iPS Cell Response to CFTR Modulators: Study of Trikafta in CF Patients Carrying Partial Function Mutations or N1303K CFTR
Phase: PHASE2
Conditions: Cystic Fibrosis
Sponsor: Emory University

Study Protocol 
 
Title: iPS Cell Response to CFTR Modulators: Study of Trikafta in CF 
Patients Carrying Partial Function Mutations or N1303K CFTR 
 
NCT Number: NCT03506061 
 
Protocol date: May 10, 2022 

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Full study title: iPS cell response to CFTR modulators: Study of TrikaftaTM in CF patients carrying partial 
function mutations or N1303K CFTR
Short study title: Partial function and N1303K mutation iPS response
Principal Investigators: Eric Sorscher, Professor, Department of Pediatrics
Rachel Linnemann, Assistant Professor, Department of Pediatrics
Sponsor(s): NIH and CFF
Protocol version number and version date: 10May2022 v.5

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1.
External (non-Emory) Collaborators (if applicable):
George M. Solomon, MD
Associate Professor of Medicine
University of Alabama at Birmingham School of Medicine
The UAB IRB will review Dr. Solomon’s engagement in human subjects research
Brian R. Davis, PhD
Professor of Molecular Medicine
University of Texas Health Science Center at Houston
The UT IRB will review Dr. Davis’s engagement in human subjects research
2.
Precis/Abstract: 
Cystic Fibrosis (CF) is a life threatening genetic disorder resulting from mutations found in the gene 
known as the cystic fibrosis transmembrane conductance regulator (CFTR). Defects in this gene prevent 
correct chloride and bicarbonate transport in and out of cells. It has become increasingly important to 
develop new in vitro model systems capable of predicting in vivo clinical effectiveness of modulator 
therapy among patients with CF. This objective represents a significant and unmet need for advancing 
personalized therapeutics in the disease. The current trial is intended to show for the first time that 
primary iPS cells differentiated to an airway epithelial phenotype can be used to predict in vivo clinical 
response for rare CF patient populations – with the long-term goal of facilitating drug access for 
individuals with unusual (or even private) CF variants. TrikaftaTM is currently approved for patients with 
CF carrying at least one copy of the common F508del variant and over 170 other CFTR abnormalities. In 
this protocol, we describe a clinical study of 42 subjects without the F508del mutation, carrying partial 
function or N1303K mutations not approved for TrikaftaTM, and who are not expected to be approved 
for CFTR modulator treatment in the immediate future. Each patient will have clinical and/or preclinical 
evidence that TrikaftaTM should offer benefit, and each will be given TrikaftaTM for approximately four 
weeks. We will monitor clinical endpoints that include FEV1 and sweat chloride. We will differentiate iPS 
cells from each subject to generate airway epithelial monolayers that can be tested for response to 
TrikaftaTM. In this way, this study will evaluate an emerging and readily accessible in vitro surrogate 
endpoint as a predictor of clinical response. Our trial will also serve as a pilot/test case for other clinical 
protocols relevant to patients with rare CFTR variants and evidence of modulator responsiveness who 
do not have an approved modulator therapy—due to rarity of their mutation. We hypothesize that a 
robust correlation will be established between in vitro TrikaftaTM responsiveness of iPS cells and in vivo 
benefit (FEV1,) in patients, and providing a powerful tool for utilizing iPS cells to identify rare CF patient 
populations most suitable for cystic fibrosis modulator therapy. 
3.
Introduction and Background: A summary of the primary hypothesis, purpose, scholarly rationale, 
and prior literature. 
CFTR variants have classically been assigned to one of six mechanistic categories, depending on the 
underlying protein defect (Rowe et al., 2005; Elborn et al., 2017; De Boeck and Amaral, 2016). Mutations 
such as G542X or W1282X are representative of class I abnormalities, in which a premature stop codon 
leads to destabilization of messenger RNA (by virtue of nonsense-mediated decay) in addition to 
markedly diminished production of foreshortened and nonfunctional polypeptide (Rowe et al., 2005). 
The common F508del CFTR variant (deletion of a phenylalanine occurring at CFTR position 508) is found 

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in at least one CFTR allele among ~90% of CF patients in the United States. This mutation is categorized 
as class II, indicating failure of proper biogenesis, maturational arrest in the endoplasmic reticulum, 
trafficking away from the plasma membrane, and premature degradation by the proteasome.  N1303K is 
another distinct class II variant. Class III mutations disrupt ability of the CFTR ion channel to open 
properly (i.e., ‘gate’), impairing transmembrane flow of chloride and/or bicarbonate. The best described 
variant in this category is G551D, in which glycine at CFTR position 551 is replaced by a charged residue 
(aspartic acid), disrupting a normal interface between two CFTR nucleotide binding domains necessary 
for channel function. Other CFTR molecular defects confer distortion of the anion selective pore (class 
IV), interrupt normal protein biosynthesis (class V), or destabilize CFTR at the plasma membrane (class 
VI). 
For many years, the CF research field pursued a conceptual approach to treatment in which 
mutation subclass was intended to match tailored, small molecule interventions. Among certain class III 
defects, for example, CFTR modulators that activate channel gating (termed ‘potentiators’) have now 
become standard of care (Accurso et al., 2010). For the common class II (maturational processing) 
variant, F508del, ‘corrector’ molecules that overcome improper biogenesis (lumacaftor, tezacaftor, 
elexacaftor) have advanced through FDA registration (Wainwright et al., 2015; Taylor-Cousar et al., 
2017; Keating et al., 2018; Middleton et al., 2019). Nonsense mutations (class I) have been viewed as 
requiring a completely distinct family of therapeutic agents, capable of enhancing translational read 
through to generate full-length CFTR and stabilize mRNA by virtue of a successful (sentinel) round of 
translation (Howard et al., 1996).
Current status of cystic fibrosis personalized medicine includes the emerging concept of 
theratype (an empiric evaluation of modulator responsiveness). The high cost of CF (and other orphan) 
pharmaceuticals and the influence of cost on commercial incentive, third party reimbursement, FDA 
registration, and patient access are acutely felt in CF patient care, although rarely described in scientific 
or medical literature.
Advances in cystic fibrosis drug discovery
A ‘personalized’ approach has underpinned remarkable progress in CF therapy. The potentiator 
ivacaftor, for example, is viewed as a safe and effective compound suitable for enhancement of CFTR 
gating and forestalling pulmonary decline. The drug is now FDA approved for numerous distinct CFTR 
variants, or ~12-13% of the cystic fibrosis population. Lumacaftor, in combination with ivacaftor (under 
the trade name Orkambi™), is registered for therapy of F508del homozygous individuals, or ~46% of 
those with the disease8. These two drugs, therefore, account for over 50% of CF patients being ‘on label’ 
for an FDA-approved modulator. Tezacaftor, a rescue molecule similar to lumacaftor but with optimized 
pharmacodynamic and safety profile, was approved in combination with ivacaftor for F508del 
homozygous individuals, as well as those who carry an F508del allele and one of several partial function 
variants at the second CFTR locus. ‘Next generation’ correctors under development by pharmaceutical, 
academic, or other groups act by mechanisms distinct from lumacaftor or tezacaftor and are designed to 
provide additional benefit when given in combination. Lung function improvement among patients with 
a single copy of F508del, for example, has been established following combined drug treatment with 
next-generation agents, irrespective of the CFTR mutation at the second locus (Herper, 2017). The 
magnitude of response for patients encoding one copy of F508del is comparable to that obtained with 
ivacaftor in G551D-associated CF. Elexacaftor is approved together with tezacaftor and ivacaftor for 
patients with at least one copy of F508del, as well as > 170 rare CFTR polymorphisms (Middleton et al., 
2019; Keating et al., 2019; CFF News, 2020).

