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A2848 - Investigation of “Read-Through” of Nonsense Mutations as a Treatment for Primary Ciliary Dyskinesia
Author Block: L. E. Ostrowski1, A. J. Smith2, N. Fregien3; 1Univ of N Carolina, Chapel Hill, NC, United States, 2Cell Biology and Physiology, Univ of N Carolina, Chapel Hill, NC, United States, 3Cell Biology, University of Miami Miller School of Medicine, Miami, FL, United States.
RATIONALE Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disease caused by mutations in genes that are required for the function of motile cilia. Individuals with PCD have impaired mucociliary clearance, and suffer from frequent infections in the oto-sino-pulmonary tract. There are no specific therapies for PCD. Many cases of PCD are caused by nonsense mutations that introduce premature termination codons (PTCs), including a c.2014C>T [p.Gln672*] mutation in the gene SPAG1. “Read-through” of nonsense mutations (insertion of an amino acid at the stop codon) can result in the production of a full-length protein with full or partial function. The goal of this work is to identify agents that promote read-through in ciliated cells and mitigate the effects of nonsense mutations.
METHODS We have designed a lentivirus based reporter system that will allow testing of agents for their ability to increase read-through specifically in the differentiated ciliated cells that are the target for treatment of PCD. The vector expresses two different reporter genes (Renilla and firefly luciferase) under the control of the ciliated cell-specific promoter, FOXJ1. The luciferase genes are linked by a segment of the SPAG1 gene containing either the WT sequence or the c.2014C>T mutation. In differentiated cells transduced with the mutant construct, only Renilla luciferase will be expressed since translation will be terminated at the c.2014C>T mutation; in cells treated with a read-through promoting agent, translation will continue and both luciferases will be expressed. This system will allow rapid screening of agents for their ability to promote read-through. The lentiviral vectors were constructed and verified by sequencing. Viral particles were produced and used to transduce undifferentiated human airway epithelial cells. Cells were plated on collagen coated Millicell filters and allowed to differentiate at the air/liquid interface. At different times, cells were harvested for luciferase assays.
RESULTS The expression of Renilla luciferase was low in the undifferentiated cells and increased in the differentiated cells transduced with either vector. The expression of firefly luciferase also increased ~60-80 fold in cells transduced with the vector containing the control SPAG1 sequence, while firefly luciferase was undetectable in cells transduced with the vector containing the c.2014C>T mutation.
CONCLUSIONS The results to date demonstrate both the specificity of the FOXJ1 promoter and the functionality of the dual luciferase vector. Future work will use the assay to identify compounds with significant ability to correct stop mutations in ciliated cells.