Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa
Abstract
:1. Introduction
2. Results
2.1. A Homozygous COL17A1 Nonsense Mutation Leads to Loss of C17 Expression
2.2. Design of AONs Capable of Exon 7 Skipping in Combination with A Liposomal Delivery System
2.3. AON-Induced Exon Skipping Restores Type XVII Collagen Expression
2.4. Treatment of 3D Skin Equivalents with AONs Results in A Stable Epidermis and Correct Localization of Type XVII Collagen
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Cell Culture
4.2. Generation of A JEB∆6∆7 Cell Line
- F1_ex6+7_fw: 5′-caggcctcgagggccggcgcgccgcATGGATGTAACCAAGAAAAACAAACG-3′
- F1_ex6+7_rv: 5′-cgaatttcactctCTTCATACGCATGGCGGG-3′
- F2_fwd: 5′-cggaaggaatttgAGAGTGAAATTCGAGTTCGAC-3′
- F2_rev: 5′-aatgccaagagccCCTGGAACACCTGGATCAC-3′
- F3_fwd: 5′-caggtgttccaggGGCTCTTGGCATTCCTAG-3′
- F3_rev: 5′-ggaggggggggggcggaatttacgtagcTCACGGCTTGACAGCAATAC-3′
4.3. AON Design
- AON1_C17_20: 5′-TTTGACTCCGTCCTCTGGTT-3′
- AON1_C17_20_Cy5: 5′-Cy5-TTTGACTCCGTCCTCTGGTT-3′
- AON2_C17_20: 5′-TCGTGTTTGACTCCGTCCTC-3′
- AON2_C17_20_Cy5: 5′-Cy5-TCGTGTTTGACTCCGTCCTC-3′
- AON3_C17_20: 5′-CTCCGTCCTCTGGTTGAAGA-3′
- AON3_C17_20_Cy5: 5′-Cy5-CTCCGTCCTCTGGTTGAAGA-3′
4.4. Splice Pattern Evaluation
4.5. DDC642 Liposome Production
4.6. Gel Retardation Assay
4.7. Cell Viability
4.8. Immunofluorescence Microscopy
4.9. Semiquantitative Real-Time PCR (sqRT-PCR)
- C17_FW: 5′-GGAGCGAGCTGATCAGCTACCTCAC-3′
- C17_RV: 5′-GCCATCCCTTGCAGTAGGCCCTG-3′
- GAPDH_FW: 5′-GCCAACGTGTCAGTGGTGGA-3′
- GAPDH_RV: 5′-CACCACCCTGTTGCTGTAGCC-3′
4.10. NGS
4.11. Western Blot Analysis
4.12. Transfection of keratinocytes with AONs
4.13. Conditioning of Cell Culture Supernatants
4.14. 3D Organotypic Skin Equivalents
4.15. Liposomal Delivery of AONs into Human Skin Explants
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Ablinger, M.; Lettner, T.; Friedl, N.; Potocki, H.; Palmetzhofer, T.; Koller, U.; Illmer, J.; Liemberger, B.; Hainzl, S.; Klausegger, A.; et al. Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa. Int. J. Mol. Sci. 2021, 22, 3326. https://doi.org/10.3390/ijms22073326
Ablinger M, Lettner T, Friedl N, Potocki H, Palmetzhofer T, Koller U, Illmer J, Liemberger B, Hainzl S, Klausegger A, et al. Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa. International Journal of Molecular Sciences. 2021; 22(7):3326. https://doi.org/10.3390/ijms22073326
Chicago/Turabian StyleAblinger, Michael, Thomas Lettner, Nicole Friedl, Hannah Potocki, Theresa Palmetzhofer, Ulrich Koller, Julia Illmer, Bernadette Liemberger, Stefan Hainzl, Alfred Klausegger, and et al. 2021. "Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa" International Journal of Molecular Sciences 22, no. 7: 3326. https://doi.org/10.3390/ijms22073326