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Patients who may be “left behind”
Because ~90% of CF patients in the United States carry at least one copy of F508del, pharmacotherapies 
(TrikaftaTM in particular) are now available to a sizable majority of those with the disease. However, 
thousands of patients harboring relatively common variants will remain without effective drug therapy 
(e.g., those with premature truncation alleles such as G542X, R553X, W1282X, etc., or refractory CFTR 
folding defects such as N1303K). Others with ultra-rare or private CFTR mutations have forms of the 
disease that are very likely to benefit from available drugs, but no access to these therapies due to 
inherent challenges associated with obtaining data for registration and third-party reimbursement (see 
below). It has been estimated that over 1,000 CFTR mutations are represented by less than 5 patients 
each (Thomas and Boyle, 2017). Establishing processes so that individuals with very rare and/or poorly 
characterized alleles can gain access to effective modulator treatment remains one of the predominant 
challenges in the field. 
Complexity of CF pathogenesis 
Cystic fibrosis personalized treatment—like other precision-type therapies—was originally envisioned as 
a strategy for directing tailored small molecules to specific protein defects. G551D CFTR, for example, 
represents an essentially pure gating problem that ivacaftor rescues robustly. However, it has become 
increasingly clear that most CFTR mutations exhibit multiple abnormalities (instability, limited 
membrane targeting, defective ion transport, etc.). A case in point, the common F508del variant has 
been shown to exhibit not only inadequate biogenesis (class II defect), but also defective gating (class III) 
and diminished cell surface stability (class VI) (Veit et al., 2016). Another example, the rare P67L CFTR 
variant (reported in ~238 individuals worldwide), has been described as a class IV abnormality (physical 
disruption of the ion conductive pore). From a ‘personalized’ medicine perspective, therefore, P67L 
might not be expected to benefit from correctors such as lumacaftor or tezacaftor. Findings from several 
groups, however, indicate P67L responds dramatically to both molecules, and the variant should be 
viewed as exhibiting characteristics of both class II (processing) and class III (gating) pathophysiology 
(Sabusap et al., 2016; Ren et al., 2013). Both ivacaftor and corrector molecules have been FDA approved 
for treatment of the P67L variant. 
Nuances that underlie the molecular disease process have become increasingly difficult to 
simplify using only a few mechanistic subcategories. Even mutations at the same position within CFTR 
show substantial difference in cellular phenotype. S549R and S549N, for example, both exhibit partial 
function and are FDA approved for gating activation with ivacaftor. In model systems, however, S549N 
leads to more abundant CFTR at the cell surface than S549R, and may be more responsive to ivacaftor in 
human subjects (Vertex Pharmaceuticals, Kalydeco full prescribing information). Preclinical and clinical 
data suggest a corrector molecule might differentially benefit patients carrying S549R versus S549N (Rab 
et al., 2017). 
Complexity of molecular mechanism is also demonstrated by variants such as W1282X (class I), 
which retains significant ion channel function even in truncated form, and is modestly responsive to 
stabilization by CFTR correctors (Wang et al., 2016; Haggie et al., 2017). Other premature stop codons 
such as E831X might be predicted to have no response whatsoever to currently available modulators 
(based on both nonsense mediated decay (attributable to the premature stop codon) and a severely 
foreshortened polypeptide). However, in part because the mutation occurs near a splice junction, a 
fraction of E831X CFTR mRNA produces a full-length protein lacking only amino acid 831 (i.e., generating 
E831del) (Haggie et al., 2017; Hinzpeter et al., 2010). The E831del variant maintains significant residual 
activity, improved clinical phenotype, and is FDA-approved for ivacaftor. Numerous CFTR class IV (splice) 
defects (e.g., 2789+5 G→A, 3272-26 A→G, 3849+10KbC→T) also retain residual activity and have 
recently become registered for modulator therapy. Overall, it has become increasingly clear that 

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modulators shown to impact one CF mutation are commonly found to benefit numerous other disease 
categories.
Challenges in concept and practice
Based on considerations such as these, two essential questions have recently emerged regarding cystic 
fibrosis therapeutics: First, how precisely can (or should) molecular mechanism dictate personalized 
treatment? In other words, if a modulator exhibits a surprising benefit against a mutation for which it 
was not designed, how should this be approached to optimize patient access? Second, as summarized 
below, how will CF patients with ultra-rare or even private variants gain access to highly effective but 
costly modulator treatments, when ‘off label’ prescribing is largely proscribed? Use of in vitro systems 
stably expressing unusual CFTR variants (e.g., the FRT model) can be used to provide data in support of 
FDA registration (Durmowicz et al., 2018), and feasibility of testing rare CFTR mutations in this fashion 
has recently been utilized for certain molecular defects which can be adequately represented by studies 
of CFTR cDNA.
Among the thousands of cystic fibrosis patients carrying low prevalence variants, it is expected 
that many would benefit from a compound such as elexacaftor, ivacaftor or tezacaftor. As with any drug, 
physicians caring for a CF patient (irrespective of genotype) may recommend these modulator therapies, 
in the same way a panoply of non-registered agents are safely and routinely given in most diseases 
without medical restriction. A difficulty arises in the case of CF modulator treatment, however, due to 
very high cost (upwards of $250,000 - $300,000 per year, which represents an economic burden for 
third-party payers). As a consequence, and in contrast to less expensive pharmaceuticals in CF and the 
vast majority of other diseases, insurance reimbursement for modulators has been limited to patients 
with an FDA-approved (‘on label’) indication. ‘On label’ variants currently comprise ~180  CFTR 
mutations from among approximately 2,000 associated with clinical disease. Therefore, although many 
patients who are ‘off label’ would likely benefit, the cost of drug vitiates patient access. On the other 
hand, an attempt to broaden label to include a new CF genotype can present a formidable challenge. 
Numbers of patients with a particular mutation are often so small that phase 3, double blind placebo-
controlled trials to promote registration are not possible. The issue is more than academic—failure to 
expand the label or otherwise augment drug access means thousands of CF patients must forego a 
potentially life-saving intervention. 
Facilitating modulator availability 
A series of innovative strategies have been devised by pharmaceutical and/or academic groups to 
address the issue of inadequate CF drug access. In part because a dramatic clinical benefit from 
modulator treatment can often be obtained within a matter of weeks, N-of-1 trials have been evaluated 
for a possible role during FDA registration (McGarry et al., 2017; Nick et al., 2014). Such studies are 
designed so that individual patients serve as their own controls, and a robust drug response can provide 
data relevant to label expansion. Variability among CF patients with the same genotype, inconsistent 
clinical findings from a single study subject, preexisting (irreversible) lung injury, disease trajectory 
(including presence of respiratory exacerbation), and practical barriers to drug approval on a case-by-
case basis have limited usefulness of this approach. As an alternative strategy, patients with similar 
molecular phenotype (e.g., residual CFTR function as judged by in vitro testing), and/or evidence of mild 
clinical disease (pancreatic sufficiency or intermediate sweat chloride values) have been evaluated as a 
group (independent of underlying gene defect or mechanistic subcategory) (Herper, 2017). In a cohort 
of patients with rare variants that can be meaningfully binned or designated based on strong scientific 
rationale, a robust clinical response to CFTR modulators can help promote label expansion to an entire 

