Allele-Specific Treatments forCystic FibrosisOctober 28thBen SpinkJohn S. Van Arnam(Ribosome) (Turmeric Root)Cystic Fibrosis:-Cystic Fibrosis (CF): autosomal recessive, 1 in 3000Caucasian births-Genetic defect in CFTR: Cystic Fibrosis transmembraneconductance regulator-Integral membrane glycoprotein-cAMP-dependent ABC (PK-A) Cl- ion transporter-ΔF508 most common mutation, but >1000 other mutationsrepresent ~2% of the total mutationsCystic Fibrosis:Pathogenesis:-Tissue-specific abnormalities in iontransport caused faulty production,processing, transport, or conductanceof CFTR-In the lung, yields increasedtransepithelial potential difference(increase in Na+ transport, decreasein Cl-)-Reduced salt likely increases mucusviscosity, stasis and low O2 contrbiuteto bacterial infection-Parallel mechanism occurs in GI lumenN Engl J Med. 2005 May 12;352(19):1992-2001.Cystic Fibrosis:Clinical FeaturesLung:-Upper respiratory tract: sinusitis/rhinorrhea, polyps-Lower respiratory tract: Excessive mucus production,airway blockage, low O2-Microbes in sputum: H. influenza, S. aureus, P.Aeruginosa, Aspergillus (biofilms)-Infection/Inflammation-End-stage events: cor pulmonale, respiratory failureCystic Fibrosis:Pathogenesis/Clinical Features outside the lung:-meconium ileus in infants-pancreatic insufficiency, often prompts furtherdiagnostic tests-genitourinary defects (inadequate nutrition/O2affectsfertility, blockage of vas deferens)CFTR in mice:Murine Pathogenesis/Clinical Features:-CFTR ΔF508: No lung pathology!-However, CFTR mice have extensive GI problems, cellularlocalization and potentiometric analysis still possible-CCSP-driven ENaC overexpression leads to CF-like lungpathologyNature Medicine 10, 452 – 454 (News and Views), 487-493 (2004)CF Therapies-Many treatments are mechanical (chest percussion,breathing exercises/apparatus, saline wash)-N-acetylcysteine for mucus clearance, _-adrenergics forairway constriction, anti-inflammatories-Antibiotics for infection-GI difficulties: lipase, supplemental vitamin E/KCystic Fibrosis: Gene Therapy-Early clinical trials demonstrated proof of principle, butwere insufficient for clinical benefit-Used cationic liposomes or adenovirus-More recent trials have used non-viral vectors w/ better success-Barriers to Gene Therapy Success:-Mucus, (literal barrier)-glycocalyx-basolateral location of receptors-These are lung-specific problems: mouse modelconsequencesPotentiometric methods of analysis-Electrical studies first used in CF research to confirmplasma membrane impermeability to Cl-, now a powerfuldiagnostic/research toolHow it works:-Differences in [salts] = voltage-Voltmeter connected to two electrodes-One electrode placed against target epithelium-Another (reference electrode) placed subcutaneously-Potential is negative to varying degreesPotentiometric methods of analysis-Probing electrode perfused with various solutions thataffect membrane potentialSaline: Baseline (a negative potential)Amiloride: Blocks Na+ absorption, reduces potentialdifferenceCl- free solution: encourages Cl- secretion, in non-CFepithelia results in hyperpolarization, this is likely Cl-secretion due to prior treatment with amilorideIsoproternol: Augments Cl- secretion (↑cAMP), even morepolarizationForskolin: Also increases cAMP, and is a bronchodilatorPotentiometric methods of analysisSaline: Baseline (anegative potential)Amiloride: Blocks Na+absorbtion, reducespotential differenceCl- free solution:encourages Cl-secretion, in non-CFepithelia results inhyperpolarization,this is likely Cl-secretion due toprior treatment withamilorideIsoproternol: AugmentsCl- secretion(↑ cAMP), evenmore polarizationForskolin: Alsoincreases cAMP,and is abronchodilatorAn example with NPD from Egan et alGrey filled square: WT Grey filled circle: Untreated ΔF508Red filled circles: 45mg/kg curcumin Orange filled circles: 15mg/kg curcuminEndoplasmic Reticulum Quality Control:-Protein biogenesis requires proper folding & modifications-Misfolded proteins are recognized by calreticulin/calnexin/others, sent back to be refolded (ERQC), or are sent to bedegraded (ERAD)-Ca2+-dependent ER chaperones are known to be responsiblefor CFTR folding and appropriate membrane translocation-SERCA (Sarcoplasmic/Endoplasmic Reticulum Ca2+)-Curcumin is a safe, relatively mild SERCA pump inhibitor-High affinity pump inhibitors (thapsigargin) are lethal forsome cellsEgan et al-Hypothesis: Could the disruption of Ca2+ levels affectCFTR degradation/processing and allow for increasedmembrane translocation and a therapeutic effect?-Egan et al use CFTR ΔF508 mice (transporter isfunctional)-Previous data suggest curcumin has therapeutic potentialdue to decrease in membrane potential-Will there be atherapeutic effect? Is the mechanism of action known? Isknowledge of the mechanism important?Egan et al• Saline: Baseline (anegative potential)• Amiloride: BlocksNa+ absorbtion,reduces potentialdifference• Cl- free solution:encourages Cl-secretion, in non-CFepithelia results inhyperpolarization,this is likely Cl-secretion due toprior treatment withamiloride• Isoproternol:Augments Cl-secretion (_cAMP),even morepolarization• Forskolin: Alsoincreases cAMP,and is abronchodilatorEgan et al• Saline: Baseline (anegative potential)• Amiloride: BlocksNa+ absorbtion,reduces potentialdifference• Cl- free solution:encourages Cl-secretion, in non-CFepithelia results inhyperpolarization,this is likely Cl-secretion due toprior treatment withamiloride• Isoproternol:Augments Cl-secretion (_cAMP),even morepolarizationEgan et al• Saline: Baseline (anegative potential)• Amiloride: Blocks Na+absorbtion, reducespotential difference• Cl- free solution:encourages Cl-secretion, in non-CFepithelia results inhyperpolarization, thisis likely Cl- secretiondue to prior treatmentwith amiloride• Isoproternol:Augments Cl-secretion (_cAMP),even more polarization• Forskolin: Alsoincreases cAMP, andis a bronchodilatorBHK=Baby HamsterKidneyER-retained CFTR ΔF508is core glycosylatedvs.WT CFTR has complexglycosylationGlycosylation is initiated in the endoplasmic reticulum (ER) bythe transfer of a core glycan, comprising two N-acetylglucosamine, nine mannose and three terminal glucoseresidues, while the polypeptide is still associated with thetranslocon. After initial folding events, the terminal glucoseresidues and one mannose residue are removed to generatea homogenous glycosylation pattern shared by