Cells Treated with serial

diluted compound and

incubated for 24 hours 

Evaluating the Effects of Small Molecule Drugs on Correcting Alternative Splicing of SMN 2 mRNA in Spinal Muscular Atrophy.

Richard F. Baier*1, Vijayalakshmi Gabbeta2, Amal Dakka2

Department of Biological Sciences, York College of Pennsylvania1

PTC Therapeutics2

IntroductionSpinal Muscular Atrophy (SMA) is an autosomal neuromuscular disorder, which causes degeneration of the alpha motor neurons in the spinal cord. A Deletion of Survival Motor Neuron 1 (DSMN1) results in Spinal Muscular Atrophy (Zheleznyakova et al. 2011). SMA is the second highest genetic cause of infant deaths (D’Amico et al. 2011) , (SMA Foundation,2012).

Survival motor neurons (SMN) are housekeeping genes that produce SMN protein. These genes are needed to provide normal biological function. Survival Motor Neuron 1 & 2 (SMN 1 and 2) are two genes important in Spinal Muscular Atrophy. SMN 1 and 2 are almost identical genes but SMN 2 has a splicing mutation in exon 7 that prevents correct splicing of exon 7 (see red block below), (Figure 1). This mutation results in a truncated protein that is not sufficient for proper biological function. In contrast, SMN 1 produces full-length protein. The Spinal Muscular Atrophy phenotype results from a deletion of SMN1. Without the SMN1 gene, the only source of SMN protein is from truncated SMN2 (Dexon7), which is not enough to support proper biological function. However, the copy number of the SMN2 gene influences the severity of the disease (Zheleznyakova et al. 2011). Previous studies have shown that replacing SMN1 through gene therapy or correcting the alternative splicing of SMN2, results in amelioration of the SMA phenotype (D’Amico et al. 2011).

PurposeThe goal of our study was to screen small molecule drug compounds that induce correct splicing of exon ∆7 in SMN2. The hypothesis of this study is that the certain small molecule drugs will improve the positive splicing signals in the area of exon ∆7 (see green block, Figure 3), causing inclusion of exon 7.

Methods

Results

Cells Lysed and RNA isolated with BIO RAD Lysis Buffer

Transgenic Mouse Tissue with human

SMN2 gene and mouse SMN

gene

RNA Isolated with RNeasy

Tissue Kit

RT-PCR with hSMN2, mSMN, and mGAPDH

Primers

PCR Products Analyzed on a

4% Agarose Gel

Figure 2. Drug Classification. A series of 6 drug compounds tested in patient cells classified for their effectiveness in inducing full-length SMN (FL-SMN) and decreasing ∆exon7 expression during a dose response. Cyclophilin was tested in the compounds to assure drug did not alter genes non-specifically.

Figure 3. Mouse muscle tissue samples treated with compounds at 3mg/kg and 10mg/kg concentrations. Samples were tested for either total SMN, hSMN2, Mouse SMN, or mGAPDH. Each test was done in a sample size of n=4. Positive and negative controls included.

Discussion• Figure 2. Class I and II showed positive results of

correcting the mRNA splicing of SMN2. Therefore, these compounds may be influencing the positive splicing signals in the splicing mechanism.

• Figure 3. Tissue samples showed a positive result but not as strong as the in vitro cell study. This may be because the drugs were administered orally to the mice instead of added directly to cells. However, these compounds may be influencing the positive splicing signals in the splicing mechanism as well.

Figure1. Splicing mechanism of spinal muscular atrophy. Red block shows the excision of exon 7 (Dexon7). Green block illustrates a small molecule drug that cause the inclusion of exon 7.

AcknowledgementsI want to thank PTC Therapeutics of South Plainfield New Jersey for this opportunity along with Theresa Natalicchio. I also want to thank Amal Dakka, Vijayalakshmi Gabbeta and Dr. Ronald Kaltreider for being my mentors and helping me along the way with this project. This work described in this poster has been part of the collaboration between PTC Therapeutics, SMA Foundation, and F. Hoffmann-La Roche.

Future Outcomes• Test Small Molecule Drug Compounds in other animals besides

mice.• Have an effective Small Molecule Drug Compound tested in Human

trials.• Have drug be released by the FDA to be the first of its kind.

Literature Cited• Bebee, T., Gladman, J., Chandler, D. 2011. Splicing regulation of the Survival Motor Neuron genes and

implications for treatment of spinal muscular atrophy. Front Biosci. 15: 1191-1204.• D’Amico, A., Mercuri, E., Tiziano, F.D., Bertini, E. 2011. Spinal Muscular Atrophy. Orphanet Journal of

Rare Diseases 6:71.• Zhelezenyakova, G.Y., Kiselev, A.V., Vakharlovsky, V.G., Rask-Anderson, M., Chavan, R., Egorova, A.A.,

Schioth, H.B., Baranov, V.S. 2001. Genetic and expression studies of SMN2 gene in Russian Patients with spinal muscular atrophy type II and III. BMC Medical Genetics. 12:96-104.

Results• Figure 2. Compounds RB- 302 and RB-808 were

classified as the best compounds tested. In a dose response FL-SMN was increased and ∆7 was decreased. Cyclophilin had no change. Class II compounds were effective but only at a higher dose response. Class III compounds were ineffective throughout the dose response.

• Figure 3. Both compounds showed a slight response at 10mg/kg in total SMN and hSMN2 by increasing FL-SMN and decreasing . The compounds did not show a response at 3mg/kg. Neither compound effected the mouse SMN or mGAPDH.

cDNA Synthesis from 5ul of

mRNA

Skin Fibroblasts Patient Cells

cDNA Synthesis from 5ul of

mRNA

Transgenic Mice treated with drugs orally.

RT-PCR of cDNA with SMN and

Cyclophilin Primers