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group of patients with the disease – despite differences in CF genotype for the cohort. While admittedly 
less ‘precise,’ the situation can be viewed as similar to conventional pharmacotherapies—where 
registration is based on compelling clinical evidence of benefit to the overall group or enriched study 
population (for further background, see Schuck et al., 2018). 
Emerging approaches 
In a recent, landmark series of approvals from FDA, ivacaftor and tezacaftor labels were extended to 
include rare variants based on clinical and/or in vitro assay data (Durmowicz et al., 2018). Trikafta™ 
approvals for new genotypes have been accomplished in the same fashion (CFF News, 2020). The notion 
that compelling in vitro data could constitute criteria for label expansion represents a paradigm shift in 
CF personalized treatment. In the same fashion that a highly predictive surrogate such as lowering blood 
cholesterol can support approval of drugs that prevent heart disease or stroke, in vitro systems capable 
of predicting CF clinical improvement (e.g., increased FEV1, fewer pulmonary exacerbations) represent a 
compelling means to rationally expand drug access among patients with ultra-rare forms of the disease.  
The nature and extent of in vitro data necessary to support registration is being evaluated for a number 
of model systems, including primary airway epithelium, organoid-type preparations, iPS cells 
differentiated to airway epithelial phenotype, and recombinant cell types, among others.  The polarizing 
FRT cell line encoding heterologously expressed mutant CFTR cDNAs has been utilized by FDA as suitable 
for this purpose (Durmowicz et al., 2018; see also above). An example showing surrogate testing 
systems for the rare N1303K variant is shown in Figure 1. Although not clinically approved for Trikafta™ 
or other modulator treatments, N1303K expressed in FRT cells exhibits activity in a range compatible 
with clinical benefit for the elexacaftor/tezacaftor/ivacaftor (Trikafta™) combination therapy. Studies of 
FRT cells encoding N1303K, as well as iPSC derived from a patient with an N1303K/W1282X genotype (in 
which airway epithelial monolayers differentiated from these cells would be viewed as essentially “null” 
for the W1282X allele) and human nasal primary airway epithelial monolayers all showed a moderate 
short circuit current (ISC) response to pharmacomodulation. The findings support the possibility of 
clinical benefit using Trikafta™ in patients encoding N1303K. In this context, it should be noted that not 
all research groups have reported this level of N1303K activity following modulator therapy. For 
example, at a recent meeting of the CFF RARE Consortium, several laboratories (Lexington, Cincinnati, 
Birmingham) viewed N1303K as a variant meriting clinical study with Trikafta™. On the other hand, 
Vertex did not include N1303K in their recent label expansion (presumably because of inadequate 
modulator responsiveness). Findings such as these indicate the importance of a formal clinical trial to 
determine whether patients with an N1303K genotype (the 5th most common cystic fibrosis mutation 
worldwide) might benefit from drugs such as Trikafta™. 
 

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Figure 1. Activity of N1303K CFTR in FRT, iPSC, and primary nasal cells. Panel A: depicts short circuit current response to 
elexacaftor/tezacaftor/ivacaftor (ETI) in FRT cells stably expressing the N1303K variant. Note that activation of ~ 10% wildtype 
level has been viewed as predictive of clinical benefit in the FRT cell system advanced by Vertex Pharmaceuticals. N1303K 
activity shown here is > 25% wildtype levels. Panel B: Western blot indicating enhancement of N1303K bands B and C by ETI.  
Panel C: Transepithelial chloride transport on skin fibroblast derived iPS cells with airway epithelial differentiation. Panel D: 
summary data using the same protocol. Triple combination therapy (TCT) using ETI indicates 5-7 µA/cm2 short circuit current, 
which is similar to the level of activity obtained using ivacaftor in G551D airway epithelial monolayers derived from iPSCs.  
Panel E: Western blot from an iPSC/N1303K experiment of this sort. Panel F: indicates ~ 5 µA/cm2 in primary nasal airway 
epithelial monolayers following ETI therapy. This level is in a range that might be predictive of clinical benefit in some settings.

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As above, poor access comprises a commonplace barrier to optimal care throughout CF clinics in the 
United States and abroad. The same issue is a major impediment to personalized treatments in diseases 
such as cancer and HIV infection (Menon et al., 2015; Evans et al., 2015; Nardini, 2014; Jabbour et al., 
2015; Lammers et al., 2014). As a result, FDA has sought in vitro model systems that could serve as 
surrogates for clinical benefit among patients with very rare (or even private) alleles and for whom 
traditional, late-phase clinical testing would be difficult. Preliminary evidence suggests differentiated iPS 
cells may be an excellent candidate in this regard (Crane et al., 2015; Mou et al., 2015).
As part of an evaluation to test the relationship between CFTR activity and modulator effectiveness, we 
worked with 43 rare CFTR variants stably expressed from a single site in the genome of the human CF 
bronchial epithelia cell line (CFBE41o-) (Han et al., 2018). The magnitude of drug response in these cell 
models was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector 
lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants 
expressed stably in FRT cells showed similar correlation (although certain variants were identified that 
demonstrate significantly higher response to modulator treatment than predicted by in vitro analysis). In 
addition, nearly all variants studied in CFBE (40/43) and FRT cells (13/16) demonstrated greater 
response to ivacaftor-lumacaftor combination therapy than either drug alone. Together, the variants 
studied here represent 87% of individuals in the CFTR2 database with at least one missense defect. Our 
results therefore indicate that most patients with CF carrying missense variants are likely to: 1) respond 
modestly to current potentiator and/or corrector therapy, while a small fraction will exhibit more 
pronounced CFTR rescue and 2) derive the greatest benefit from combination treatment. 
Overview FDA has indicated new in vitro 
model systems capable of predicting in vivo 
clinical benefit may represent a significant 
and unmet need for advancing personalized 
CF therapeutics. The present study is 
designed to specifically and prospectively test 
iPS cells taken from patients encoding rare CF 
variants as a means to determine in vitro 
thresholds for CFTR rescue. Using iPS cells 
differentiated to exhibit a respiratory 
epithelial phenotype, we will determine 
whether the cells can be used to predict FEV1 
response among individuals carrying rare, 
partial function mutations who receive 
TrikaftaTM. Substudy 1 will comprise an open-label, two center trial of orally administered elexacaftor, 
tezacaftor and ivacaftor (TrikaftaTM) that will enroll 22 patients (11 at Emory) with rare/orphan 
genotypes (Figure 2 summarizes trial endpoints); a detailed schedule of events is provided under 
“Subject Involvement and Characteristics”. We will also enroll subjects who encode the N1303K variant 
as emblematic of a mutation not approved for Trikafta™, but likely to respond (Substudy 2). As 
described above, N1303K CFTR is relatively common, not on the Trikafta™ label, and has been shown by 
our laboratory and others to exhibit activation following Trikafta™ in a number of cell-based systems. 
We plan to enroll 20 subjects (10 at Emory) that carry N1303K and test these individuals for lung 
function, iPSC/airway epithelial CFTR current, and other clinically important endpoints when the drug is 
administered in vivo. 
Figure 2. Summary of trial design.
0
4
2
-1
Visit Week
Screening
two tablets (elexacaftor 100 mg/tezacaftor 50 
mg/ivacaftor 75 mg) and one tablet (ivacaftor 
150 mg) added to prescribed medications
Follow 
up
Sweat Chloride
NPD
CFQ-R Questionnaire
Spirometry
Safety Labs / EKG
Weight Gain

