<scp>O</scp>rkambi® and amplifier co‐therapy improves function from a rare <i><scp>CFTR</scp></i> mutation in gene‐edited cells and patient tissue

Molinski, Steven; Ahmadi, Saumel; Ip, Wan; Ouyang, Hong; Villella, Adriana; Miller, John P.; Lee, Po‐Shun; Kulleperuma, Kethika; Du, Kai; Paola, Michelle Di; Eckford, Paul D. W.; Laselva, Onofrio; Huan, Ling Jun; Wellhauser, Leigh; Li, Ellen; Ray, Peter N.; Pomès, Régis; Moraes, Theo J.; Gonska, Tanja; Ratjen, Félix; Bear, Christine E.

doi:10.15252/emmm.201607137

Cited by 94 publications

(78 citation statements)

References 71 publications

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“…Safety and tolerability data is typically a primary goal of early phase clinical trials, and will hopefully provide the necessary data to advance this strategy that has the potential to impact modulator therapies across a variety of CF populations. One published report supports the hypothesis of amplifier bioactivity in cells derived from a CF patient with a rare disease‐causing CFTR variant …”

Section: Additional Modulator Strategiesmentioning

confidence: 73%

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Rapid therapeutic advances in CFTR modulator science

Clancy

2018

Pediatric Pulmonology

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Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by variants in the gene encoding the cystic fibrosis transmembrane conduction regulator (CFTR) protein. Loss of CFTR function disrupts chloride, bicarbonate and regulation of sodium transport, producing a cascade of mucus obstruction, inflammation, pulmonary infection, and ultimately damage in numerous organs. Established CF therapies treat the downstream consequences of CFTR dysfunction and have led to steady improvements in patient survival. A class of drugs termed CFTR modulators has recently entered the CF therapeutic landscape. These drugs differ fundamentally from prior therapies in that they aim to improve the function of disease‐causing CFTR variants. This review summarizes the science behind CFTR modulators, including their targets, mechanism of action, clinical benefit, and future directions in the field. CFTR modulators have dramatically changed how CF is treated, validated CFTR as a therapeutic target, and opened the door to truly personalized therapies and treatment regimens.

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Section: Additional Modulator Strategiesmentioning

confidence: 73%

“…One published report supports the hypothesis of amplifier bioactivity in cells derived from a CF patient with a rare disease-causing CFTR variant. 89…”

Section: Additional Modulator Strategiesmentioning

confidence: 99%

Rapid therapeutic advances in CFTR modulator science

Clancy

2018

Pediatric Pulmonology

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“…23 More recently, potentiometric sensors have also been used in human airway epithelial cell cultures to test CFTR modulators against ΔF508-CFTR and other CFTR mutations. 24,25 A biochemical synergy screen of ΔF508-CFTR surface expression was developed using an engineered extracellular epitope 14 to discover second-generation corrector combinations that target distinct ΔF508-CFTR folding defects. Novel ΔF508-CFTR therapeutics with two correctors and a potentiator are in clinical development.…”

Section: Discussionmentioning

confidence: 99%

ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

et al. 2018

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The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (ΔF508). The ΔF508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ΔF508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ΔF508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ΔF508-CFTR modulators that target the NBD1 domain.

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“…hCFTR was detected with hCFTR‐specific murine mAb 596 (1:5000) or rabbit Ab Anti‐GFP (1:5000). The blots were developed with ECL (Amersham) on the Li‐Cor Odyssey Fc (LI‐COR Biosciences) in a linear range of exposure …”

Section: Methodsmentioning

confidence: 99%

“…The blots were developed with ECL (Amersham) on the Li-Cor Odyssey Fc (LI-COR Biosciences) in a linear range of exposure. [44][45][46][47] To evaluate protein glycosylation status, HEK293 cells were transiently transfected with WT-zCftr-GFP or F507del-zCftr-GFP. The cells were grown at 37°C for 24 hours and subsequently lysed in modified RIPA buffer as described above.…”

Section: Immunoblottingmentioning

confidence: 99%

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis‐causing mutation

Laselva

Erwood

et al. 2019

FASEB BioAdvances

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F508del‐cystic fibrosis transmembrane conductance regulator (CFTR) is the major mutant responsible for cystic fibrosis (CF). ORKAMBI®, approved for patients bearing this mutant, contains lumacaftor (VX‐809) that partially corrects F508del‐CFTR's processing defect and ivacaftor (VX‐770) that potentiates its defective channel activity. Unfortunately, the clinical efficacy of ORKAMBI® is modest, highlighting the need to understand how the small molecules work so that superior compounds can be developed. Because, human CFTR (hCFTR) and zebrafish Cftr (zCftr) are structurally conserved as determined in recent cryo‐EM structural models, we hypothesized that the consequences of the major mutation and small molecule modulators would be similar for the two species of protein. As expected, like the F508del mutation in hCFTR, the homologous mutation in zCftr (F507del) is misprocessed, yet not as severely as the human mutant and this defect was restored by low‐temperature (27°C) culture conditions. After rescue to the cell surface, F507del‐zCftr exhibited regulated channel activity that was potentiated by ivacaftor. Surprisingly, lumacaftor failed to rescue misprocessing of the F507del‐zCftr at either 37 or 27°C suggesting that future comparative studies with F508del‐hCFTR would provide insight into its structure: function relationships. Interestingly, the robust rescue of F508del‐zCftr at 27°C and availability of methods for in vivo screening in zebrafish present the opportunity to define the cellular pathways underlying rescue.

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Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue

Cited by 94 publications

References 71 publications

Rapid therapeutic advances in CFTR modulator science

Rapid therapeutic advances in CFTR modulator science

ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis‐causing mutation

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