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News & Blogs » Molecular Biology News » Correcting Human Disease Mutations with CRISPR/Cas9-AAV Strategies

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July 22, 2021 11:00 AM-6:00 PM DET

Correcting Human Disease Mutations with CRISPR/Cas9-AAV Strategies

The Porteus lab is developing gene editing strategies to correct disease causing mutations. His lab focuses on modifying stem cells through the combined use of CRISPR/Cas9 gene editing tools and AAV vectors for payload delivery. Following ex vivo gene editing, transplantation of modified stem cells into preclinical animal models and in vitro assays allows them to test their strategies' efficacy and potential clinical application.

Preclinical Strategies to Rectify CFTR Mutations

Cystic fibrosis is an autosomal recessive disease that results from mutations in the CFTR gene (cystic fibrosis transmembrane conductance regulator). Over 2,000 mutations have been identified which affect CFTR’s expression or function through different mechanisms. Disease severity and time of onset depend on the specific underlying mutations and the extent of CFTR loss of function. A hallmark of cystic fibrosis is the increased sweat salt concentration, which serves as a diagnostic criterion (i.e., sweat chloride analysis). Phenotypes associated with severe disease and directly responsible for cystic fibrosis morbidity include increased accumulation of thick mucus secretions in airways, chronic inflammation, and susceptibility to recurrent respiratory infections (De Boeck, 2020).

CFTR is an ATP-gated anion channel critical for transepithelial fluid and electrolyte transport in respiratory, gastrointestinal, and pancreas tissues, among others (Hwang et al. 2018). CFTR localizes to the apical epithelium of exocrine glands, where it helps maintain ion concentration balance. Besides its chloride conductance, CFTR modulates the epithelial sodium channel activity (ENaC) (Berdiev et al. 2009). As it occurs due to mutations associated with cystic fibrosis, the malfunction of CFTR results in thickening of glandular secretions and consequent duct obstruction and atrophy (e.g., airways).

Reprinted from “Cystic Fibrosis Airways”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates

The most prevalent CFTR mutation, F508del, leads mostly to complete loss of channel function due to protein misfolding and defective channel gating (Patel et al. 2019). There is no cure for cystic fibrosis, and available therapies traditionally have only ameliorated disease symptoms. More recently, small molecules have been used that target CFTR and enhance its function. Nevertheless, the effectiveness of these modulators is highly dependent on the specific CFTR mutation(s) and other genetic and epigenetic factors, which may impact individual responses. Therefore, these therapies do not provide relief to all patients.

Recognizing the need for curative therapies to restore CFTR function effectively, the Porteus lab has initiated studies combining the use of CRISPR/Cas9 and AAV vectors to achieve gene editing of airway basal stem cells. In previous work, their efforts focused on repairing the most prevalent mutation associated with cystic fibrosis, the F508del (Vaidyanathan et al. 2020). Nevertheless, because of the heterogeneity of mutations underscoring cystic fibrosis, Porteus and colleagues have envisioned a strategy for targeted replacement of the full CFTR sequence at its endogenous locus. Once edited ex vivo, airway basal stem cells derived from cystic fibrosis patients may be used for autologous cell therapy. By using two different AAV vectors, each carrying part of the CFTR sequence, Vaidyanathan and colleagues at the Porteus lab have been able to bypass the payload size limitations of AAVs (Vaidyanathan et al. 2021). This approach also allowed the team to include sequences corresponding to homology arms to enable homologous recombination events and a specific tag sequence to enrich gene-edited airway basal stem cells.

Following this strategy, Vaidyanathan et al. successfully inserted the full CFTR sequence, modifying airway basal stem and human bronchial epithelial cells derived from eleven cystic fibrosis patients. Correction of CFTR expression was confirmed by immunoblotting, and functional recovery was demonstrated by improved transepithelial ion transport in differentiated epithelial sheets derived from edited cells (Li et al. 2004). Furthermore, recovery of CFTR chloride transport was as high as 150% compared to non-disease control cells.

Therefore, the Poarteus lab has developed a CRISPR/Cas9-AAV method that supports the correction of all CFTR mutations and thus may provide a solution to a broader patient population through autologous airway stem cell therapy. Nevertheless, the team acknowledges the significant challenges ahead for developing methods to implant CFTR corrected stem cells into airway epithelia, a process that they are prepared to initiate by using biomaterial vessels in animal models of cystic fibrosis sinus disease.

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Berdiev, B. K., Qadri, Y. J. & Benos, D. J. Assessment of the CFTR and ENaC association. Mol. Biosyst. (2009) doi:10.1039/b810471a.

De Boeck, K. Cystic fibrosis in the year 2020: A disease with a new face. Acta Paediatrica, International Journal of Paediatrics (2020) doi:10.1111/apa.15155.

Hwang, T. C. et al. Structural mechanisms of CFTR function and dysfunction. Journal of General Physiology (2018) doi:10.1085/jgp.201711946.

Li, H., Sheppard, D. N. & Hug, M. J. Transepithelial electrical measurements with the Ussing chamber. J. Cyst. Fibros. (2004) doi:10.1016/j.jcf.2004.05.026.

Patel, S. D., Bono, T. R., Rowe, S. M. & Solomon, G. M. CFTR targeted therapies: Recent advances in cystic fibrosis and possibilities in other diseases of the airways. Eur. Respir. Rev. (2020) doi:10.1183/16000617.0068-2019.

Vaidyanathan, S. et al. High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia. Cell Stem Cell (2020) doi:10.1016/j.stem.2019.11.002.