D - Oligonucleotide Therapeutics (including siRNAs, aptamers, antagomirs, miRNAs, shRNA, antisense, and splice switching oligos, plasmids)
729: Antisense Oligonucleotide Targeting FUS-DDIT3 Oncogene for RNase H Degradation
Type: Poster Session
Poster Board Number: 729
Presentation Details
Session Title: Wednesday Posters: Oligonucleotide Therapeutics
Introduction: Myxoid Liposarcoma (MLSc) is soft-tissue sarcoma caused by a translocation between the FUS and DDIT3 genes on chromosomes 12 and 16 respectively (96% of cases) or more rarely between the EWSR1 and DDIT3 genes (4% of cases). The aberrant fusion protein FUS-DDIT3 is thought to act as a transcription factor inhibiting adipocyte differentiation. Tumor growth is slow in MLSc and prognosis is typically good for patients with low grade MLSc, however the disease often becomes chemotherapy resistant upon metastasis. Primary tumors typically occur in the limbs. There are 11 documented fusion transcript variants of myxoid liposarcoma, with most transcripts consisting of the N-terminus of FUS and the entire coding sequence of DDIT3. Type 1 and 2 are the most common fusion transcripts, with breakpoints at FUS exon 7 and 5 respectively. Our lab has been working to develop antisense oligonucleotides (ASO) for type 1 and type 2 MLSc chimeric FUS-DDIT3 mRNA that will target the transcript for RNase H degradation, resulting in terminal differentiation of MLSc tumor cells.
Methods: To screen ASOs for knockdown efficiency, we acquired the tumor-derived MLSc type 1 DL221 cell line. To model the type 2 MLSc cell line, we generated a stable HEK-293T cell line expressing the type 2 FUS-DDIT3 fusion by infecting cells with a lentivirus expressing the type 2 fusion transcript. ASOs were designed by tiling across the type 1 and type 2 breakpoints between FUS and DDIT3 sequences in the chimeric mRNA to engage specifically the chimeric mRNA without affecting the normal transcripts. We screened several different ASO chemistries in our cell lines, including 2’O-Methoxyethyl (2’-MOE) and Locked Nucleic Acid (LNA) using lipid transfection.
Results: We have identified a top 2’-MOE and candidate in our type 2 cell line. The 2’-MOE ASO resulted in 37% average knockdown compared to a non-targeting control. We have experiments ongoing to identify a top LNA ASO in our type 2 cell line. We also have experiments ongoing in NSG mice to select the lead ASO candidate for type 1 MLSc using the DL221 cell line. Looking towards the clinic, we are planning future human clinical trials for both type 1 and type 2 patients.
Erin F. Hall1, Simon Wentworth2, Jillian Gallagher1, Toloo Taghian1, Yvette Diaz Lopez1, Abigail W. McElroy1, Hector Ribeiro Benatti1, Sarah Nath1, Neeta Somaiah3, Gina D'Amato4, Jonathan K. Watts5, Miguel Sena-Esteves6, Heather L. Gray-Edwards1
1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA,2Department of Neurology, University of Massachusetts Medical School, Worcester, MA,3Department of Sarcoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX,4Sylvester Comprehensive Cancer Center, University of Miami Health System, Miami, FL,5RNA Therapeutics Institute, UMass Chan Medical School, Worcester, MA, Canada,6Department of Neurology, UMass Chan Medical School, Worcester, MA"
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