scholarly journals Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis

2022 ◽  
Vol 119 (3) ◽  
pp. e2114886119
Author(s):  
Wren E. Michaels ◽  
Cecilia Pena-Rasgado ◽  
Rusudan Kotaria ◽  
Robert J. Bridges ◽  
Michelle L. Hastings
Keyword(s):  
Cystic Fibrosis ◽  
Protein Function ◽  
Antisense Oligonucleotides ◽  
Messenger Rna ◽  
Gene Mutations ◽  
Severe Form ◽  
Open Reading Frame ◽  
Common Mutation ◽  
Reading Frame ◽  
Cftr Protein

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

scholarly journals Open reading frame correction using antisense oligonucleotides for the treatment of cystic fibrosis caused by CFTR-W1282X

2021 ◽  
Author(s):  
Wren E. Michaels ◽  
Cecilia Pena-Rasgado ◽  
Rusudan Kotaria ◽  
Robert J. Bridges ◽  
Michelle L. Hastings
Keyword(s):  
Cystic Fibrosis ◽  
Protein Function ◽  
Antisense Oligonucleotides ◽  
Gene Mutations ◽  
Severe Form ◽  
Open Reading Frame ◽  
Common Mutation ◽  
Reading Frame ◽  
Protein Levels ◽  
Cftr Protein

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR mRNA expression as a result of nonsense mediated mRNA decay (NMD), as well as production of a non-functional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations, due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs) designed to induce skipping of exons in order to restore the mRNA open reading frame have shown therapeutic promise pre-clinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the 5th most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from homozygote CFTR-W1282X patients. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

scholarly journals Cystic Fibrosis: Improving quality of life

2021 ◽  
Vol 8 (2) ◽  
pp. 91-96
Author(s):  
Sunil Chaudhry
Keyword(s):  
Quality Of Life ◽  
Cystic Fibrosis ◽  
Sweat Glands ◽  
Common Mutation ◽  
Close Monitoring ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Estimate Frequency ◽  
Cftr Protein

Cystic Fibrosis (CF) or Mucoviscidosis is an inherited condition. In cystic fibrosis transmembrane conductance regulator (CFTR) protein does not functions properly i.e regulation of fluids and salts outside the cells. Cystic fibrosis affects exocrine glands eg., the mucus-secreting and sweat glands in the respiratory and digestive systems. The frequency of common mutation F508del (deletion of phenylalanine residue at position 508) in children is between 19% and 34%. The estimate frequency of CF as 1:10,000 to 1:40,000 in children. There is no cure for cystic fibrosis, but treatment can reduce symptoms and complications to improve quality of life. Close monitoring and early, aggressive intervention is recommended to slow the progression of CF, which can lead to possible longer life.

Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes

1992 ◽  
Vol 263 (6) ◽  
pp. C1147-C1151 ◽  
Author(s):  
R. D. Krauss ◽  
G. Berta ◽  
T. A. Rado ◽  
J. K. Bubien
Keyword(s):  
Cell Cycle ◽  
Cystic Fibrosis ◽  
Antisense Oligonucleotides ◽  
T84 Cells ◽  
Cftr Protein ◽  
Low Levels ◽  
B Lymphoid ◽  
Cycle Dependent ◽  
Cl Permeability

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.

scholarly journals GM1 as Adjuvant of Innovative Therapies for Cystic Fibrosis Disease

2020 ◽  
Vol 21 (12) ◽  
pp. 4486 ◽  
Author(s):  
Giulia Mancini ◽  
Nicoletta Loberto ◽  
Debora Olioso ◽  
Maria Cristina Dechecchi ◽  
Giulio Cabrini ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Anion Channel ◽  
Apical Plasma Membrane ◽  
Multiple Drug ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Starting Point ◽  
Innovative Therapies ◽  
Cftr Protein

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.

scholarly journals Nucleotide sequence analysis of a gene from Burkholderia (Pseudomonas) cepacia encoding an outer membrane lipoprotein involved in multiple antibiotic resistance.

1996 ◽  
Vol 40 (2) ◽  
pp. 307-313 ◽  
Author(s):  
J L Burns ◽  
C D Wadsworth ◽  
J J Barry ◽  
C P Goodall
Keyword(s):  
Cystic Fibrosis ◽  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Open Reading Frame ◽  
Pseudomonas Cepacia ◽  
Multiple Antibiotic Resistance ◽  
Reading Frame ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Antibiotic Efflux

Antibiotic-resistant Burkholderia (Pseudomonas) cepacia is an important etiologic agent of nosocomial and cystic fibrosis infections. The primary resistance mechanism which has been reported is decreased outer membrane permeability. We previously reported the cloning and characterization of a chloramphenicol resistance determinant from an isolate of B. cepacia from a patient with cystic fibrosis that resulted in decreased drug accumulation. In the present studies we subcloned and sequenced the resistance determinant and identified gene products related to decreased drug accumulation. Additional drug resistances encoded by the determinant include resistances to trimethoprim and ciprofloxacin. Sequence analysis of a 3.4-kb subcloned fragment identified one complete and one partial open reading frame which are homologous with two of three components of a potential antibiotic efflux operon from Pseudomonas aeruginosa (mexA-mexB-oprM). On the basis of sequence data, outer membrane protein analysis, protein expression systems, and a lipoprotein labelling assay, the complete open reading frame encodes an outer membrane lipoprotein which is homologous with OprM. The partial open reading frame shows homology at the protein level with the C terminus of the protein product of mexB. DNA hybridization studies demonstrated homology of an internal mexA probe with a larger subcloned fragment from B. cepacia. The finding of multiple antibiotic resistance in B. cepacia as a result of an antibiotic efflux pump is surprising because it has long been believed that resistance in this organism is caused by impermeability to antibiotics.

