Advancing lentiviral gene therapy for cystic fibrosis.

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Project Summary Cystic fibrosis (CF) is a common, life-shortening genetic disease. Mutations in the cystic fibrosis conductance regulator (CFTR) gene lead to defective or absent CFTR that is unable to effectively transport anions in many tissues, but pulmonary complications are the most common cause of death. Recent advances in CF treatment include drugs able to rescue protein function. However, these treatments are mutation-class specific and are ineffective for some mutations. Thus, there is a persistent need to develop mutation agnostic treatments that can benefit all people with CF, such as gene addition. Lentiviral vectors are well suited for CF gene therapy because they have sufficient carrying capacity, and their ability to integrate into the host’s genome ensures long-term benefits. Currently available vectors, however, produce low vector titers or inefficiently transduce airway epithelial cells from the apical surface. Despite these limitations, treatment of newborn CF pigs with a lentiviral vector carrying a functional copy of CFTR partially rescued CF phenotypes, suggesting that achieving therapeutic CFTR expression is within reach. My long-term goal is to design a lentiviral vector suitable for the effective treatment of pulmonary CF in humans. The overall objective of his proposal is to evaluate two independent but complementary strategies, to improve lentiviral delivery of CFTR to human airway epithelial cells. Lentiviral vectors can accommodate envelopes from other viruses, termed pseudotyping, which can lead them to preferentially transduce specific cell types. For the first strategy, I screened seven viral envelopes and identified two derived from baboon endogenous retrovirus (BaEV) that produce high vector titers and efficiently transduce primary human airway epithelial cells from the apical surface. The second strategy is to increase CFTR expression in transduced cells. Because airway epithelial cells are electrochemically coupled through gap junctions, a few cells expressing high levels of CFTR may perform sufficient CFTR-mediated anion transport to correct CF phenotypes. Increased gene expression can be achieved through codon optimization. I compared three codon optimized CFTR (coCFTR) sequences and identified one that significantly increases CFTR-mediated transepithelial Cl- transport. My central hypothesis is that increasing transduction efficiency and transgene expression will be sufficient to achieve wild type levels of CFTR-mediated anion transport. To test my hypothesis, I propose the following Specific Aims: 1) Determine if BaEV pseudotyped lentiviral vectors can efficiently deliver CFTR to primary human airway epithelial cells, and 2) Determine if coCFTR delivery reduces the proportion of complemented cells needed to achieve wild type levels of CFTR-mediated anion transport. These strategies will be tested in vitro using human CF donor-derived airway epithelia, and in vivo using the CF pig model. These experiments will provide valuable preclinical data that will guide vector design and inform future clinical trials using lentiviral vectors to treat CF.