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4.
Objectives: Primary and secondary aims and outcome measures. 
Primary Objective: We propose a clinical study of subjects carrying either partial function mutations, or 
encoding N1303K CFTR. Each patient will be given TrikaftaTM for approximately four weeks. We will 
monitor clinical endpoints that include FEV1, sweat chloride, quality of life questionnaire, and weight. 
We will also evaluate cutaneous punch biopsy material and/or blood samples from each subject so that 
iPS cells can be differentiated into airway epithelial monolayers and tested for response to TrikaftaTM.
Substudy 1 (Partial Function)
Primary Endpoints: FEV1, response of iPS cells to TrikaftaTM treatment
 
Secondary Endpoints: Sweat chloride, CFQ-R (quality of life questionnaire), weight gain, safety 
laboratory evaluation/EKG
OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR ENDPOINTS
Primary
To determine whether iPS cells 
differentiated to primary airway 
epithelia -- and treated with 
ivacaftor/tezacaftor/elexacaftor in 
vitro -- can predict clinical 
responsiveness to TrikaftaTM in vivo.
•
Functional and biological 
correction of CFTR expressed in 
iPS cells as judged by short 
circuit current measurements in 
monolayers, and western blot 
analysis
•
Forced expiratory volume in one 
second (FEV1)
•
These in vitro tests represent 
standard functional and 
biochemical analysis that 
address CFTR rescue, with direct 
relevance to the in vivo 
situation.
•
FEV1 is a conventional endpoint 
and provides a direct 
measurement of patient health 
benefit among individuals with 
cystic fibrosis 
Secondary
To verify safety and establish 
efficacy of TrikaftaTM in enrolled 
patients. 
•
Sweat chloride testQuality of life 
survey 
•
Standard laboratory tests and 
other measurements, as well as 
history and physical 
examination 
•
Provide supportive information 
regarding study intervention 
effect using in vivo measures 
known to indicate CFTR rescue
•
A well-established tool for 
monitoring patient benefit in 
cystic fibrosis
•
Confirm safety of the FDA-
approved drug 
Substudy 2 (N1303K)
Identical to Substudy 1, except that sweat chloride will serve as primary endpoint with other endpoints 
(shown in the table above) designated as secondary.
 

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5.
Study design and methods: 
Human Subjects Involvement
Minor protocol changes for Substudy 2 are described in text. 
The study is an open-label clinical trial of orally-administered TrikaftaTM in subjects with CF and 
carrying certain mutations that are without FDA approved modulator treatment. Subjects will be 
given the study drug elexacaftor 100 mg/tezacaftor 50 mg/ivacaftor 75 mg (2 pills once daily in the 
morning) and ivacaftor (150 mg) once daily in the evening, as the FDA-registered agent, TrikaftaTM. 
Dose and schedule will be identical to what has already been FDA-approved for the F508del CF 
genotype. Enrollment is planned at two centers, Emory University and University of Alabama at 
Birmingham. In Substudy 1, patients with residual CFTR function (based on sweat chloride < 80 
meq/L or pancreatic sufficiency) will be tested. In Substudy 2, patients with CF encoding the N1303K 
variant will be enrolled as a “case in point” – emblematic of variants with in vitro evidence of 
Trikafta™ responsiveness who are not approved (i.e., not “on label” for the drug). iPSC-derived 
airway epithelial monolayers from these patients will be examined for their ability to predict FEV1 
and sweat chloride response and other clinical endpoints. Our study will not significantly increase the 
risks (or decrease the acceptability of risks) associated with the use of TrikaftaTM in CF.  
Procedures
Screening/
Enrollment
Day -28 to -1
Baseline
Visit 1, Day 1
Study Visit 2
(phone call) 
Day 7  +/-2 
days
Study Visit 3
Day 14 +/- 2 
days
Study Visit 4
Day 28 +1/-2 
days
Final Study 
Visit Day 56 
+/-2 days
Informed consent
X
Medical history
X
X
X
X
X
X
Physical exam
X
X
X
X
X
Cutaneous punch biopsy and/or blood sampleA
X
XE
CFTR genotype
X
Spirometry
X
X
X
X
X
Oxygen saturation 
X
X
X
X
X
Sweat chloride testing
X
X
X
X
X
Safety laboratory assessmentB
X
X
X
X
X
Pregnancy TestC
X
X
X
EKG
X
X
Ophthalmologic examD
X
Drug accountability 
X
X
X
Questionnaire (CFQ-R)
X
X
X
X
X
Administer study drug
X
X
Concomitant medication
X
X
X
X
X
X
Adverse event assessment
X
X
X
X
X
A Substudy 1: cutaneous punch biopsy. Substudy 2: blood sample with optional cutaneous punch biopsy.  
B Renal and liver panels, CBC/Diff urinalysis
CFemales of reproductive age
DSlit lamp ophthalmologic exam if not done within 3 months before screening for those ages 12-17 at the time of 
screening
EIf necessary to produce additional iPS cells

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Human subject participation will consist of a screening day (-28), day 1 (+/-2), day 7 (+/- 2) [phone 
call visit], day 14(+/-2), day 28 (+1/- 2) and day 56 (+/-2) follow up (wash-out). The visits will include 
patient history with concomitant medication review, physical exam, safety laboratory assessments 
(renal and liver panels, CBC/Diff, urinalysis), serum and urine pregnancy testing, CFTR genotype, 
spirometry, O2 saturation, sweat chloride test, ophthalmology exam in children, cutaneous punch 
biopsy and/or blood sample for iPSC derivation, EKG, drug accountability and questionnaire (CFQ-R 
assessment), and adverse event assessment.
We anticipate the screening and enrollment visit will require 4-6 hours to complete the informed 
consent, medical history, O2 saturation, concomitant medication review, physical exam, safety 
laboratory assessments (CBC/Diff, RENAL/LFT chemistry panel, and urinalysis), EKG, CFTR genotyping, 
questionnaire, serum pregnancy test for females, sweat chloride test, and spirometry.
On day 1 (when subject returns), the visit will require approximately 6 hours to complete the 
concomitant medication review, adverse event assessment, interval medical history, physical exam, 
O2 saturation, safety laboratory assessments (CBC/Diff, RENAL/LFT chemistry panel, and urinalysis), 
questionnaire, sweat chloride, urine pregnancy test for females, spirometry, cutaneous punch biopsy 
(3mm) and/or blood sample, drug dispensing, and drug administration.  The cutaneous punch biopsy 
may occur at any time between the Screening Visit and Day 1 based on dermatology clinic schedule 
availability.

