Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing

Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies.

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Recent Advances in Targeted Genetic Medicines for Cystic Fibrosis

10.5772/intechopen.92434 ◽

2021 ◽

Author(s):

Salsabil Elboraie ◽

Konstantinos N. Kafetzis ◽

Rajeev Shrivastava ◽

Aristides D. Tagalakis

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Genome Editing ◽

Therapeutic Options ◽

Cftr Gene ◽

Transmembrane Conductance Regulator ◽

Nonviral Vectors ◽

Transmembrane Conductance ◽

Therapy Strategies ◽

Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) gene was discovered just over 30 years ago, and soon after, gene therapy for cystic fibrosis (CF) has been rapidly and continually developing. Recently, novel gene therapy strategies have been developed, including mRNA delivery, genome editing, and mRNA repair; all these strategies are collectively named “genetic medicines.” The last quarter of the century showed a significant boost in the development of viral and nonviral vectors to deliver genetic treatment. This chapter will provide a brief overview of the CFTR gene and its different classes of mutations as well as a review of the different genetic therapeutic options that are under research. Later in this chapter, drugs that target different CFTR mutation classes and are currently approved to treat CF patients will be briefly presented.

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Therapeutic genome editing with engineered nucleases

Hämostaseologie ◽

10.5482/hamo-16-09-0035 ◽

2017 ◽

Vol 37 (01) ◽

pp. 45-52 ◽

Cited By ~ 6

Author(s):

Simone Haas ◽

Viviane Dettmer ◽

Toni Cathomen

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Genetic Disorders ◽

Zinc Finger Nucleases ◽

New Era ◽

Engineered Nucleases ◽

Genetic Interventions ◽

Type Gene ◽

Programmable Nucleases ◽

Cancer Immunotherapies

SummaryTargeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.

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Advances of gene therapy for primary immunodeficiencies

F1000Research ◽

10.12688/f1000research.7512.1 ◽

2016 ◽

Vol 5 ◽

pp. 310 ◽

Cited By ~ 11

Author(s):

Fabio Candotti

Keyword(s):

Gene Therapy ◽

Primary Immunodeficiency ◽

Genetic Diseases ◽

Severe Combined Immunodeficiency ◽

Granulomatous Disease ◽

Combined Immunodeficiency ◽

Recent Past ◽

Adenosine Deaminase Deficiency ◽

Primary Immunodeficiency Diseases ◽

Therapy Field

In the recent past, the gene therapy field has witnessed a remarkable series of successes, many of which have involved primary immunodeficiency diseases, such as X-linked severe combined immunodeficiency, adenosine deaminase deficiency, chronic granulomatous disease, and Wiskott-Aldrich syndrome. While such progress has widened the choice of therapeutic options in some specific cases of primary immunodeficiency, much remains to be done to extend the geographical availability of such an advanced approach and to increase the number of diseases that can be targeted. At the same time, emerging technologies are stimulating intensive investigations that may lead to the application of precise genetic editing as the next form of gene therapy for these and other human genetic diseases.

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Gene Therapy for Cystic Fibrosis Utilizing a Replication Deficient Recombinant Adenovirus Vector to Deliver the Human Cystic Fibrosis Transmembrane Conductance Regulator cDNA to the Airways. A Phase I Study. Children's Hospital Medical Center, Cincinnati, Ohio

Human Gene Therapy ◽

10.1089/hum.1994.5.8-1019 ◽

1994 ◽

Vol 5 (8) ◽

pp. 1019-1057 ◽

Cited By ~ 15

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Phase I Study ◽

Recombinant Adenovirus ◽

Medical Center ◽

Adenovirus Vector ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Recombinant Adenovirus Vector ◽

Cystic Fibrosis Transmembrane

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Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing

Nature Communications ◽

10.1038/s41467-019-11454-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 19

Author(s):

Giulia Maule ◽

Antonio Casini ◽

Claudia Montagna ◽

Anabela S. Ramalho ◽

Kris De Boeck ◽

...

Keyword(s):

Cystic Fibrosis ◽

Genome Editing ◽

Genetic Diseases ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Gene Correction ◽

Splicing Mutations ◽

Mutant Sequence ◽

A Genome ◽

Allele Specific

Abstract Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272–26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272–26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

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Reverse Transcription-Competitive Multiplex PCR Improves Quantification of mRNA in Clinical Samples—Application to the Low Abundance CFTR mRNA

Clinical Chemistry ◽

10.1093/clinchem/45.5.619 ◽

1999 ◽

Vol 45 (5) ◽

pp. 619-624 ◽

Cited By ~ 11

Author(s):

