Ivacaftor and CFTR Splice site mutations

little note (09-18): Trial for residual cftr function mutations, including some splice site mutations, has finally started; click here: Clinicaltrials.gov

First of all, the intent of this page is just to give some scientific informations about what, in the world, scientists, users, doctors say about the use of Ivacaftor on other CFTR classes of mutation. Obviously we will focus on splicing mutations.
Ivacaftor is the first drug that treats the basic defect in Cystic fibrosis, potentiating the mutated (and also the wild type/normal) protein already on the cell surface. Many mutations in fact produce truncated proteins (Class I mutations) or misfolded proteins (Class II mutations like DF508 or N1303K)that actually cannot reach the cell surface, and need another molecule (a corrector) to rescue the CFTR protein and let it positionate in the right place.
Some splicing mutations (621+1G->T, 1717-1G->A,1078delT, 711+1G->T, 1525-1G->A, 2751+2T->A, 296+1G->C, 1717-9T->C from (C. Castellani et al. / Journal of Cystic Fibrosis 7 (2008) 179–196) , even if categorized in the severe Class I, may be able to produce some aberrant/incorrect CFTR. This difference distinguishes those defects from the stop mutations that are also in Class I.
Class III mutations are the actual target of Ivacaftor because it is certain, after many clinical trials, its activity among these "gating" mutations such as G551D. Another 9 gating mutations are now under clinical trial tests(ClinicalTrials.gov).
Class IV mutations have a defect in conductance. Some of those have a gating and a conductance defect together. R117H mutation, has an ongoing clinical trial and Ivacaftor's activity on this mutation is almost certain (ClinicalTrials.gov), for some others (in the same class IV) this activity is not so obvious. More trials will be needed to investigate it. A number between 20 to 30 mutations in this Class resulted in a meaningfull improvement in Chloride Efflux during laboratory tests on Fischer Rat Thyroid cells, while other class IV mutations resulted in few increase in vitro.
Class V mutations (3849+10kbC->T, 2789+5G->A, A455E, 3272-26A->G,IVS8-T5, 4006-1G->A, 621+3A->G, 711+3A->G from C. Castellani et al. / Journal of Cystic Fibrosis 7 (2008) 179–196) are the next target that should be investigated for Ivacaftor. This is why:

Not everyone knows that in the "famous" phase II trial to test vx-770 (ivacaftor) efficacy, clinical investigators' plan was to test it on 2 groups of patients: patients with G551D and patients with G551D, R117H (conductance) or 2789+5G->A (Splicing)
[...]Inclusion Criteria:
- Confirmed diagnosis of Cystic Fibrosis
- Part 1: G551D mutation in at least 1 allele (any known or unknown mutations allowed in second allele with the exception of R117H or 2789+5G-->A mutation).
- Part 2: must have the G551D, R117H, or 2789+5G-->A mutation in at least 1 allele (any known or unknown mutations allowed in the second allele)[...].
After about one year from the starting date, investigators (maybe because of burocracy or whatever) decided to change their mind and modify the trial settings

taken from:Clinicaltrials.gov
These are the final clinical trial settings:
[...]Inclusion Criteria:
Confirmed diagnosis of Cystic Fibrosis
G551D mutation in at least 1 allele (any known or unknown mutations allowed in second allele with the exception of R117H or 2789+5G-->A mutation)[...]
From: Clinicaltrials.gov

Investigators also stated in several clinical trial description that:
"VX-770 is a potent and selective potentiator of wild-type, G551D, F508del, and R117H forms of human CFTR. Based on in vitro studies and pharmacologic, pharmacokinetic (PK), and safety profiles, VX-770 has been selected for clinical development as a possible treatment for patients with CF"
i.e. Phase III trial on G551D from clinicaltrials.gov

In the scientific perspective, we can find several interesting publications about Ivacaftor and its activity on wild type.
The most important ones are from the scientist that for first understood vx-770's importance. Van Goor.
In "Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770" (Fredrick Van Goor, Sabine Hadida, Peter D. J. Grootenhuis, Bill Burton, Dong Cao, Tim Neuberger,Amanda Turnbull, Ashvani Singh, John Joubran, Anna Hazlewood, Jinglan Zhou, Jason McCartney,Vijayalaksmi Arumugam, Caroline Decker, Jennifer Yang, Chris Young, Eric R. Olson, Jeffery J. Wine ,Raymond A. Frizzell, Melissa Ashlock, and Paul Negulescu)
Proc. Natl Acad. Sci. USA 106, 18825–18830 (2009)

