Safety and Viability of an E. coli Nissle Colibactin Knockout in Healthy Volunteers: A Randomised Controlled Safety Study

E. coli Nissle 1917 (EcN) is a well-established, safe human probiotic. Over
the past years, many bioengineering strategies for this strain have been
developed and functionally enhanced E. coli Nissle strains have been used in
several clinical trials. However, it has been shown that EcN harbors a cluster
of genes coding for the biosynthesis of hybrid nonribosomal
peptide-polyketide(s), so-called pks-islands, ultimately leading to the
production of colibactin. St udies have shown that colibactin causes cell cycle
arrest, DNA double-strand breaks, and senescence in mammalian cells. Moreover,
colibactin-producing E. coli accelerate tumor progression in multiple mouse
models. To create a safe platform strain for future advanced microbiome
therapeutics (AMT) development, the colibactin peptidase (ClbP) gene has been
removed from EcN. In mice models, safety of this EcN colibactin knockout
(EcNΔClbP) has been shown, as well as a reduced capacity to colonise the gut,
which can be beneficial in therapeutic settings . To assess the safety of the
EcNΔClbP strain in humans and compare its kinetics compared to the wildtype
EcN, we here propose a randomised controlled safety study in healthy
volunteers.

Assess the safety and tolerability of the EcNΔClbP strain relative to the EcN
wildtype strain following a 1-week daily administration, through
questionnaires, medical examination and measurement of inflammatory markers.

Double-blind, randomised, controlled, safety study.

To study the above objectives, a double-blind randomised controlled
intervention study will be performed within the department of Internal and
Vascular Medicine of the Amsterdam UMC, location AMC. The complete study
duration is 2 weeks (1 week of intervention, followed by 1 week of follow-up).
During this period, subjects are requested to visit the AMC 3 times (and once
for the screening). A more detailed overview of the study visits can be found
below, followed by a schematic overview of the study design (Figure 1).
3.2 Study visits
Subjects will visit the AMC four times in total. Each visit will take 30-60
minutes (maximum of 4 hours over 2 weeks). A more detailed description of all
measurements and procedures can be found in section 7.3 Study procedures.

Visit 1: screening
During the first visit, oral and written information on the study will be given
to the participant after which informed consent will be obtained. Thereafter,
subjects will be screened for eligibility through examination of their medical
history, a physical examination and analysis of blood safety parameters.
After inclusion, subjects will receive the necessary material and instructions
regarding the faeces collection and the nutritional diary. Before visit 2,
subjects will be randomly assigned to either the EcNΔClbP strain or the
wildtype EcN strain. One week before visit 2, subjects will start wearing the
CGM device, collect a faeces sample and start recording their dietary habits
using an online dietary booklet (https://mijn.voedingscentrum.nl/nl/eetmeter).

Visit 2: baseline visit
Within 3 months of the screening, subjects will come fasted to the AMC,
bringing the 24h faeces and a fresh faecal sample with them. These will be
stored at -80° C until analysis. During the visit, subjects will undergo a
physical examination (length, weight, blood pressure, body impedance analysis)
and fasting venous blood will be drawn. Subjects will complete questionnaires
on the Bristol stool chart and GI comfort, dietary habits/eating behaviour and
QoL questionnaires, after which they receive the allocated study intervention.
Subjects will ingest the EcN study product daily around the same time for 7
days. Subjects are asked to collect faecal samples, wear a CGM device and to
record their dietary habits over the next 14 days using an online dietary
booklet (https://mijn.voedingscentrum.nl/nl/eetmeter).

Visit 3: intervention visit
After one week, subjects will come fasted to the AMC, bringing the 24h faeces
and a fresh faecal sample with them and any unused EcN study product. The same
measurements and questionnaires from visit 2 will be repeated. There will be
special attention for clinically relevant (serious) adverse events ((S)AE).
This will be the last day of the study intervention, which is followed by a
one-week washout period.

Visit 4: follow-up visit
After one week, subjects will come fasted to the AMC, bringing the 24h faeces
and a fresh faecal sample with them. The same measurements and questionnaires
as during visit 2 and 3 will be repeated and again, there will be special
attention for clinically relevant (serious) adverse events ((S)AE). This is the
last study visit.

The investigational products are the EcNΔClbP strain and the active comparator
is the wildtype EcN strain. The wildtype EcN strain is a well-established, safe
human probiotic and has been used for over a century [3]. Participants will be
randomised to the intervention or control group in CASTOR. Subjects in the
intervention group will receive 1011 CFU of the EcNΔClbP strain daily for 7
days. In the control group, subjects will receive 1011 CFU of the wildtype EcN
strain daily for 7 days. Both strains will be produced under HACCP guidelines
by NIZO, Ede, the Netherlands

Subjects will visit the AMC (fasted) four times (30-60 min per visit): visit 1
= screening; visit 2 = baseline measurements + start intervention; visit 3 =
follow-up at week 1; visit 4 = follow-up at week 2. During these visits a
physical examination will be performed, questionnaires will be filled out and
blood is drawn, 14-32 ml per visit (110 ml in total). In addition, subjects
will be asked to collect faeces at home, wear a CGM device during the study and
keep a food diary during 2 weeks. Subjects will receive ¤200 as compensation
for their participation as well as reimbursement of their travel expenses.
The risks associated with participation can be considered negligible. The
potential side-effects of the EcNΔClbP strain are expected to be equal or less
prominent compared to the wildtype EcN. The wildtype EcN strain is a
well-established, safe human probiotic and has been used for over a century.
The most common side effect that has been reported is flatulence after
initiation of treatment, whereas abdominal pain, gut noises, loose stools,
diarrhoea, nausea and vomiting may very rarely occur. The amount of blood drawn
is below the maximum 500 mL per day; other study procedures are considered
harmless and will not cause any physical or psychological discomfort.