exon 7 inclusion and SMN protein. Our results identify novel regulators of <i>SMN2</i> exon 7 inclusion, revealing potential targets for SMA therapeutics.</p></div

Splicing factors tested for activity in <i>SMN2</i> exon 7 splicing.

<p>Splicing factors tested for activity in <i>SMN2</i> exon 7 splicing.</p

SR proteins regulate <i>SMN2</i> exon 7 splicing in a cell-free splicing assay.

<p>(<b>A</b>) Semi-quantitative radiolabeled RT-PCR analysis of <i>in vitro</i> transcribed <i>SMN1</i> and <i>SMN2</i> RNA isolated from cell-free splicing reactions with or without the addition of the indicated recombinant SR proteins or a cellular fraction enriched in SR proteins (total SR). (<b>B</b>) Quantification of <i>SMN2</i> exon 7 splicing following addition of the indicated SR proteins. Splicing is normalized to <i>SMN2</i> exon 7 splicing in nuclear extract without additional SR protein (dashed line indicates basal exon 7 splicing). Only experiments repeated more than once are included in the graph. Error bars represent the standard error of the mean (SEM); total SR (2.5 ng n = 2, 5 ng n = 4, 10 ng n = 4), SRSF1 (0.36 pmol n = 4, 0.72 pmol n = 4, 1.08 pmol n = 2), SRSF2 (0.8 pmol n = 2, 1.6 pmol n = 4, 3.2 pmol n = 4). Asterisks indicate p value (two-tailed) ≤0.05 as determined by a one sample t test with a theoretical value of 1.0 for normalization to basal <i>SMN2</i> exon 7 splicing. (<b>C</b>) Analysis of <i>in vitro</i> transcribed β-globin RNA splicing in a cell-free splicing reaction with or without (–) the addition of total SR proteins (10 ng), SRSF1 (0.72 pmol) or SRSF2 (1.6 pmol) to verify recombinant protein activity. The graph shows the percent of RNA that is spliced: (spliced/(unspliced+spliced)*100), error bars are SEM, n = 2.</p

Overexpression of SR proteins modulates endogenous <i>SMN2</i> exon 7 inclusion in cells.

<p>(<b>A</b>) Semi-quantitative radiolabelled RT-PCR of endogenous <i>SMN2</i> mRNA following selective over-expression of SR proteins in HeLa cells. Reaction products were digested with DdeI to distinguish between <i>SMN1</i> and <i>SMN2</i> transcripts, digestion products are indicated on the left. Mock samples were exposed to the transfection reagent in the absence of siRNA. (<b>B</b>) The graph represents quantification of <i>SMN2</i> exon 7 inclusion: (Exon 7 included/skipped). Asterisks indicate a statistically significant decrease in exon 7 inclusion p≤0.05 and **p≤0.01, by unpaired Student’s t tests. Error bars represent SEM. In all cases, SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF9 n = 6; SRSF11 n = 5. (<b>C</b>) Immunoblot analysis of protein lysates from HeLa cells transfected with the indicated expression vectors. Immunoblots of T7-tagged SRSF1, SRSF2, SRSF3, SRSF7, and SRSF9 probed with a T7-specific antibody, endogenous SRSF4, 5 and 6 probed with SRSF4-, SRSF5- and SRSF6-specific antibodies and FLAG-tagged SRSF11 probed with a FLAG-specific antibody. β-actin and β-catenin were analyzed for loading control.</p

Splicing factors with experimentally validated affects on <i>SMN2</i> exon 7 inclusion are shown at their predicted binding sites.

<p>Exon 7 (capital letters within the black box) and 50 nucleotides of introns 6 and 7 (lower case letters) are shown. Binding motifs and references are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115205#pone.0115205.s001" target="_blank">S1 Table</a>.</p

Knockdown of SRSF2 and SRSF3 in SMA cells increases <i>SMN2</i> exon 7 inclusion and SMN protein abundance.

<p>(<b>A</b>) Immunoblots of SMN and β-actin following knockdown of SR proteins in GM00232 type 1 SMA patient-derived fibroblast cell line. • indicates a putative SMN protein lacking exon 5. (<b>B</b>) Quantification of protein abundance: (SMN/β-actin). (<b>C</b>) Semi-quantitative radiolabelled RT-PCR showing endogenous <i>SMN2</i> exon 7 inclusion following knockdown of SR proteins in GM00232 cells. (<b>D</b>) Quantification of <i>SMN2</i> exon 7 inclusion (included_{All isoforms including exon 7}/skipped_{All isoforms skipping exon 7}) corrected for cytosine content. (E) Quantitation of RT-PCR experiments to assess mRNA expression of indicated SR proteins following siRNA treatment. The dashed line represents the level of expression in control-treated cells. Error bars represent SEM. SRSF1, SRSF4, SRSF5 and SRSF6 n = 3, SRSF2 and SRSF11 n = 4, SRSF3 n = 5. Asterisks indicates a statistically significant change in SMN abundance or <i>SMN2</i> exon 7 inclusion, *p≤0.05, **p≤0.01 and ***p≤0.001, unpaired Student’s T-tests. Control cells were transfected with a scrambled control siRNA.</p

SRSF3 regulates <i>SMN2</i> expression in SMA patient iPS cells.

<p>(<b>A</b>) Immunoblot of SMN and β-actin following knockdown of SRSF3 with siRNA in SMA patient-derived iPS cells. • indicates a putative SMN protein lacking exon 5. Quantitation is shown on the right (SMN/β-actin). Asterisks indicate p value (two-tailed) ≤0.05 as determined by a one sample t test with a theoretical value of 1.0 for normalization to SMN in control-treated cells. The dashed line represents the level of expression in control-treated cells. (<b>B</b>) Semi-quantitative radiolabelled RT-PCR of endogenous <i>SMN2</i> mRNA after knockdown of SRSF3 using in SMA patient iPS cells or a scrambled siRNA (Control). Products are indicated on the left. Quantification of <i>SMN2</i> exon 7 inclusion (inclusion/skipped). Error bars represent SEM, n = 3. Asterisk represents a statistically significant increase in exon 7 inclusion where p≤0.05, unpaired Student’s T-tests.</p

Knockdown of SR and hnRNP proteins modulates endogenous <i>SMN2</i> exon 7 inclusion.

<p>(<b>A</b>) Semi-quantitative radiolabelled RT-PCR of endogenous <i>SMN2</i> mRNA following RNAi-mediated knockdown of individual SR or hnRNP proteins in HeLa cells. Reaction products were digested with DdeI. Digestion products are labeled. (<b>B</b>) Graphs show the quantification of <i>SMN2</i> exon 7 inclusion: (included/skipped). Asterisks indicate a statistically significant change in exon 7 inclusion, *p≤0.05, **p≤0.01 and ***p≤0.001, unpaired Student’s T-tests. (<b>C</b>) Quantification of RT-PCR confirmed effective, selective knockdown of SR proteins and hnRNPs. The dashed line represents the level of control expression. In all cases, error bars represent SEM. For SRSF1 and SRSF2 n = 3; SRSF4, SRSF6, hnRNP A2/B1, ELAVL1 and hnRNP D n = 4; SRSF7, SRSF10, and hnRNP U n = 5; SRSF9, hnRNP C, hnRNP H1 and hnRNP F n = 6, SRSF5 n = 7, SRSF3 and SRSF11 n = 8; Control cells were transfected with a scrambled control siRNA. (<b>D</b>) Immunoblot analysis of indicated endogenous protein from lysates of cells treated with siRNAs targeting the indicated SR protein or a non-specific control siRNA. β-actin is a loading control.</p