Development of individual therapy for cystic fibrosis using primary epithelial tissue cultures

Cystic fibrosis is characterized by a mucosal immunodeficiency, resulting from
genetic mutations in CFTR of which almost 2000 are known 1. CF disease (and
response to therapy) is highly heterogeneous, which is caused by mostly unknown
interactions between the
mutant CFTR, the patient-specific genetic background and the environment. For
many mutations, the functional impact of the mutation on CFTR remains largely
unknown. In addition, variability in CFTR expression and function in subjects
with similar CFTR mutations has also been clearly observed but relations with
disease severity and underlying mechanisms are mostly unknown, albeit suggested
in previous studies by us and others 2-4.

CF subjects are meticulously clinically phenotyped (lung function, BMI,
colonization with pathogens, bronchial alveolar lavages, CFTR function
measurements in rectal biopsies of newborns or subjects with rare mutations) as
to start symptomatic treatments as early as needed, all as part of standard
care. Currently, new drugs that can directly restore the CFTR protein - the
underlying cause of disease - cause a paradigm shift in the field 5-7. These
novel treatments can achieve life-changing effects, but act in a mutation and
patient-specific fashion. The majority of subjects do not yet receive these
therapies, but it is expected that many more subjects will receive these type
of treatments in the near future, and that treatment should start as early as
possible as to prevent disease expression. Approximately 5% of subjects in our
center now receive these treatments, and a larger group (~50%) is expected to
receive treatment in the near future depending on regulatory approval. As for
treatment, earlier treatment is expected to most effectively limit disease.

Biomarkers that accurately predict long-term individual treatment efficacy are
lacking 8,9. Current biomarkers used for these drugs are based on experiences
with symptomatic treatments (e.g. change in lung function) or are individual
readouts suited for CF diagnosis (e.g. sweat chloride concentration, nasal
potential). Their value for CFTR targeting treatments has been demonstrated at
the group level. These biomarkers can only be measured after in vivo
application of drugs, and are associated with significant intra- and
intersubject variation that prevent accurate measurement of individual drug
efficacy 8. Especially for young children biomarkers are lacking. Novel
biomarkers that facilitate a more accurate quantitation of treatment on
individual CFTR function are needed, as to select and develop optimal treatment
strategies in young children to prevent disease expression on the long run.

We have generated proof-of-concept that CF subjects can be preclinically
selected for treatment by studying their drug response in in vitro intestinal
stem cell cultures, and observed that responses of drugs in intestinal cells at
the group level correlate with published outcome measures of in vivo clinical
trials 2 (and Dekkers et al, 2016, Science Translational Medicine). However, we
also observed that the prediction of the pulmonary response to treatment in
individual patients using intestinal cells can be difficult, and we hypothesize
that similar approaches using primary airway cultures may be more suited for
the prediction of individual drug efficacy in the airways. A collection of
paired epithelial tissue samples (rectal, nasal, bronchial) from individuals
will be essential to directly compare their in vitro assay performance as well
as to study their potential value for prognostic typing of individual disease
severity and drug efficacy. Furthermore, in order to study intra-subject
variability, we aim to collect rectal biopsies and generate intestinal
organoids of 10 subjects with CF. Responses to drugs between the *new* and
*old* culture of organoids will be compared.

In addition to in vitro testing of pure drugs on an individual tissue, we also
observed that the activity of these drugs can be measured in plasma of patients
upon treatment 10. This approach will help to study human variability in the
pharmacokinetic properties of the treatment, i.e. which subjects have high
circulating and low circulating levels of drugs, and how does this relate to in
vivo treatment. By including plasma samples from patients, and test these
before and after treatment start, we have the ability to study how the
individual tissue in the lab responds to pure drugs, and how these drug
responses compare to drug levels in the circulation after in vivo treatment,
and how tissues from individuals that do not receive treatment respond to
circulating drug levels from subjects that already receive these drugs. This
combined pharmacokinetic and pharmacodynamic modelling is already helping
clinical decision-making in clinical trials and for off-label treatments of
CFTR-modulating compounds, facilitating the adaptation, continuation or
cessation of treatments at an individual basis. This process may significantly
improve by using individual airway cultures.

