Patterns of single nucleotide polymorphisms (SNPs) in eukaryotic DNA are traditionally attributed to selective pressure, drift, identity descent, or related factors-without accounting for ways in ...which bias during de novo SNP formation, itself, might contribute. A functional and phenotypic analysis based on evolutionary resilience of DNA points to decreased numbers of non-synonymous SNPs in human and other genomes, with a predominant component of SNP depletion in the human gene pool caused by robust preferences during de novo SNP formation (rather than selective constraint). Ramifications of these findings are broad, belie a number of concepts regarding human evolution, and point to a novel interpretation of evolving DNA across diverse species.
Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon ...(PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.
Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new ...therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl-) ...secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.
Primary murine nasal septal epithelial (MNSE) wild type (WT) and transgenic CFTR(-/-), human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium Ca(2+)i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.
Sinupret-mediated Cl- secretion ΔISC(µA/cm(2)) was pronounced in WT MNSE (20.7+/-0.9 vs. 5.6+/-0.9(control), p<0.05), CFTR(-/-) MNSE (10.1+/-1.0 vs. 0.9+/-0.3(control), p<0.05) and HSNE (20.7+/-0.3 vs. 6.4+/-0.9(control), p<0.05). The formulation activated Ca(2+) signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl- secretion. Sinupret also enhanced CBF and ASL depth.
Sinupret stimulates CBF, promotes transepithelial Cl- secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca(2+)-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl- secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.
The mechanisms underlying cystic fibrosis (CF) lung disease pathogenesis are unknown.
To establish mechanisms linking anion transport with the functional microanatomy, we evaluated normal and CF ...piglet trachea as well as adult swine trachea in the presence of selective anion inhibitors.
We investigated airway functional microanatomy using microoptical coherence tomography, a new imaging modality that concurrently quantifies multiple functional parameters of airway epithelium in a colocalized fashion.
Tracheal explants from wild-type swine demonstrated a direct link between periciliary liquid (PCL) hydration and mucociliary transport (MCT) rates, a relationship frequently invoked but never experimentally confirmed. However, in CF airways this relationship was completely disrupted, with greater PCL depths associated with slowest transport rates. This disrupted relationship was recapitulated by selectively inhibiting bicarbonate transport in vitro and ex vivo. CF mucus exhibited increased viscosity in situ due to the absence of bicarbonate transport, explaining defective MCT that occurs even in the presence of adequate PCL hydration.
An inherent defect in CF airway surface liquid contributes to delayed MCT beyond that caused by airway dehydration alone and identifies a fundamental mechanism underlying the pathogenesis of CF lung disease in the absence of antecedent infection or inflammation.
Patients with cystic fibrosis (CF) exhibit pronounced respiratory damage and were initially considered among those at highest risk for serious harm from SARS‐CoV‐2 infection. Numerous clinical ...studies have subsequently reported that individuals with CF in North America and Europe—while susceptible to severe COVID‐19—are often spared from the highest levels of virus‐associated mortality. To understand features that might influence COVID‐19 among patients with cystic fibrosis, we studied relationships between SARS‐CoV‐2 and the gene responsible for CF (i.e., the cystic fibrosis transmembrane conductance regulator, CFTR). In contrast to previous reports, we found no association between CFTR carrier status (mutation heterozygosity) and more severe COVID‐19 clinical outcomes. We did observe an unexpected trend toward higher mortality among control individuals compared with silent carriers of the common F508del CFTR variant—a finding that will require further study. We next performed experiments to test the influence of homozygous CFTR deficiency on viral propagation and showed that SARS‐CoV‐2 production in primary airway cells was not altered by the absence of functional CFTR using two independent protocols. On the contrary, experiments performed in vitro strongly indicated that virus proliferation depended on features of the mucosal fluid layer known to be disrupted by absent CFTR in patients with CF, including both low pH and increased viscosity. These results point to the acidic, viscous, and mucus‐obstructed airways in patients with cystic fibrosis as unfavorable for the establishment of coronaviral infection. Our findings provide new and important information concerning relationships between the CF clinical phenotype and severity of COVID‐19.
Several studies have suggested a mechanistic relationship between CFTR and COVID‐19.
In contrast to previous reports, we found no association between CF carrier status (mutational heterozygosity) and clinical severity of SARS‐CoV‐2 infection among nearly 3000 patients with COVID‐19 and ~6000 controls.
SARS‐CoV‐2 production in primary airway cells was not altered by homozygous absence of functional CFTR.
Our results point to acidic, viscous, and mucus‐obstructed airways in patients with cystic fibrosis as unfavorable for the propagation of the coronavirus.
Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent ...posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis.
The treatment of patients with advanced-stage solid tumours typically involves a multimodality approach (including surgery, chemotherapy, radiotherapy, targeted therapy and/or immunotherapy), which ...is often ultimately ineffective. Nucleic acid-based drugs, either as monotherapies or in combination with standard-of-care therapies, are rapidly emerging as novel treatments capable of generating responses in otherwise refractory tumours. These therapies include those using viral vectors (also referred to as gene therapies), several of which have now been approved by regulatory agencies, and nanoparticles containing mRNAs and a range of other nucleotides. In this Review, we describe the development and clinical activity of viral and non-viral nucleic acid-based treatments, including their mechanisms of action, tolerability and available efficacy data from patients with solid tumours. We also describe the effects of the tumour microenvironment on drug delivery for both systemically administered and locally administered agents. Finally, we discuss important trends resulting from ongoing clinical trials and preclinical testing, and manufacturing and/or stability considerations that are expected to underpin the next generation of nucleic acid agents for patients with solid tumours.
Making precision medicine personal for cystic fibrosis Manfredi, Candela; Tindall, Janice M; Hong, Jeong S ...
Science (American Association for the Advancement of Science),
07/2019, Letnik:
365, Številka:
6450
Journal Article
Recenzirano
Odprti dostop
Molecular defects in the cystic fibrosis gene prompt creative approaches to treatment
Cystic fibrosis (CF) is an inherited, life-threatening disease that primarily involves exocrine tissues (such as ...lungs, pancreas, and liver) for which highly active pharmacotherapies have recently emerged. More than 1700 disease-associated variants are described in the CF transmembrane conductance regulator (
CFTR
) gene, which encodes an epithelial cell ion channel that is defective in patients with CF. On the basis of classifying CFTR mutant proteins according to pathogenic mechanisms, the disease has been viewed as a model for personalized therapeutics. However,
CFTR
variants may have pleiotropic effects, which complicates assignment of specifically tailored drugs to discrete mechanistic subcategories. In addition, the cost of new CFTR modulators constrains third-party reimbursement and has delayed drug availability for certain patient groups, including individuals with ultrarare
CFTR
variants for which the treatments are not formally approved but may still be effective. Issues such as these are being addressed by innovative and powerful approaches to promote CF precision medicine.
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