scholarly journals Design of in vitro Transcribed mRNA Vectors for Research and Therapy

2019 ◽  
Vol 73 (5) ◽  
pp. 391-394
Author(s):  
Marina Tusup ◽  
Lars E. French ◽  
Mara De Matos ◽  
David Gatfield ◽  
Thomas Kundig ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Open Reading Frame ◽  
Purification Method ◽  
Reading Frame ◽  
Mrna Purification ◽  
Functional Regions ◽  
In Cells

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

Molecular cloning and characterization ofEchinostoma caproniheat shock protein-70 and differential expression in the parasite derived from low- and high-compatible hosts

Parasitology ◽  
2008 ◽  
Vol 135 (12) ◽  
pp. 1469-1477 ◽  
Author(s):  
M. HIGÓN ◽  
C. MONTEAGUDO ◽  
B. FRIED ◽  
J. G. ESTEBAN ◽  
R. TOLEDO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Messenger Rna ◽  
Distinct Pattern ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Echinostoma Caproni ◽  
Hsp70 Protein ◽  
Polyclonal Antisera ◽  
Secretory Products ◽  
Hydrophobic Portion

SUMMARYWe cloned and expressedEchinostoma caproniHSP70 inEscherichia coli. This molecule presents an open reading frame (ORF) of 655 amino acids, and a theoretical molecular weight of 71 kDa.E. caproniHSP70 protein showed a high homology to other helminth molecules, major differences being located in the C-terminal region of the molecule, with a hydrophobic portion. Studies of protein and messenger RNA (mRNA) expression revealed a distinct pattern, depending on the host (low- or high-compatible). Specific polyclonal antisera raised against the recombinant protein expressed inEscherichia colidemonstrated its selective presence in excretory/secretory products (ESP) of adult parasites obtained from high-compatible hosts. Immunological studies showed clearly the association of HSP70 with the parasite surface and other structures, including eggs.

Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy

2011 ◽  
Vol 50 ◽  
pp. 233-248 ◽  
Author(s):  
Patrick Kim Chiaw ◽  
Paul D.W. Eckford ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecule ◽  
Molecular Basis ◽  
Structural Changes ◽  
Common Mutation ◽  
Channel Activity ◽  
Wild Type ◽  
In The Wild ◽  
Cftr Protein ◽  
Small Molecule Modulators

Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway obstruction with recurrent airway inflammation and infection. Pulmonary obstruction in CF has been linked to the loss of CFTR function as a regulated Cl− channel on the lumen-facing membrane of the epithelium lining the airways. We have learned much about the molecular basis for nucleotide- and phosphorylation-dependent regulation of channel activity of the normal (wild-type) version of the CFTR protein through electrophysiological studies. The major CF-causing mutation, F508del-CFTR, causes the protein to misfold and be retained in the ER (endoplasmic reticulum). Importantly, recent studies in cell culture have shown that retention in the ER can be ‘corrected’ through the application of certain small-molecule modulators and, once at the surface, the altered channel function of the major mutant can be ‘potentiated’, pharmacologically. Importantly, two such small molecules, a ‘corrector’ (VX-809) and a ‘potentiator’ (VX-770) compound are undergoing clinical trial for the treatment of CF. In this chapter, we describe recent discoveries regarding the wild-type CFTR and F508del-CFTR protein, in the context of molecular models based on X-ray structures of prokaryotic ABC (ATP-binding cassette) proteins. Finally, we discuss the promise of small-molecule modulators to probe the relationship between structure and function in the wild-type protein, the molecular defects caused by the most common mutation and the structural changes required to correct these defects.

scholarly journals Gene Therapy Approach for Treating DeltaF508, G542X and R553X Cystic Fibrosis Mutations Through the Usage Of CRISPR Prime Editing

2021 ◽  
Author(s):  
Mira Tafa ◽  
Sevim Naz Karışık ◽  
Ece Begüm Aksoy ◽  
Rüya Aslan
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Animal Model ◽  
Ex Vivo ◽  
Low Cost ◽  
Cationic Liposome ◽  
Chloride Ions ◽  
Common Mutation ◽  
Cftr Protein

Cystic Fibrosis is a rare genetic disease that affects the transmission of chloride ions due to mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Even though there are nearly 2000 mutations identified to be related to the condition, the most common mutation is F508del; deletion of a phenylalanine residue at 508. On the other hand, G542X which is a Class I mutation is also found very commonly and there are no modulator treatments available for it. Furthermore, it was investigated that R553X mutation can as well be corrected simultaneously with G542X mutation. Therefore, the main focus is on designing a gene therapy project that can correct all these three mutations at once by utilizing the prime editing technique via lipid-based delivery. In this way, the mutations can be edited through plasmids that were designed containing 2 pegRNAs and the Cas enzyme. To implement such an approach efficiently, both ex vivo, an animal model, and in vivo steps are to be designed. For the cell line, fibroblasts are selected due to their simplicity and low cost. The animal model of the experiment is determined to be a ferret concerning the high similarity to the human's CFTR protein and finally, the procedure will follow on a direct application in human Cystic Fibrosis patients. The plasmids are thought to be delivered through a cationic liposome that will reach the lungs with the aid of a nebulizer. At the last stage of the experimental procedure, Sanger Sequencing will be done to see if the desired edit within the CFTR has been performed successfully, and Next Generation Sequencing will be executed to see if there has been an off-target mutation in the remainder of the genome. Whereas for detecting the presence and expression of CFTR protein in humans, immunodetection with flow cytometry will be conducted.

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