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Dissertation
Advances in lentiviral gene therapy for cystic fibrosis
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2023
DOI: 10.25820/etd.007885
Abstract
Despite significant advances in cystic fibrosis (CF) treatments, a one-time treatment for this life-shortening disease remains elusive. Stable complementation of the disease-causing mutation with a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR gene could fulfill that goal. Lentiviral vectors are attractive for this purpose because they can accommodate large genes like CFTR. They transduce non-dividing cells, and they integrate into the host’s genome, leading to long-term expression. Previous proof of principle studies demonstrated that lentiviral delivery of CFTR to the airways of CF pigs partially rescues phenotypic defects. The objective of this work was to advance lentiviral gene therapy to achieve full correction of CF phenotypes in the airways by two distinct but complementary strategies.
For the first strategy, we investigated promoter choice and CFTR codon optimization to increase CFTR expression. We evaluated two promoters, phosphoglycerate kinase (PGK) and elongation factor 1-α (EF1α), that have been proven safe in clinical trials. We also compared the wildtype human CFTR sequence to three alternative codon optimized (coCFTR) sequences generated by different algorithms. Using CFTR-mediated anion current in primary human CF airway epithelia to quantify channel expression and function, we determined that EF1α produced greater currents than PGK, and identified a coCFTR sequence that conferred significantly increased CFTR-mediated anion currents.
The second approach we investigated was increasing the transduction efficiency through improved pseudotyping. We studied four different viral envelope glycoproteins and determined their cell tropism for airway cells, including ciliated, secretory, and basal cell types. Of these, a modified baboon endogenous retrovirus (BaEVRless), and the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-CoV-2-S) were identified as promising candidates. Both produced greater transgene expression than baculovirus glycoprotein 64 (GP64) after apical transduction in primary human airway epithelia. BaEVRless displayed broad cell tropism, while SARS-CoV-2-S transduced ciliated and secretory cells, but basal cell transduction was rare.
Future studies will focus on evaluating the combination of the most promising coCFTR expression cassettes and pseudotypes. Validation in CF animal models will also be required. We are encouraged by the improvements in vector design presented here, as they advance lentiviral vectors towards CF gene therapy clinical trials.
Details
- Title: Subtitle
- Advances in lentiviral gene therapy for cystic fibrosis
- Creators
- Laura I Marquez Loza
- Contributors
- Paul B McCray (Advisor)John F Engelhardt (Committee Member)Julia Klesney-Tait (Committee Member)Aloysius J Klingelhutz (Committee Member)Mark A Stamnes (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Molecular Medicine)
- Date degree season
- Spring 2023
- DOI
- 10.25820/etd.007885
- Publisher
- University of Iowa
- Number of pages
- xiii, 97 pages
- Copyright
- Copyright 2023 Laura Isabel Marquez Loza
- Language
- English
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 76-97).
- Public Abstract (ETD)
- Cystic fibrosis (CF) is a common, life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a channel or pore that allows anion movement. Although many organs are affected in CF, lung disease causes the most debilitating symptoms, and often requires a lung transplant. Currently, there is no treatment that is effective for all CFTR disease-causing mutations. Delivery of a working copy of the mutated gene to airway cells in people with CF could treat all mutations. Lentiviral vectors can be used to deliver genes to human cells, and because they insert into the host genome, can provide long-term gene expression. This strategy was tested in a CF pig model with promising results, but to make this an effective treatment in people, delivery and expression need to be further optimized. We tested two strategies to accomplish this: 1) Increase the amount of CFTR channels that are produced by a single copy of the delivered gene, and 2) Deliver the gene to more cells. We identified a modified CFTR sequence that, without changing the final protein, increases the abundance of protein in human airway cells. We also identified two alternative viral envelope glycoproteins that can coat lentiviral vectors, and allow the vector to enter more airway cells to deliver the working CFTR gene. We are encouraged by these results, as they represent significant advances towards developing a long-lasting treatment for all people with CF.
- Academic Unit
- Biomedical Science Program
- Record Identifier
- 9984831231802771
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