1 Cutaneous 3 mm punch biopsy (performed here (and on day 56 if necessary to produce 
additional iPS cells)): In this procedure, a circular blade is rotated from epidermis to 
subcutaneous fat, yielding a ~ 3 mm cylindrical core of tissue. Extending the skin 
perpendicular to the incision line allows for facile closure, which can typically be 
accomplished with a single suture. Pain is well-managed by local subcutaneous anesthetic. A 
small fibrotic scar can be minimized by linear closure of the biopsy. Significant pain, bleeding, 
or infection are very rare.
The subject will complete a phone call visit on day 7 (+/-2) that will require approximately 30 minutes  
to complete concomitant medication review, interval medical history, and adverse event assessment.
On day 14 (+/-2) the subject will return for a visit that will require approximately 5-6 hours to 
complete concomitant medication review, interval medical history, adverse event assessment, 
physical exam, drug dispensing and drug return, questionnaire, safety laboratory assessments 
(CBC/Diff, RENAL/LFT chemistry panel, and urinalysis), sweat chloride test, O2 saturation and 
spirometry. EKG will be performed on day 14.
On day 28 (+1/-2) we will conduct interval medical history, adverse event assessment, concomitant 
medications, physical exam, sweat chloride test, spirometry, O2 saturation, safety laboratory 
assessments (CBC/Diff, RENAL/LFT chemistry panel, and urinalysis), questionnaire, and drug return. 
This visit should require 4-6 hours to complete.  This visit should occur no later than Day 29 since a 28-
day supply of Trikafta™ will be provided. 
The ‘wash-out’ visit will be conducted on day 56 (+/-2) which will require approximately 4-6 hours to 
complete concomitant medication review, interval medical history, adverse event assessment, physical 
exam, safety laboratory assessments (CBC/Diff, RENAL/LFT chemistry panel, and urinalysis), 

Emory IRB Version 10May2022 v.5
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questionnaire, sweat chloride, urine pregnancy test for females, cutaneous punch biopsy (3mm) 
and/or blood sample, O2 saturation, and spirometry.  The cutaneous punch biopsy may occur within ± 
14 days of the day 56 visit based on dermatology clinic schedule availability.  
Spirometry may be performed either pre- or post-bronchodilator at Screening.  At all other visits, 
spirometry should be performed pre-bronchodilator.  However, if patient is unable to withhold 
bronchodilator use at the Day 1 visit, then the spirometry assessment will be considered ‘post-
bronchodilator’ and attempts should be made to collect spirometry post-bronchodilator at remaining 
visits.  
In order for a spirometry assessment to be considered pre-bronchodilator, subjects should adhere to 
the following:
• Subjects should refrain from using short-acting bronchodilators (e.g., albuterol) or anticholinergic
(e.g., ipratropium bromide [Atrovent®]) for more than 4 hours before the spirometry assessment.
• Subjects should refrain from using long-acting bronchodilator (e.g., salmeterol) for more than 12 
hours before the spirometry assessment; and
• Subjects should refrain from using once-daily, long-acting bronchodilator (e.g., tiotropium bromide
[Spiriva®]) for more than 24 hours before the spirometry assessment.
Sources of Patient-Derived Material
•
Review of patient medical records and demographic information
•
History and physical exam

Blood and urine for safety studies (<6 tablespoons (depending on weight) of blood per visit)
•
Sweat chloride results
•
Questionnaire data
•
Spirometry (FEV1, FVC, and FEF25-75%) calculated per Hankinson equation
•
Cutaneous punch biopsy (3 mm) and/or blood sample /iPS cell derivation 
•
O2 saturation
•
EKG
•
Ophthalmologic exam
•
CFTR genotype
•
Pregnancy test
•
Drug accountability
6.
Participant selection: 
Inclusion Criteria: 

Provision of signed and dated informed consent form, and when appropriate, assent form

Stated willingness to comply with all study procedures and availability for the duration of the 
study

Male or Female age ≥12

A clinical diagnosis of CF or CFTR-related disease* and either: 1) evidence for a partial function 
mutation not currently covered or likely to be covered for FDA treatment with a CFTR modulator 
(Substudy 1), or 2) N1303K CFTR and a minimal function mutation (Substudy 2).

Sweat Chloride < 80 mmol/L and/or pancreatic sufficiency (no exogenous pancreatic enzyme 
supplement therapy) or carrying the N1303K CFTR variant

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
Able to perform spirometry meeting ATS criteria for acceptability and repeatability 

Clinically stable in the past 4 weeks with no evidence of CF exacerbation (prior to Screening AND 
Day 1)

Willingness to use at least one form of acceptable birth control including abstinence or condom 
with spermicide. This will include birth control for at least one month prior to screening and 
agreement to use such a method during study participation for an additional four weeks after 
the last administration of study drug. 

Ability to take TrikaftaTM.

Agreement to adhere to all current medical therapies as designated by the CF care center 
physician.
* A diagnosis of CF as defined by current guidelines: one or more clinical features consistent with the CF 
phenotype or positive CF newborn screen AND one or more of the following criteria: sweat chloride ≥ 60 
mmol/L by quantitative pilocarpine iontophoresis or 2 well characterized disease-causing CFTR gene 
mutations 
OR 
With the consensus of two study co-investigator physicians, a clinical diagnosis of CF or CFTR-related 
disorder with documented evidence of chronic pulmonary disease (chronic cough with sputum 
production, persistent pulmonary radiographic abnormalities (e.g bronchiectasis), chronic abnormality 
in ppFEV1, and/or persistent colonization with a typical CF pathogen)
Exclusion Criteria

Documented history of drug or alcohol abuse within the last year

Subjects should not have a pulmonary exacerbation or changes in therapy for pulmonary 
disease in the 4 weeks prior to screening

Listed for lung or liver transplant at the time of screening

Cirrhosis or elevated liver transaminases > 3X ULN

Pregnant or breastfeeding

Inhibitors or inducers of CYP3A4, including certain herbal medications and grapefruit/grapefruit 
juice, or other medicines known to negatively influence TrikaftaTM administration 

History of solid organ transplant

Active therapy for non-tuberculosis mycobacterial infection or any plan to initiate non-
tuberculosis mycobacterial therapies during the study period 

Known allergy to TrikaftaTM 

Treatment in the last 6 months with an approved CFTR modulator 

Any other condition that in the opinion of the lead investigators might confound results of the 
study or pose an additional risk from administering study drug

Treatment with another investigational drug or other intervention within one month prior to 
enrollment, throughout the duration of study participation, and for an additional four weeks 
following final drug administration.

Evidence of cataract/lens opacity determined to be clinically significant by an ophthalmologist 
at or within 3 months prior to the Screening Visit

