Exon-skipping antisense oligonucleotides for cystic fibrosis therapy.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

LPLRFamide causes anorexigenic effects in broiler chicks and Bobwhite quail.

Although LPLRFamide was the first member of the RFamide family to be isolated from a vertebrate species, its effects on hunger and satiety-related processes are poorly documented. Thus, we intracerebroventricularly administered LPLRFamide (3.0-15.0 nmol) to both Cobb-500 (a broiler type of Gallus gallus) and Bobwhite quail (Colinus virginianus) chicks and measured their food intake. The threshold of anorexigenic response was 7.0 nmol in Cobb-500 chicks and the effect had diminished by 30 min post-injection. In Bobwhite quail all doses of LPLRFamide tested caused anorexia that remained throughout the 60 min observation period. A comprehensive behavior analysis was conducted and Cobb-500 chicks had increased food pecks early in the observation period and spent a greater amount of time in deep rest. Although food pecks were increased pecking efficiency was decreased. In Bobwhite quail, feeding pecks and the number of jumps were reduced after LPLRFamide treatment. We judged that these behaviors in both species were likely not competitive with ingestion and thus did not secondarily contribute to anorexia. These results demonstrate that LPLRFamide is associated with satiety-related processes in Cobb-500 chicks and Bobwhite quail, while threshold of responses are different.

Central calcitonin exerts anorectic effects via the hypothalamus in chicks.

Calcitonin (CT) causes satiety in mammals, but the mechanisms that mediate this effect are poorly understood. Additionally, there are no reports on CT-induced satiety within the avian class. Therefore, the purpose of this study was to elucidate some of the central mechanisms regulating CT-induced satiety in a non-mammalian vertebrate, the chick. Broiler-type chicks, at 4 days of age, responded to central CT (0.3, 1.0 and 3.0 nmol) with both reduced food and water intake. The effect on water intake was secondary to that of food. An increased number of c-Fos immunoreactive cells were found in hypothalamic nuclei associated with satiety including the arcuate nucleus, dorsomedial nucleus and ventromedial hypothalamus after central CT injection. Increased jumps, distance traveled and time spent perching on food containers were also observed, and these behaviors are likely not competitive with ingestion. Also, central CT injection was associated with reduced food pecks, but increased pecking efficiency. Blockage of corticotrophin releasing factor receptors did not prevent central CT-induced satiety. Central CT appears to be a regulator of satiety in chicks and this effect is likely mediated via interactions within the hypothalamus.