Modification of Bcl-x and Mcl-1 pre-mRNA splicing using splice-switching oligonucleotides Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • Bauman, John A.
    • Affiliation: School of Medicine, Department of Pharmacology
Abstract
  • Over 90% of multi-exon pre-mRNA transcripts undergo alternative splicing and up to one-half of disease-causing mutations affect splicing. Thus, alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) are chemically modified antisense oligonucleotides that hybridize to pre-mRNA sequences involved in splicing and block access to the transcript by splicing factors. As a result the splicing machinery is redirected to alternative splice sites thereby modifying the pattern of splicing and influencing protein expression. The efficacy of SSOs has been established in various animal disease models and clinical trials for Duchenne Muscular Dystrophy. However, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. Bcl-x pre-mRNA is alternatively spliced to express anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL expression is up-regulated in many cancers and confers chemoresistance, distinguishing it as an important target for cancer therapy. It was previously demonstrated that redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS induced apoptosis in breast and prostate cancer cells. In the research presented herein, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. Delivery to tumor cells in vivo was achieved by encapsulating the SSO in targeted lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. These findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing. Like Bcl-x, Mcl-1 pre-mRNA is alternatively spliced to produce proteins with opposing functions. Splicing of all three Mcl-1 exons produces anti-apoptotic Mcl-1L, which is highly expressed in many malignancies and confers resistance to chemotherapeutic drugs. Skipping of exon 2 leads to expression of Mcl-1S, a potent pro-apoptotic protein expressed at low levels in most cells. I designed and screened SSOs targeted to the 5' and 3' splice sites of exon 2 in Mcl-1 pre-mRNA, which resulted in redirection of Mcl-1 pre-mRNA from Mcl-1L to -1S. Mcl-1 splice-switching resulted in PARP cleavage and cell death in HeLa cells, indicative of apoptosis. Mcl-1 splice-switching also sensitized cells staurosporine. These findings validate Mcl-1 as a target for SSOs to induce apoptosis and sensitize cancer cells to chemotherapy.
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  • In Copyright
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  • " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology."
Advisor
  • Kole, Ryszard
Degree granting institution
  • University of North Carolina at Chapel Hill
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Place of publication
  • Chapel Hill, NC
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  • Open access
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