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Subject recruitment plan
Under Substudy 1, we plan to recruit 22 subjects (11 subjects per site at Emory and the University of 
Alabama at Birmingham) with cystic fibrosis carrying a partial function mutation genotype not covered 
or expected to be covered by an FDA approved treatment with a CFTR modulator. Under Substudy 2, 
we plan to recruit 20 subjects encoding N1303K CFTR to Emory and UAB (10 subjects per site). In both 
substudies we intend to include ~50% female and ~50% male subjects age > 12. Subjects will first be 
recruited from the cities participating in the trial (Atlanta and Birmingham), and CF physicians have 
previously expressed interest in discussing the proposed clinical trial with eligible individuals. Study 
investigators also have immediate access to a robust national patient registry through the Cystic 
Fibrosis Foundation therapeutic development network, and (following IRB approval) will contact cystic 
fibrosis physicians caring for individuals with the appropriate genotypes to ask whether these patients 
might be interested and/or willing to discuss possible study participation (including consent) with one 
of the lead investigators on the proposed trial (Sorscher, Linnemann, Solomon, Stecenko). 
All individuals who consent to participation will be evaluated at one of two clinical CF care centers 
(Emory, University of Alabama in Birmingham). Travel, meals, accommodations, etc. for study visits will 
be covered by funds available through the project, and an incentive payment of $150 per visit will be 
provided as a gift card or check. For minors who participate in the study, travel expenses, etc. will also 
be provided for one parent and the gift card or check for each visit provided to that parent (i.e., not 
the minor).  For adult subjects accompanied by a caregiver, travel expenses, etc. will also be provided 
for the caregiver. 
7.
Informed Consent Process:
Informed consent will be obtained by well-trained study coordinators or investigators using an 
Institutional Review Board approved document. Parents/guardians/participants will be asked to sign 
the informed consent documents only after the study has been fully described to the subject and all 
questions have been answered. Participants will be offered at least 24 hours to review the 
consent/assent before the research is to begin, although consent may be obtained on the day of 
screening if preferred by study subjects (eg those traveling from significant distance). 
Assent for minors will be obtained by experienced study personnel with many years expertise in this 
area. Minors will be informed about the study using age appropriate, and easily understood language. 
Care will be taken to assure minors: 1) Are very comfortable with the study, 2) Are under no pressure to 
enroll or participate, and 3) Understand they can withdraw at any time without penalty or change in 
their medical care. The investigator obtaining consent/assent will provide a written summary of the 
interaction for inclusion with the patient’s research file.
The participant's parent/guardian will be asked to read and review the document. The investigator will 
explain the research study to the participant and parent/guardian and answer any questions that may 
arise. A verbal explanation will be provided in terms suited to the parent/guardian and participant’s 
comprehension of the purposes, procedures, and potential risks of the study and of a subject's rights as 
a participant. Participants and parent/guardian will have the opportunity to carefully review the written 
consent form and ask questions prior to signing. The participants and parent/guardian will have the 
opportunity to discuss the study with their family and/or think about the consent document prior to 
agreeing to participate. Participants and parent/guardian will be informed that participation is voluntary 

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and that they may withdraw from the study at any time, without prejudice. A copy of the informed 
consent/assent document will be given to the parent/guardian/participants for their records. The 
informed consent process will be conducted and documented in the source document (including the 
date), and the form signed, before the participant undergoes any study-specific procedures. The rights 
and welfare of the participants will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study. Parents of subjects 
under the age 18 will sign the consent form with separate assent signed by children participating in the 
study, as applicable.
CF is a life long illness, and patients/families are typically very sophisticated regarding the nature of CF 
care -- including clinical trial participation. The disease is not associated with cognitive or other 
intellectual limitations. No vulnerable adult subjects are intended for enrolment in the trial. If, in the 
opinion of the study investigator, a prospective participant lacks sufficient understanding and is 
therefore not able to provide informed consent, that subject will not be enrolled. The ability to obtain 
consent represents criteria for inclusion in the trial.
8.
Compensation for time and effort:
Travel, meals, accommodations, etc. for study visits will be covered by institutional or other funds and 
an incentive payment of $150 per in-person visit and $30 at the Day 7 telephone call will be provided 
as a gift card or check. All payments will be pro-rated. For minors who participate in the study, travel 
expenses, etc. will also be provided for one parent and the gift card or check for each visit provided to 
that parent (i.e., not the minor).  For adult subjects accompanied by a caregiver, travel expenses, etc. 
will also be provided for the caregiver.  An incentive payment of $150 will also be provided if an 
unscheduled visit is needed (for multiple repeat assessments or cutaneous punch biopsy occurring on 
another day).   If the unscheduled visit is needed just for a repeat laboratory sample collection, an 
incentive payment of $30 will be provided. 
9.
Study Discontinuation
Criteria for discontinuing the study intervention (e.g., halting rules), may include any monitoring test(s) 
and associated clinical decision point(s) which in the opinion of the clinical investigator, qualified 
designee, or Data Safety Monitoring Board are viewed as placing the patient at significant risk if 
TrikaftaTM administration is to continue. This could include, for example (but is not limited to) 
worsening liver function or abnormalities associated with TrikaftaTM administration. Discontinuation 
from TrikaftaTM does not mean discontinuation from the study, and remaining study procedures should 
be completed as indicated by the study protocol. If a clinically significant finding is identified (including, 
but not limited to changes from baseline) after enrollment, the investigator or qualified designee, 
and/or Data Monitoring Committee (DMC) (also referred to as Data Safety Monitoring Board, DSMB) 
will determine if any change in participant management is needed. Any new clinically relevant finding 
will be reported as an adverse event (AE).  
The data to be collected at the time of study intervention discontinuation will include:

All measurements described for the day 56 follow-up/washout visit
Participants are free to withdraw from participation in the study at any time upon request.
An investigator may discontinue or withdraw a participant from the study for the following reasons:

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
If any clinical adverse event, laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best interest of the 
participant

Disease progression which requires discontinuation of the study intervention

If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation

Participant is unable to receive TrikaftaTM for 3 days.

Pregnancy

Significant study intervention non-compliance 
The reason for participant discontinuation or withdrawal from the study will be recorded. An attempt 
will be made to replace subjects who sign the informed consent form and receive TrikaftaTM, and 
subsequently withdraw, or are withdrawn or discontinued from the study.
A participant will be considered lost to follow-up if he or she fails to return for any scheduled visit 
and is unable to be contacted by the study site staff. 
The following actions will be taken if a participant fails to return to the clinic for a required study 
visit:

The site will attempt to contact the participant and reschedule the missed visit within 2-5 days, 
and counsel the participant on the importance of maintaining the assigned visit schedule and 
ascertain if the participant wishes to and/or should continue in the study.

Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, 
a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the participant’s medical record or study file. 

Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up.
10. Data Safety Monitoring Plan
Oversight for this trial will be performed by a DSM Chair. A Data Safety Monitoring Board (DSMB) will be 
selected in consultation with the Chair to specifically oversee the clinical study. The DSMB will consist of 
at least two physicians experienced in treating patients with cystic fibrosis, a DSMB coordinator and a 
biostatistician. The DSMB Chair will convene a protocol conference call with committee members so 
that the DSMB can approve the monitoring plan and review any concerns regarding the current protocol 
prior to initiation of study enrollment.
The DSMB is primarily responsible for reviewing safety data which will be provided on a continual basis 
throughout the performance of the study as well as reviewing interim analyses report after four patients 
have completed the 28-day study drug administration, and again following 50% enrollment (completion 
of 11 subjects in Substudy 1 and 10 subjects in Substudy 2). The DSMB may recommend early 
termination or modification of either trial substudy for reasons of study subject safety. Subjects will be 
monitored for adverse events (AEs) and serious adverse experiences (SAEs) by the Investigators, the 
study sites’ Institutional Review Boards, and the DSMB. The DSMB will also provide recommendations 
needed to assist the investigative team.