Stefan M Loitsch ◽

Stefan Kippenberger ◽

Nurlan Dauletbaev ◽

Thomas OF Wagner ◽

Joachim Bargon

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Multiplex Pcr ◽

Reverse Transcription ◽

Coefficient Of Variation ◽

High Variability ◽

Clinical Samples ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Cystic Fibrosis Transmembrane

Abstract Background: To monitor gene therapy, we wished to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. We developed a PCR-based method to measure CFTR mRNA in clinical samples. Methods: Expression was determined by reverse transcription-competitive multiplex PCR (RCMP) for CFTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, and for serial dilutions of two internal cDNA standards consisting of CFTR and GAPDH mutants containing short deletions. The RCMP used simultaneous amplification of the gene of interest with a reporter gene in one reaction tube. The expression of CFTR was calculated with reference to the amount of GAPDH to correct for variations in initial RNA loading. Results: Amplification of cDNAs derived from different amounts of RNA (1–4 μg) gave similar GAPDH/CFTR ratios, with a coefficient of variation (CV) below 7.5%. RCMP was applied on nasal and bronchial brushings and shows a high variability of CFTR expression in non-cystic fibrosis donors. Conclusion: This method is precise and reproducible and advantageous for use with limited amounts of tissue, such as from biopsies or from nasal or bronchial brushings.

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Potential of helper-dependent Adenoviral vectors in CRISPR-cas9-mediated lung gene therapy

Cell & Bioscience ◽

10.1186/s13578-021-00662-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ranmal Avinash Bandara ◽

Ziyan Rachel Chen ◽

Jim Hu

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Lung Disease ◽

Viral Vectors ◽

Gene Editing ◽

Genetic Diseases ◽

Adenoviral Vectors ◽

The Status

AbstractSince CRISPR/Cas9 was harnessed to edit DNA, the field of gene therapy has witnessed great advances in gene editing. New avenues were created for the treatment of diseases such as Cystic Fibrosis (CF). CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Despite the success of gene editing with the CRISPR/Cas9 in vitro, challenges still exist when using CRISPR/Cas9 in vivo to cure CF lung disease. The delivery of CRISPR/Cas9 into lungs, as well as the difficulty to achieve the efficiency required for clinical efficacy, has brought forth new challenges. Viral and non-viral vectors have been shown to deliver DNA successfully in vivo, but the sustained expression of CFTR was not adequate. Before the introduction of Helper-Dependent Adenoviral vectors (HD-Ad), clinical trials of treating pulmonary genetic diseases with first-generation viral vectors have shown limited efficacy. With the advantages of larger capacity and lower immunogenicity of HD-Ad, together with the versatility of the CRISPR/Cas9 system, delivering CRISPR/Cas9 to the airway with HD-Ad for lung gene therapy shows great potential. In this review, we discuss the status of the application of CRISPR/Cas9 in CF gene therapy, the existing challenges in the field, as well as new hurdles introduced by the presence of CRISPR/Cas9 in the lungs. Through the analysis of these challenges, we present the potential of CRISPR/Cas9-mediated lung gene therapy using HD-Ad vectors with Cystic Fibrosis lung disease as a model of therapy.

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Can enhancing the expression and activity of the Cystic Fibrosis Transmembrane Regulator improve efficacy of gene therapy for Cystic Fibrosis?

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.802.22 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Maximillian Woodall ◽

Robert Tarran ◽

Deborah Baines

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Cystic Fibrosis Transmembrane Regulator ◽

Cystic Fibrosis Transmembrane ◽

Expression And Activity

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Innovative therapies in genetic diseases: Cystic fibrosis

Romanian Journal of Pediatrics ◽

10.37897/rjp.2021.1.3 ◽

2021 ◽

Vol 70 (1) ◽

pp. 16-20

Author(s):

Elena-Silvia Shelby ◽

◽

Florina Mihaela Nedelea ◽

Tanser Huseyinoglu ◽

Relu Cocos ◽

...

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Clinical Trials ◽

Antisense Oligonucleotides ◽

Viral Vectors ◽

Genetic Diseases ◽

Wild Type ◽

Gene Encoding ◽

Innovative Therapies ◽

Type Gene

Cystic fibrosis, also named mucoviscidosis, is the most frequent hereditary pulmonary disease and is produced by mutations in the CFTR gene, encoding an anionic channel for chloride and bicarbonate involved in the regulation of salt and bicarbonate metabolisms. Currently, about half of the patients with cystic fibrosis can benefit personalized therapy consisting in modulators, drugs which restore or improve the functionality and stability of CFTR. Moreover, presently, other therapies, such as gene therapy using the CRISP/CAS-9, modified antisense oligonucleotides or the insertion of the wild-type gene using nanolipidic particles or viral vectors, are being developed. This article aims to take stock of the principal types of cystic fibrosis therapies which have been approved or are in clinical trials.

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