Identification of NMD-inhibiting ASOs and assessment of their specificity. a Schematic of NMD reporters. The numbers show the CFTR exons in the NMD reporters. The red asterisk (*) indicates the location of the W1282X mutation. b Schematic of ASO screening. 19 MOE-PS-modified 15-mer ASOs (yellow and magenta bars) were designed to cover the presumptive EJC binding sites on exons 24, 25, and 26 at 1-nt resolution. U2OS cells stably expressing each NMD reporter were transfected with individual ASOs targeting EJC binding regions on CFTR exon (c) 24, (d) 25, or (e) 26, respectively. Reporter mRNA levels were measured by radioactive RT-PCR, using primers (red bars above the target exon) listed in Supplementary Table 5. f Effect of cycloheximide on CFTR expression in 16HBE-W1282X and DLD1-W1282X cells. g Effect of the 15-mer ASO cocktail LC15-1 on CFTR expression in 16HBE-W1282X cells. h Comparison between the effects of LC15-1 or LC18 on CFTR expression in 16HBE-W1282X cells. i Effects of cycloheximide on CFTR expression in DLD1-WT, 16HBE-G551D, and 16HBE-F508del cells. jCFTR mRNA levels in DLD1-WT, 16HBE-G551D, and 16HBE-F508del transfected with Sc15 or LC15-1 at a nominal total concentration of 120 nM. k Endogenous NMD-sensitive mRNA levels in 16HBE-W1282X cells treated with cycloheximide, 120 nM Sc15 or LC15-1. All mRNA levels in (f)–(k) were measured by RT-qPCR; CFTR mRNA levels were measured using forward and reverse primers targeting exon 22 and exon 23, respectively. RPL32 served as internal reference for all panels except h, in which HPRT served as internal reference. NT = No treatment; Dox: doxycycline 1 μg/mL; Sc15/18 = 15/18-mer Scramble ASO; CC15 = ASO cocktail C488/C507/C526; LC15-1=ASO cocktail C478/C495/C515; LC18 = 18-mer ASO cocktail C24/25/26; CHX = 1-h incubation with 100 μg/mL cycloheximide. Data are represented as mean values ± SD. All data points represent independent biological replicates. c–e (n = 1). f–k (n = 3 for all treatments, except n = 2 in LC18-mer 120 nM in h). For all statistical tests, n.s. P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. f, h (LC15-1 vs LC18), i, k two-tailed Student’s t-test. g, h One-way ANOVA with Dunnett’s post-test, versus Sc15. j One-way ANOVA. Credit: Nature Communications (2022). DOI: 10.1038/s41467-022-30668-y
Antisense oligonucleotides, or ASOs, are molecules that can be used to control protein levels in cells. Cold Spring Harbor Laboratory Professor Adrian Krainer leveraged ASO technology to develop the first FDA-approved treatment for spinal muscular atrophy called Spinraza. The drug has helped over 11,000 patients make more of a protein that certain neurons in the spine need.
Since then, Krainer has been searching for more ways ASOs can help treat other disorders. He has zeroed in on cystic fibrosis (CF), where patients do not make enough of a protein called CFTR. His team discovered how to use ASOs to make more of an imperfect but still functional version of CFTR. The discovery sets the stage for a new therapeutic approach that may help reduce CF symptoms and improve patients' quality of life.
The imperfect CFTR protein is a result of a gene mutation. It causes cells to receive the wrong instructions for making the protein. The faulty instructions are eliminated and the protein isn't made, since in general, imperfect proteins may be disruptive. Krainer's ASOs trick cells into following the faulty instructions and making the imperfect CFTR protein. His team found that, in this case of CF, having an imperfect version of the protein is better than having none at all. Their method improved the function of lung cells, suggesting the ASO strategy could improve symptoms in CF patients with this mutation.
Published in Nature Communications, the team's discovery spotlights a new way ASOs can be used to treat disease. The study was led by Young Jin Kim, a former M.D.-Ph.D. student in the Krainer laboratory. Krainer hopes to continue expanding the potential of ASO technology in therapeutics. He thinks in the future ASOs may increasingly become a way to tailor therapies specific to an individual's unique genetic mutations. "If more of this type of drug, ASOs, are approved," Krainer says, "I wouldn't be surprised if in the not-so-distant future ASOs become a routine way to make personalized medicines."
More information:
Young Jin Kim et al, Gene-specific nonsense-mediated mRNA decay targeting for cystic fibrosis therapy, Nature Communications (2022). DOI: 10.1038/s41467-022-30668-y
Citation:
A new treatment approach for cystic fibrosis (2022, July 14)
retrieved 18 April 2024
from https://medicalxpress.com/news/2022-07-treatment-approach-cystic-fibrosis.html
This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no
part may be reproduced without the written permission. The content is provided for information purposes only.