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One goal of the present study is to provide clinical data that will support a correlation between IPS CFTR 
activity and clinical benefit of a CFTR modulator. In the present study, the highest level of stringency will 
be applied. This will include: 1) DSMB, as described above, 2) detailed interim evaluations, 3) generation 
of case report forms for all study subjects, 4) detailed statistical analysis, tabular summaries, and leading 
edge evaluation to test clinical benefit, 5) compiling data in SAS or other well-accepted formatting, 6) 
discussions with interested parties (e.g. Vertex and CF Foundation) to provide updates regarding clinical 
trial progress, and 7) timely publication of data, etc. Logistical support and resources of the type 
intended here are provided by the Institution and the Department of Pediatrics for physician-sponsored 
clinical trials at Emory. Our intent will be to furnish a clinical profile that provides high stringency and 
rigor. We recognize that for the present clinical trial data will not be used for submission to FDA or 
towards label expansion of TrikaftaTM. However, by providing a compelling data set and offering this 
information early and openly to Vertex, interested third-party insurers, CFF, as well as CF patients and 
their families, we will furnish information that will help guide more extensive studies of IPS cell analysis 
among patients with rare forms of the disease. 
Study Monitoring: The Emory University self-monitoring tool will be customized to fit the needs of the 
current study.  This tool will be utilized by the study team for monitoring the progress of the trial, and 
assuring data accuracy and protocol compliance. Study monitoring will take place at least once a year. 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s). The self-monitoring process and study team will visually inspect study records 
collected since each previous monitoring visit. The scope of the monitoring shall include the informed 
consent process, eligibility, CRFs, biological specimen tracking and AE reporting. 
Quality control (QC) procedures will be implemented beginning with the data entry system. Data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated to the site(s) for clarification/resolution. 
A designated member of the study team will provide the completed self-monitoring tool to the study PI 
and team within 7 days of the monitoring visit. The study PI will document receipt and review of the 
monitoring report, resolutions and/or corrective actions to findings on the Site Monitoring Log. The 
monitoring reports will be shared between sites, with the Emory Clinical Trials Audit and Compliance 
(CTAC) office, and with the DSMB. Protocol deviations/violations will be reported to the Emory IRB per 
reporting guidelines and will be reviewed by the DSMB in a timely fashion or during regular meetings, as 
needed.
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party to study participants, investigators, NIH, and any other 
regulatory authorities. If the study is prematurely terminated or suspended, the Principal Investigator 
(PI) will promptly inform study participants, the Institutional Review Board (IRB), and will provide the 
reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be 
informed of changes to study visit schedule.
 

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Circumstances that may warrant termination or suspension include, but are not limited to:
•
Determination of unexpected, significant, or unacceptable risk to participants
•
Insufficient compliance to protocol requirements
•
Data that are not sufficiently complete and/or evaluable
•
Determination that the primary endpoint has been met
•
Determination of futility
11. Statistical Methods For a proposed open-label prospective 4-week trial of TrikaftaTM.
Substudy 1: Patients with CF or CFTR-dependent illness who do not have access to modulator therapy 
but exhibit a clinical phenotype suggestive of partial/residual CFTR activity. 
We will serially evaluate clinical outcomes of interest prior to and after the start of therapy. Change in 
clinical measurements (spirometry, weight/BMI, quality of life, sweat chloride) will be monitored. Our 
main goals are to determine the clinical response to TrikaftaTM and to determine if in vitro 
responsiveness of iPS cells predicts clinical response. The primary outcome is response to Trikafta™ 
after 4 weeks of treatment. In this study a clinical response to TrikaftaTM at week 4 of treatment will be 
defined as an improvement, from baseline, in FEV1 > 5% predicted and each subject will be classified a 
responder or non-responder. In this study, if at least 40% of subjects meet the definition of responder, 
the drug will be considered efficacious (i.e., comparable to the benefit which led to OrkambiTM 
approval by FDA following trials in F508del homozygotes). Overall response rate will be accompanied 
by an approximate binomial 95% confidence interval.  To determine the role of iPS in-vitro response to 
TrikaftaTM, we will compare the Δ lsc (change in lsc) among TrikaftaTM responders and TrikaftaTM non-
responders using a two-sample t-test or a Wilcoxon rank sum test if distributions are skewed. 
Additionally, we will correlate the Δ lsc obtained from in vitro experiments with the change in FEV1% 
predicted (post minus pre) using Spearman’s rank-order correlation with an associated 95% 
confidence interval. We will also test whether the iPS response reaches a CFTR functional threshold of 
40% wild-type levels (a magnitude of activity in primary airway epithelial cells viewed as potentially 
relevant to clinical benefit). In a sensitivity analysis, we will replicate the FEV1 analysis with sweat 
chloride responses, and determine the in vivo response rate. 
For other outcomes of interest including sweat chloride levels, quality of life, and weight/BMI, we will 
apply a mixed effect model to examine the change in these measures over the study duration. Models 
will include a subject-specific intercept, to account for variability among subjects prior to the start of 
the treatment, and a categorical variable for time (baseline, day 14, day 28, day 56). A Dunnet’s post-
hoc comparisons procedure will be used to determine which time points demonstrated a significant 
change from baseline. Using the same mixed effects models, we will include Δ Isc as predictors of in 
vivo improvement. We will also test the interaction between time and Δ lsc to determine when in-vitro 
response is most associated with change in clinical response. Adverse events and other safety 
endpoints will be tabulated and reported using counts and percentages or means and standard 
deviations, as appropriate. 
In addition, because clinical parameters may vary among patients prior to the start of therapy, we will 
also approach the analysis using a modified N of 1 design configured for the purpose of comparing iPS 
studies in contrast to the aggregate analysis described above. Using the framework of single subject 
research, we will evaluate therapeutic response in individual subjects by an interrupted time series 
(ITS) approach. Briefly, in the absence of an independent control group, ITS analysis is a quasi-
experimental design in which each subject acts as their own control and is followed serially in time 
prior to and after an interruption (in this case, the start of treatment). If the treatment has causal 

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impact, the post-intervention time series will have a different level or slope than the pre-intervention 
series. Time series models also have the power to test and correct for possible cyclical patterns and 
outliers. Using segmented regression analysis, we will conduct ITS models for each of the subjects 
enrolled in the trial. A significant change in slope from the pre-treatment period will indicate a 
significant treatment effect. ITS models will be constructed for each outcome of interest where 
historical and pre-treatment data are available. The change in slope will also be correlated with the in-
vitro response using Spearman’s rank-order correlation coefficient. 
All analyses will be conducted using the intention to treat principal, where all subjects will be included 
in analysis regardless of how long they took the study medication. Statistical significance will be 
assessed at the 0.05 level and analyses conducted using SAS v. 9.4 (SAS Institute; Cary, NC).
Sample size
The FDA has suggested that scientifically meaningful ‘binning’ of patients based on clinical 
characteristics can help guide future approval of specific patient groups for drug treatment. The present 
study is not intended for FDA submission or label expansion of TrikaftaTM, but as a means to determine 
whether IPS cells differentiated to respiratory phenotype can be used to predict clinical response to 
CFTR modulators. For the purposes of this protocol, the subjects enrolled, although potentially of 
different genotypes, will represent a scientifically meaningful cohort or ‘bin’ of individuals with partial 
function mutations, sweat chloride < 80 mmol/L, and/or pancreatic sufficiency. This protocol is designed 
to compare in vitro responsiveness of differentiated iPS cells to in vivo clinical activity of TrikaftaTM, and 
will test the null hypothesis that the true TrikaftaTM response rate of CFTR individuals with partial 
function mutation genotypes is ≤ 10% vs. the alternative hypothesis that the true TrikaftaTM response 
rate is ≥ 40%. We will initially enroll four subjects, and expect all of these individuals will show a clinical 
response (e.g., > 5% improvement of % predicted FEV1 from baseline). If none of the first four subjects 
exhibit a response, the study will be terminated. Otherwise, we intend to enroll a total sample size of 22 
individuals. Subjects who do not complete the study will be replaced. In this design, if TrikaftaTM is 
ineffective, the probability of early termination after four subjects is 66% and only a 5% chance of 
concluding the drug is effective (type I error). If TrikaftaTM is effective, there is an 86.4% chance of 
reaching this conclusion. If fewer than four patients show a clinical effect, TrikaftaTM will be considered 
ineffective. To demonstrate the ability of iPS cells to predict clinical benefit, 22 patients will provide at 
least 80% power to detect a significant correlation of at least r = 0.55 between the changes in clinical 
outcomes with changes in short circuit current as measured in iPS cells. Tabular details of this analysis 
are provided below. Power calculations were performed using PASS v. 14 (Kaysville, UT) with a two-
sided Z-test and a significance level of 0.05.
Cumulative # of 
responses
Decision
95% Confidence Interval (CI)
Stage 1: Enter 4 
subjects
0
Terminate the trial because 
the agent is ineffective.
Response rate is ≤10%
LB of 95% CI is < 10%
At least 1
Inconclusive result, continue 
trial (proceed to stage 2).
95% CI contains 10% and the 
UB is < 40%

