Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis
Abstract
:1. Introduction
2. Results
2.1. FUS Common Variant Analysis
2.2. Allele Selectivity of MOE and TMO-Modified FUS-Targeted AOs
2.3. TMO Reduces SFPQ Positive Protein Aggregation
2.4. Effect of Allele-Selective TMOs on FUS Protein Expression
2.5. Effect of Reducing Total Length and Gap Length on Efficacy and Selectivity
2.6. Hybridization-Dependent Off-Target Gene Knockdown
3. Discussion
4. Materials and Methods
4.1. Antisense Oliginucleotide Synthesis
4.2. Cell Culture and Transfection
4.3. RNA Extraction
4.4. ddPCR Analysis
4.5. Protein Isolation and Western Blot
4.6. Immunocytochemistry
4.7. cDNA Synthesis and qPCR
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Populations | CV1 | CV2 | CV1 and/or CV2 | CV1 and CV2 |
---|---|---|---|---|
European | 42.9 | 45.9 | 56.5 | 32.3 |
American | 45.8 | 49.9 | 57.9 | 37.8 |
Asian | 41.1 | 41.4 | 42.6 | 39.9 |
African | 5.3 | 34.2 | 38.8 | 0.7 |
AO Name | Gapmer Configuration | Complementary to | Sequence (5′-3′) |
---|---|---|---|
CV1a1 | 20 (5-10-5) | CV1 Allele 1 | TCTGGCCATATCCTGAAGTG |
CV1a1-1 | 20 (6-8-6) | CV1 Allele 1 | TCTGGCCATATCCTGAAGTG |
CV1a1-2 | 20 (7-6-7) | CV1 Allele 1 | TCTGGCCATATCCTGAAGTG |
CV1a1-3 | 18 (5-8-5) | CV1 Allele 1 | CTGGCCATATCCTGAAGT |
CV1a1-4 | 16 (5-6-5) | CV1 Allele 1 | TGGCCATATCCTGAAG |
CV1a2 | 20 (5-10-5) | CV1 Allele 2 | TCTGGCCATAGCCTGAAGTG |
CV1a2-1 | 20 (6-8-6) | CV1 Allele 2 | TCTGGCCATAGCCTGAAGTG |
CV1a2-2 | 20 (7-6-7) | CV1 Allele 2 | TCTGGCCATAGCCTGAAGTG |
CV1a2-3 | 18 (5-8-5) | CV1 Allele 2 | CTGGCCATAGCCTGAAGT |
CV1a2-4 | 16 (5-6-5) | CV1 Allele 2 | TGGCCATAGCCTGAAG |
CV1a2b | 20 (5-10-5) | CV1 Allele 2 | GCCATAGCCTGAAGTGTCCG |
CV2a1 | 20 (5-10-5) | CV2 Allele 1 | CATAGCCAGGGTAGGAGGAC |
CV2a1-1 | 20 (6-8-6) | CV2 Allele 1 | CATAGCCAGGGTAGGAGGAC |
CV2a1-2 | 20 (7-6-7) | CV2 Allele 1 | CATAGCCAGGGTAGGAGGAC |
CV2a1-3 | 18 (5-8-5) | CV2 Allele 1 | ATAGCCAGGGTAGGAGGA |
CV2a1-4 | 16 (5-6-5) | CV2 Allele 1 | TAGCCAGGGTAGGAGG |
CV2a2 | 20 (5-10-5) | CV2 Allele 2 | CATAGCCAGGATAGGAGGAC |
CV2a2-1 | 20 (6-8-6) | CV2 Allele 2 | CATAGCCAGGATAGGAGGAC |
CV2a2-2 | 20 (7-6-7) | CV2 Allele 2 | CATAGCCAGGATAGGAGGAC |
CV2a2-3 | 18 (5-8-5) | CV2 Allele 2 | ATAGCCAGGATAGGAGGA |
CV2a2-4 | 16 (5-6-5) | CV2 Allele 2 | TAGCCAGGATAGGAGG |
Scramble Control | 20 (5-10-5) | None | ACCTTATCCAATAGCGCCTC |
Selectivity Control | 20 (5-10-5) | Both alleles | CCACTGTAACTCTGCTGTCC |
Ulefnersen mimic | 20 (5-10-5) | Both alleles | GC*A*A*T*GTCACCTTTCA*T*ACC |
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Mejzini, R.; Caruthers, M.H.; Schafer, B.; Kostov, O.; Sudheendran, K.; Ciba, M.; Danielsen, M.; Wilton, S.; Akkari, P.A.; Flynn, L.L. Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis. Int. J. Mol. Sci. 2024, 25, 8495. https://doi.org/10.3390/ijms25158495
Mejzini R, Caruthers MH, Schafer B, Kostov O, Sudheendran K, Ciba M, Danielsen M, Wilton S, Akkari PA, Flynn LL. Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis. International Journal of Molecular Sciences. 2024; 25(15):8495. https://doi.org/10.3390/ijms25158495
Chicago/Turabian StyleMejzini, Rita, Marvin H. Caruthers, Balazs Schafer, Ondrej Kostov, Kavitha Sudheendran, Marija Ciba, Mathias Danielsen, Steve Wilton, Patrick Anthony Akkari, and Loren L. Flynn. 2024. "Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis" International Journal of Molecular Sciences 25, no. 15: 8495. https://doi.org/10.3390/ijms25158495