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1.  Antisense-based therapy for the treatment of spinal muscular atrophy 
The Journal of Cell Biology  2012;199(1):21-25.
One of the greatest thrills a biomedical researcher may experience is seeing the product of many years of dedicated effort finally make its way to the patient. As a team, we have worked for the past eight years to discover a drug that could treat a devastating childhood neuromuscular disease, spinal muscular atrophy (SMA). Here, we describe the journey that has led to a promising drug based on the biology underlying the disease.
doi:10.1083/jcb.201207087
PMCID: PMC3461520  PMID: 23027901
2.  Antisense-mediated exon inclusion 
Exon skipping induced by gene mutations is a common mechanism responsible for many genetic diseases. A practical approach to correct the aberrant splicing of defective genes is to use antisense oligonucleotides (ASOs). The recognition of splice sites and the regulation of splicing involve multiple positive or negative cis-acting elements and trans-acting factors. Base-pairing of ASOs to a negative element in a targeted pre-mRNA blocks the binding of splicing repressors to this cis-element and/or disrupts an unfavorable secondary structure; as a result, the ASO restores exon inclusion. For example, we have recently shown that appropriate 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASOs can efficiently correct survival motor neuron 2 (SMN2) exon 7 splicing in a cell-free splicing assay, in cultured human cells—including patient fibroblasts—and in both peripheral tissues and the CNS of SMA mouse models. These ASOs are promising drug leads for SMA therapy.
doi:10.1007/978-1-61779-767-5_20
PMCID: PMC3390937  PMID: 22454070
Exon skipping; antisense oligonucleotide; MOE; splicing; SMN2; SMA; ESS; ISS; cis-acting element; in vitro splicing assay; minigene; exon 7 inclusion; RT-PCR; ICV; ICV infusion; mouse tissue; spinal cord; CNS
3.  Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides 
Open Biology  2012;2(10):120133.
Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.
doi:10.1098/rsob.120133
PMCID: PMC3498831  PMID: 23155487
alternative splicing; antisense oligonucleotides; cancer
4.  THE SPLICING FACTOR SRSF1 REGULATES APOPTOSIS AND PROLIFERATION TO PROMOTE MAMMARY EPITHELIAL CELL TRANSFORMATION 
The splicing-factor oncoprotein SRSF1 (also known as SF2/ASF) is upregulated in breast cancers. We investigated SRSF1’s ability to transform human and mouse mammary epithelial cells in vivo and in vitro. SRSF1-overexpressing COMMA-1D cells formed tumors, following orthotopic transplantation to reconstitute the mammary gland. In 3-D culture, SRSF1-overexpressing MCF-10A cells formed larger acini than control cells, reflecting increased proliferation and delayed apoptosis during acinar morphogenesis. These effects required the first RNA-recognition motif and nuclear functions of SRSF1. SRSF1 overexpression promoted alternative splicing of BIM and BIN1 isoforms that lack pro-apoptotic functions and contribute to the phenotype. Finally, SRSF1 cooperated specifically with MYC to transform mammary epithelial cells, in part by potentiating eIF4E activation, and these cooperating oncogenes are significantly co-expressed in human breast tumors. Thus, SRSF1 can promote breast cancer, and SRSF1 itself or its downstream effectors may be valuable targets for therapeutics development.
doi:10.1038/nsmb.2207
PMCID: PMC3272117  PMID: 22245967
5.  A generalizable pre-clinical research approach for orphan disease therapy 
With the advent of next-generation DNA sequencing, the pace of inherited orphan disease gene identification has increased dramatically, a situation that will continue for at least the next several years. At present, the numbers of such identified disease genes significantly outstrips the number of laboratories available to investigate a given disorder, an asymmetry that will only increase over time. The hope for any genetic disorder is, where possible and in addition to accurate diagnostic test formulation, the development of therapeutic approaches. To this end, we propose here the development of a strategic toolbox and preclinical research pathway for inherited orphan disease. Taking much of what has been learned from rare genetic disease research over the past two decades, we propose generalizable methods utilizing transcriptomic, system-wide chemical biology datasets combined with chemical informatics and, where possible, repurposing of FDA approved drugs for pre-clinical orphan disease therapies. It is hoped that this approach may be of utility for the broader orphan disease research community and provide funding organizations and patient advocacy groups with suggestions for the optimal path forward. In addition to enabling academic pre-clinical research, strategies such as this may also aid in seeding startup companies, as well as further engaging the pharmaceutical industry in the treatment of rare genetic disease.
doi:10.1186/1750-1172-7-39
PMCID: PMC3458970  PMID: 22704758
Orphan disease therapy; Preclinical drug development; Generalizable screening methods; Translational toolbox
6.  ONCOGENIC SPLICING FACTOR SRSF1 IS A CRITICAL TRANSCRIPTIONAL TARGET OF MYC 
Cell reports  2012;1(2):110-117.
The SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here we show that SRSF1 is a direct target of the transcription-factor oncoprotein MYC. These two oncogenes are significantly co-expressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two non-canonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC’s oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC, and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.
doi:10.1016/j.celrep.2011.12.001
PMCID: PMC3334311  PMID: 22545246

Results 1-6 (6)