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Substudy 2: Patients with N1303K CFTR. 
The primary analysis will be to estimate the mean absolute change in sweat chloride concentration from 
baseline through day 28 among the adolescents and adults with at least one copy of an N1303K 
mutation and a minimal function mutation who are not currently modulator eligible. A one-sided 0.025 
alpha level single sample t-test comparing this estimate to no change will be reported, along with 
corresponding confidence intervals. A complementary analysis evaluating the percentage of participants 
with ≥15 mmol/L decline will also be reported. With a previously estimated 9.7 mmol/L standard 
deviation of the change in sweat chloride for successful modulator trials previously (Boyle et al., 2014; 
Vermeulen et al., 2017), an anticipated 6% of responders would be expected by chance alone. 
Therefore, the treatment response prevalence will be compared to a reference rate of 6%. For 
longitudinal sweat chloride through day 28 and for secondary endpoints (absolute change in ppFEV1, 
CFQ-R respiratory domain, and weight/BMI from baseline through day 28), means at each time point 
and changes over time will be summarized along with corresponding confidence intervals estimated by 
mixed effects models to account for within-subject correlation.
Changes in sweat chloride following treatment with ETI will be correlated with iPS cell responses in vitro, 
in addition to secondary clinical outcomes measures. A non-linear Spearman’s correlation coefficient 
will be estimated for each measure paired with iPS cell responses in vitro, with corresponding fitted 
curves displayed graphically along with coordinates for participant’s paired in vivo and in vitro 
responses. We expect variation both in sweat chloride and in iPS cell short circuit current measurements 
(Isc) due to heterogeneity in genotypic profiles among participants in the study. Sample selection for iPS 
cell experiments will be coordinated across studies to optimize power and unbiasedness as specified in a 
sample selection plan.
Sample Size
With a sample size of 20 participants, we anticipate 99% power to detect a mean sweat chloride 
decrease of 10 mmol/L assuming a standard deviation of 9.7 mmol/L (Boyle et al., 2014; Vermeulen et 
al., 2017) under a one-sided 0.025 alpha-level t-test. With a higher standard deviation of 15 mmol/L, the 
power decreases to 81% to detect a mean decrease of 10, but we retain 98.9% power to detect the 
hypothesized mean decrease of 15 mmol/L.
SAEs and AEs will be tabulated using standard coding terms sorted by System Organ Class (SOC). The 
incidence of AEs will be tabulated by seriousness and severity. The number of SAEs and AEs will be 
Stage 2: Enter 
18 additional 
subjects
4 or less
(stage I + stage II)
Consider that the agent is 
ineffective. Response rate is 
≤10%
LB of 95% CI is less than 10%
5 or more
(stage I + stage II)
Drug may be effective
Response rate is greater than 
10% and not different from 
40%
95% CI contains 40% and LB 
is > 10%
14 or more
(stage I + stage II)
Drug is effective
Response rate is at least 40%
95% CI LB ≥ 40%
LB = Lower Bound, UB = Upper Bound

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summarized as follows: (i) The proportion of subjects with at least one (S)AE, (ii) The average number of 
(S)AEs per subject, and (iii) The rate of (S)AEs per subject week of follow-up.
Expected Outcomes Subjects will be recruited by study investigators as above, including through the CF 
Foundation Therapeutic Development Network resource. Please note that based on considerable past 
experience conducting clinical trials involving CF subjects at both participating institutions, we do not 
have concerns regarding our ability to perform the patient-oriented study described here, or to 
propagate and characterize iPS cells (Crane et al., 2015; Mou et al., 2015; Hawkins et al., 2017; Jacob et 
al., 2017). The protocol will furnish one test of our hypothesis that thresholds of CFTR activity in 
differentiated iPS cells encoding partial function mutations in vitro or in vivo predict benefit following 
TrikaftaTM in vivo. If a strong correlation can be established between TrikaftaTM response in vitro and in 
vivo (e.g., FEV1) in both substudies, the clinical results will provide early evidence for possible usefulness 
of in vitro surrogate models in this setting. 
Scientific Rigor All in vitro and in vivo studies will be conducted with appropriate “n” so that 
therapeutically relevant conclusions can be drawn. Multiple biologic or technical replicates will be 
performed and evaluated in blinded fashion by an experienced statistician. CFTR mutations are equally 
prevalent in both sexes, and we expect iPS cells studied in human subjects carrying partial function 
mutations will reflect male and female individuals equally. 
Summary Many CF patients who might substantially benefit from new and precise modulator 
treatments do not have access to these drugs. Off-label prescribing of expensive new agents is often 
not reimbursed by third-party payers. This project will test new in vitro predictive thresholds using iPS 
cells for CFTR rescue in vivo, and furnish a body of knowledge relevant to such issues among individuals 
with rare or even private CF alleles. We will also specifically evaluate patients with CF encoding 
N1303K for evidence of clinical benefit attributable to Trikafta™. 
12. Confidentiality:

Participant confidentiality and privacy will be strictly held in trust by the participating 
investigators, their staff, and others responsible for the intended interventions. This 
confidentiality is extended to cover testing of biological samples and other tests in addition to 
the clinical information relating to participants. Therefore, the study protocol, documentation, 
data, and all other information generated will be held in strict confidence. No information 
concerning the study or the data will be released to any unauthorized third party without prior 
written approval. 

All research activities will be conducted in as private a setting as possible.

The clinical investigators, other authorized representatives, representatives of the Institutional 
Review Board (IRB), or regulatory agencies may inspect all documents and records required to 
be maintained by the investigator, including but not limited to, medical records (office, clinic, or 
hospital) and pharmacy records for the participants in this study. The clinical study site will 
permit access to such records.

The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 
secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
other requirements.

Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored securely. This will not include the participant’s 
contact or identifying information. Rather, individual participants and their research data will be 

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identified by a unique study identification number. The study data entry and study management 
systems used by clinical sites research staff will be secured and password protected. At the end 
of the study, all study databases will be de-identified and archived by the PI. With regard to 
other patient related aspects of confidentiality, all interviews and procedures will be held in a 
private room.

Identifiers will be destroyed 3 years from the date of the Federal Financial Report (FFR).

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