CF is the most prevalent lethal genetic disease in Caucasians caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We are studying two molecular genetic mechanisms which act as modifiers on disease severity and response to therapy: the splicing machinery and the homeostatic mechanisms nonsense mediated mRNA decay (NMD) pathway and the unfolded protein response (UPR).

I. Splicing Modulation

A significant fraction of CF-causing mutations (10-15%) affects pre-mRNA splicing (class V mutations). These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between the level of correct transcripts and disease severity, suggesting a role for splicing regulation as a genetic modifier. We are now developing a targeted modulation approach aimed to correct the splicing pattern of CFTR transcripts, according to the specific mutation carried. Increasing the level of normal CFTR mRNA, transcribed from CFTR allele carrying splicing mutations can lead to activation of the CFTR channel and restoration of its function. The approach is based on antisense oligonucleotides (AOs) administration, which is one of the most new and promising treatments for genetic disorders caused by splicing mutations. AOs are short synthetic RNA-like molecules chemically modified, which can specifically anneal to motifs predicted to be involved in the pre-mRNA splicing. Their binding to selected sites mask the targeted region and promote normal splicing. We hypothesize that this strategy will interfere with the aberrant CFTR splicing in patients carrying splicing variants/mutations leading to restoration of the CFTR function.

II. Read-through modulation by nonsense mediated mRNA decay

A fraction of 10-15% of CF-causing mutations are nonsense mutations which lead to premature termination codon (PTC) (class I mutations). Transcripts carrying PTCs are known to undergo degradation by the NMD pathway.  Aminoglycosides (e.g. gentamicin antibiotics) and other small molecules as ataluren can read-through PTCs, permitting translation of full-length proteins and can be used as a treatment for patients carrying this type of mutations. We have found variable clinical response to read-through modulation in CF patients, carrying the W1282X nonsense mutation, due to variable efficiencies of the NMD pathway. A good response was found only in patients with high levels of CFTR nonsense transcripts, reflecting less efficient NMD.  We further showed that down-regulation of NMD increased the level of CFTR nonsense transcripts and led to an enhanced CFTR chloride channel activity in response to gentamicin. We are currently investigating another major regulatory feedback loop involving the NMD pathway and the UPR. We have found that different levels of UPR activation can affect NMD efficiencies and can influence the response of patients carrying PTCs to read-through modulation. 

Our research has profound impact on the understanding of disease severity in CF patients and on potential therapeutic approaches.