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Abstract: PO1304

Treatment with Antisense-Oligonucleotide or Splicing Regulating Proteins for X-Linked Alport Syndrome Cases with Deep Intronic Variant

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Horinouchi, Tomoko, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
  • Yamamura, Tomohiko, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
  • Nagai, Sadayuki, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
  • Aoto, Yuya, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
  • Sakakibara, Nana, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
  • Nagano, China, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
  • Iijima, Kazumoto, Hyogo Prefectural Kobe Children's Hospital, Kobe, Hyogo, Japan
  • Nozu, Kandai, Kobe University Graduate School of Medicine, Pediatrics, Kobe, Hyogo, Japan
Background

X-linked Alport syndrome (XLAS) is a hereditary progressive renal disease caused by mutation in COL4A5. Some cases of XLAS are caused by deep intronic variants which cause aberrant splicing and produce cryptic exon inclusion. Preventing translation of such cryptic exon has the potential to be an effective therapy. We reported that exon skipping therapy with antisense-oligonucleotide (ASO) was very effective in the XLAS mice model with a truncating mutation. However, an ASO needs very high sequence specificity and few patients can be treated by the same ASO. Therefore, we attempted to modify the splicing pattern not only by ASO but also by proteins important for splicing regulation. U2AF65 is one of the important splicing related proteins binding to polypyrimidine tracts promoting exonization. It has been reported that overexpression of the U2AF65 promotes or suppresses exonization in some circumstances.

Methods

We identified four cases of XLAS caused by the production of the same cryptic exon inclusion (r.[384_385ins385-764_385-617]) by different deep intronic variants: three cases (c.385-756C>G, c.385-749T>A and c.385-645T>A) were ours and 1 (c.385-719G>A) was a reported variant. For these cases, we introduced ASO that could skip cryptic exon. Moreover, using in vitro splicing evaluation system (minigene assay), we attempted to reduce the exonization of cryptic exon by overexpression of U2AF65.

Results

We succeeded in preventing the cryptic Exon insertion by introducing ASO treatment for patient’s urine derived cells. In addition, in all patients, overexpression of U2AF65 in the minigene splicing analysis system successfully reduced the cryptic exon inclusion.

Conclusion

The cryptic exonization in deep intron region by nucleotide change is often difficult to identify, but there may be more such cases. In addition to ASO treatment, splicing related proteins such as U2AF65 has a potential for the treatment splicing abnormalities with splicing modifications.

Funding

  • Government Support – Non-U.S.