We will collect airway tissue during diagnostic bronchoscopy of subjects with
CF: nasal cells via brushing, bronchial cells via brushings and pulmonary
biopsies. For most subjects, intestinal cultures have been already collected
and biobanked in collaboration with HUB. If rectal biopsies are not available,
we will collect these as well. For 10 subjects with CF with known differences
in response to therapy based on previous organoid measurements, additional
rectal tissue is collected to address intra-subject variability. In addition,
10 non-CF patients (0-18 yrs) will be included as a control group.
It is essential to focus on young subjects as these studies aim to develop
prospective diagnostic tools for young children, and the adult CF airway
epithelium is affected by long-term disease, leading to potential bias of
results.

Materials are coded to study correlations between data generated in the
laboratory and observations during clinical follow up in the presence or
absence of treatments. People are approached to biobank surplus material for
future scientific studies using a separate TcBio protocol and informed consent.
Without biobank consent, all patient*s material collected for this study will
be destroyed at the end of the study.

Airway epithelial stem cell cultures are generated using published protocols
that allow the apparent unlimited expansion in vitro of patient-specific airway
cells (from nasal and bronchial sources) 11. These culture models facilitate
the study of CFTR expression and function using conventional molecular,
electrophysiological and fluid secretion assays which are all operational in
the lab.

We will also use the generated cell cultures and the bronchial biopsies from
these subjects for proof of concept studies that aim to characterize mechanisms
associated with variability in cystic fibrosis disease and response to therapy.
Our previous studies in intestinal cells clearly demonstrate that the
patient-specific genetic background impact the CF phenotype (e.g. swelling of
organoids) and response to therapy independent of the CF-causing mutations 2
(and Dekkers et al 2016, Science Translational Medicine). In this project we
focus on the functional characterization of CFTR-dependent epithelial cell
functions (epithelial barrier function, electrophysiology and fluid secretion),
and how these are modified by pathogens, or pharmacological and genetic
interventions that modify CFTR function or (potential) modifier genes such as
alternative ion channels or transcription factors that control CFTR expression.
This will involve DNA, RNA, protein and metabolic expression profiling in the
context of laboratory interventions (DNA gene editing and therapy, RNA editing,
and pharmacological interventions such as siRNA and small molecule treatments).
It implies that we may identif

The overall aim of the study is to improve diagnostic and therapeutic options
for people with CF, especially in the context of young children and novel
CFTR-targeting drugs.

Primary Objective:
1. to demonstrate relations between CFTR genotype, residual function and
response to therapy in vitro using airway and intestinal biopsies and cultures,
and to correlate these to clinical disease development before and after
initiation of CFTR-targeting treatments.

Secondary Objective(s):
1. to establish proof of concept for novel mechanisms that contribute to
disease variability between people with CF
2. to establish novel epithelial cell culture technologies for the study of CF
disease

This is a monocenter, invasive observational study. 20 children with CF will be
included in the study as well as 10 non-CF subjects.
The total study duration is expected to be 4 years. Study duration for each
individual patient will be 1 day, consisting of 1 visit at the hospital for
their bronchoscopy. An interim analysis will be performed after the first 10
subjects with CF have participated, to determine whether we are able to culture
the pulmonary, nasal and intestinal materials.

Subjects who are undergoing a bronchoscopy as part of their care will be asked
to participate in this study. In addition to their scopy the physician and
designated study team member will collect all samples required for the conduct
of this study (lung biopsies, lung brushings, nasal brushings, rectal biopsies
and blood).

Subjects will be asked to store their airway tissues and cultures in the UMCU
lung biobank (in progress) and the rectal organoids in HUB-CF biobank

Collecting lung and intestinal biopsies from a minor involves the risk of
temporary bleeding in a limited amount of subjects. Brushing of the nasal
cavity can cause irritation of the nasal mucosa, which will disappear within a
few hours. These materials provide the opportunity to develop individual-based
biomarkers to investigate pulmonary disease, and individualized medicine, and
is especially relevant for young children for whom these are currently lacking.
Lung disease and therapy is highly heterogeneous in CF, and subject are
expected to benefit most from individually tailored therapies that already
start at young age. These studies provide opportunities to discover new
biomarkers and drug targets for treatment of CF. Although we do not expect
direct benefits of this study for the patients, the validation of airway
cultures for prognostic drug efficacy testing and the potential collection of
their tissue in a biobank may help to establish new diagnostic and therapeutic
options for CF. At later stages, personalized treatment strategies using these
biobanked materials may be realized at an improved accuracy as compared to
current procedures using individual intestinal cultures.
The extension of the time under anesthesia is minimal (around 10 minutes
extra). The study has a minimal risk and minimal burden in total.