They stated that "VX-770 also increased the Po of F508del- and wild-type CFTR by 5-fold and 2-fold, respectively" where Po is the open probability of the CFTR Channel

In this other work

Pharmacological Rescue of Mutant CFTR Function for the Treatment of Cystic Fibrosis
Fredrick Van Goor • Sabine Hadida • Peter Grootenhuis
Top Med Chem (2008) 3: 91–120

suggested that the potential targets for the monotherapy of this molecule could be wider than just class III and potentially expand to class IV, and V "[...]Other mutant classes that results in low to moderate levels of CFTR in the apical membrane[...]"

This idea was further discussed even in the latter 2 NACF conferences; i.e. at the NACF 2010 conference poster 280 reported:
"In cultured CF HBE, VX-770 potentiated 10 μM forskolin-stimulated chloride transport in HBE isolated from CF bronchi carrying the F508del mutation on both alleles or F508del on one allele and a Class III (G551D), Class IV (R117H-5T), or Class V (2789 + 5G ->A, 3272-26A ->AG) mutation on the other allele. The results of this in-vitro study support ongoing evaluation of VX-770 monotherapy in CF patients with a variety of CFTR mutant forms"

Another main scientist, Yu, investigated the effect of Vx-770 on wild type.
From: Yu H et al, Ivacaftor potentiation of multiple CFTR channels with gating mutations, J Cyst Fibros (2012), doi:10.1016/j.jcf.2011.12.005
With the following clips taken from that paper, he confirmed Van Goor's prevision of ivacaftor's effect on the open probability of wild type (2fold)

Doubling the open probability, for Yu, wouldn't translate into a 2 fold potentiation of Chloride Transport

Anyway the author suggests that "other CFTR gene mutations, associated with residual CFTR function [...] and some Splice mutations (3849+10Kbc)" should be investigated to "evaluate the ivacaftor response and potential clinical benefit for CFTR mutations beyond CFTR gating mutations"

Same conclusion for Sloane and Rowe

Cystic fibrosis transmembrane conductance regulator protein repair as a therapeutic strategy in cystic fibrosis Peter A. Sloane,d and Steven M. Rowe
Curr Opin Pulm Med 16:591–597
2010 Wolters Kluwer Health | Lippincott Williams & Wilkins

Anyway Sloane and Rowe again, in 2012 forsee a 2fold ivacaftor potentiation in CFTR activity of Wild-type (and not just 1.5 fold as in Yu's prevision)

A pharmacologic approach to acquired cystic fibrosis transmembrane conductance regulator dysfunction in smoking related lung disease. Sloane PA, Shastry S, Wilhelm A, Rowe S. et al.
PLoS One 2012; 7(6):e39809.

More over, in a FDA document submitted by Vertex with APPLICATION NUMBER: 203188Orig1s000 to the CENTER FOR DRUG EVALUATION AND RESEARCH, we find:

Many more authors are sure about the effect of Ivacaftor on splicing mutations, just to cite some:

"small amounts of CFTR may reach the cell surface in class II mutations, and this could further increase with correctors raising the hypothesis that potentiating the activity of class II mutated CFTR (or classes IV, V and VI) could lead to clinical benefits"

Thursfield RM, Davies JC. Cystic Fibrosis: therapies targeting specific gene defects. Paediatr. Respir.
Rev. (2012), http://dx.doi.org/10.1016/j.prrv.2012.04.003

"The CFTR 3849+10kb C->T point mutation in intron 19 of CFTR gene reduces mRNA splicing efficiency to ˜8% of normal.[99] Subjects with this mutation have phenotypically mild CF,NPD profiles, and interestingly can have normal sweat chloride concentrations.[99] A functionally similar mutation,2789+5kb G->A, which occurs at the splice donor site of exon 14b, reduces mRNA splicing efficiency to ˜4% of normal,similarly associated with phenotypically mild CF.[100] Hypothetically, function of these mutants may be amenable to enhancement either by use of CFTR potentiators, or by therapies aimed at increasing the expression of the mRNA or the efficiency at which mRNA splices"
from: Targeted Therapy for Cystic Fibrosis. Rubenstein et alii
Cystic Fibrosis Transmembrane Conductance Regulator Mutation-Specific Pharmacologic Strategies
Mol Diag Ther 2006; 10 (5): 293-301

Dr. Amaral from Portugal, at the recent ECRD 2012, about Splicing mutations hypothesized some therapeutic paths among which:

Published online free.

Even Dr De_Jonge from Holland showed this slides in a conference:
• Klasse 3+4+5 mutaties komen voor in 26% van alle CF patiënten in Nederland
• In buiten het lichaam gekweekte cellen is stimulatie van de kanaalactiviteit van deze klasse CFTR mutaties door Ivacaftor recent aangetoond
• Fase 2 studies van Ivacaftor bij patiënten met deze mutaties zijn in de USA en in Europa (ook in Nederland) in voorbereiding"

Same thing for the French CF association:

Or the italian one:

and http://www.fibrosicisticaricerca.it/Fibrosi-Cistica/Domande-e-risposte/Kalydeco-ivacaftor--VX-770-situazione-attuale-e--futuro

Peter Mueller from Vertex, finally, on March the 21st of 2012, had a presentation at IOM.edu about future scenarios for Ivacaftor.
This slide (31) is taken from his work and ... splicing mutations are included(for further investigation).

There are, moreover, experiences of 3 off-label users that even if without a G551D mutation, could benefit from Ivacaftor. These 3 girls have SPLICING MUTATIONS.

"February 24, 2012 FEV1...1.31
March 2, 2012 FEV1..........1.48
March 7, 2012 FEV1...........1.57
March 14, 2012 FEV1..........1.55

may 9th : I have been off of it now for three weeks and my PFTs have fallen back down :( So, I was staying in the 1.55-1.58 range (on Kalydeco) and it promptly fell to 1.42 within two weeks.."



19years old

"PFTs- From my previous appointment on February 21: FVC 34%, FEV1 22%, FEV25/75 8%, PEF 56%
PFTs- From today's appointment May 8 - FVC 47%, FEV1 32%, FEV25/75 11%, PEF 77%

There hasn't been that kind of improvement in my numbers in at least 4 years. I haven't felt this well in 4 years. 4 years. 4 years"


(Even if on april the 6th, she said her previous hospital appointment gave her these numbers:
(FVC- 39% FEV1- 28% FEV1/FVC- 75% PEF- 53% FEF2575- 13% ) )

DF508 and 1717-1G>A
21years old

"After just 28 days on Kalydeco, her sweat chloride dropped from 107 to 79 and her FEV1 increased 11% to 57%--the highest FEV1 she has seen in 3 years--with NO antibiotics"

then after a while:
"She had a repeat sweat test last week and she is back up to 99 and down to 48% FEV1" (August 8th)"

from: http://forums.cysticfibrosis.com/messageview.cfm?catid=4136&threadid=618522&enterthread=y&STARTPAGE=2


14 years old

Obviously these informations are subjective.


Maybe this paper can help us to better understand that as little as 5% function can allow people with CF, slower their CF course and maybe conduct a "normal" life.

"Five Percent of Normal Cystic Fibrosis Transmembrane Conductance Regulator mRNA Ameliorates the Severity of Pulmonary Disease in Cystic Fibrosis"
Anabela S. Ramalho*, Sebastian Beck*, Michelle Meyer, Deborah Penque, Garry R. Cutting,and Margarida D. Amaral

Final Notes:
In this Patent application number: 20110288122, there's a list of mutations on which a Ivacaftor could potentially be usefull. Some are Gating mutations, some others are conductance ones, and there are many Splicing mutations too.
Here is the list:
G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+1G->A, 405+1G->A, 406-1G->A, 4005+1G->A, 1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+1G->T, 3850-1G->A, 2789+5G->A, 3849+10 kbC->T, 3272-26A->G, 711+5G->A, 3120G->A, 1811+1.6 kbA->G, 711+3A->G, 1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T, 4005+2T->C and 621+3A->G.

taken from: http://www.faqs.org/patents/app/20110288122

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