Encapsulated Faecal Microbiota Transplantation to Preserve Residual Beta Cell Function in Patients with Recently-Diagnosed Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by a
progressive beta cell destruction and subsequent insulin dependence in the
first 2-3 decades of life. In the last thirty years, the incidence of T1D has
tripled, leading to a lifelong increase of considerable morbidity and mortality
in those affected compared to healthy subjects. In fact, T1D diagnosed in
childhood leads to an almost 20 year loss of life-expectancy, which is more
than most childhood cancers. Notwithstanding decades of intensive research in
animals, the environmental factors driving T1D are still unknown and
therapeutic strategies have invariably failed to halt disease progression.

As the increased T1D incidence is primarily observed in subjects who are not
genetically predisposed, environmental factors including altered diet,
antibiotic use as well as mode of birth have been suggested to play a role, and
these factors have invariably been linked to changes in the gut microbiome.
Indeed, an altered composition of the faecal microbiota composition has been
observed in adolescent T1D patients. Moreover, an increased amount of
pathogenic bacterial species has been identified in faecal samples of T1D
patients at the time of diagnosis. Interestingly, this altered faecal
microbiota is already present before the clinical onset of T1D and is related
to islet autoantibodies.

Non-obese diabetic (NOD) mouse studies suggest that interaction of intestinal
microbes with the innate immune system is a critical factor in developing T1DM,
most likely by inducing an altered T-helper cell type 17 (Th17) population in
the small intestinal lamina propria. One of the current hypotheses linking the
gut microbiome to immunological tone is production of microbial metabolites
such as the short-chain fatty acids (SCFAs). Production of these compounds is
indeed altered in T1D, and butyrate (one of the best known SCFA) is known to
stabilize T-cell function in mice. Furthermore, irritation of the pancreatic
duct by microbiota in the proximal gut may contribute to beta cell
inflammation. By introducing beneficial intestinal microbiota to the proximal
gut, the organisms or their metabolites that alter immunological tone and
irritate the pancreatic duct may be attenuated, resulting in improved beta cell
function and restoration.

We hypothesized that if one is able to shape the (small) intestinal microbiota
with faecal microbiota transplantation (FMT), it may be possible to stabilize
or even reverse β-cell destruction, thereby reducing exogenous insulin needs
and subsequently associated complications in T1D. Previously, we showed that an
FMT can significantly alter the intestinal microbiota composition for a period
of 7-8 weeks. Additionally, it is a safe procedure that has been performed >500
times in our institute without any procedure-related adverse events. Therefore,
we initiated in 2013 a randomized pilot trial comparing the efficacy of
repetitive allogenic (healthy donor) vs. autologous (own) FMT on residual β-
cell function in new-onset T1D (DIMID trial). Newly-diagnosed T1D patients were
included and randomized to the allogenic (n=10) or autologous FMT group (n=10).
Moreover, healthy age and sex matched donors were used for the allogenic FMT.

Surprisingly, autologous FMT had a significant (p<0.01) effect on preserving
residual β-cell function as determined by stimulated C-peptide response upon a
mixed-meal test after 12 months. Interestingly, the allogenic FMT from a
healthy donor had a less obvious beneficial effect and showed overall a similar
β-cell decline as seen in other trials with placebo use (see figure 1) [19]. We
have found several changes induced by both donor and autologous FMT on gut
microbiome composition and identified several bacterial strains and plasma
metabolites and T-cell signatures that predicted response to FMT.


The positive impact of the autologous FMT on the beta cell function provides an
interesting opportunity to stabilize the beta-cell destruction and extend or
even bring back the honeymoon period (wherein individuals with recently
diagnosed T1D remain well-regulated with minimal doses of insulin). Especially
since the use of autologous FMT comes with even less risks than the use of
healthy donor FMT (transmission of potential unknown pathogens). However, the
current FMT procedure whereby the faecal suspension is administered through a
nasoduodenal tube is quite invasive, time consuming and causes patient
discomfort. In contrast, the use of encapsulated autologous FMT is
non-invasive, causes no discomfort and can be simply administered in the home
setting. If successful, the use of autologous FMT capsules provides a safe and
feasible option to treat recently-diagnosed T1D patients on a daily basis to
contribute to the preservation of beta cells in recently diagnosed T1D patients.

In the last years we worked on setting up the production of lyophilized FMT
(LFMT) capsules at Amsterdam UMC. We optimized the freeze-drying process and
determined the viability of the faecal microbes throughout the processing steps
and during storage. The lyoprotectant trehalose 10% in combination with
maltodextrin 5% appeared to provide the highest bacterial viability and
preserved the microbial composition best during freeze-drying. The optimal
freeze-drying time was determined to be 48 hours at -80°C. As expected, during
preparation of the LFMT capsules the bacterial viability decreased slightly,
especially after freeze-drying and encapsulation. This small decrease in
viability after freezing and freeze-drying is a known phenomenon and impossible
to avoid. Fortunately, upon restitution of the microbes in the small intestine
the viable microbes are able to multiply and engraft, which has been shown in
previous studies. Moreover, we found the faecal bacteria in frozen FMT and LFMT
capsules were stable during storage in the freezer, without any relevant
decreases in bacterial viability.

We thus propose to combine our data from the previous fresh autologous FMT
study with our current developments in preparing autologous LFMT capsules to
stabilize further beta cell degradation in T1D subjects with residual beta cell
function. We will investigate whether the beneficial effect of autologous FMT
extents towards recently-diagnosed (0.5-3.5 years) T1D patients, as it is known
that beta cell degradation and loss of function is progressive for the first
5-7 years and thereafter stabilises. During this time-period individuals lose
their so-called honey-moon period where they become increasingly dependent on
intensive insulin use and glucose monitoring. Therefore, it is conceivable that
this patient group benefits from beta cell function preserving therapies and
makes this application a careful but important step towards a beta-cell
preserving therapy in T1D based on modulation of the gut microbiome.

In this study we will confirm that a microbial intervention based on capsules
containing autologous (own) lyophilized faecal matter (LFMT), taken daily for 3
month has beneficial effects on residual beta cell function (C-peptide
secretion upon MMT) in recently diagnosed type 1 diabetes mellitus (0.5-3.5
years). A parallel objective is to assess the impact on glycaemic control and
see which small- as well as large intestinal (faecal samples) microbiota are
associated with these clinical changes over time.

Study Design
This is an open label single-arm, single-centre study. During a run-in period
of 3 months, stools of the participants will be collected and processed into
freeze-dried faecal microbiota capsules. After the run-in period, participants
will ingest the autologous FMT capsules daily for 3 months, which will be
followed by a follow-up period of 3 months. In total, participants will be
followed for 9 months after inclusion.

Study visits
Subjects will visit the AMC 4 times in total. Each visit will take 180 minutes
(maximum of 12 hours over 9 months). A more detailed description of all
measurements and procedures can be found in section 7.3 Study procedures.

Visit 1: Screening and run-in visit (-3 months)
Patients will be screened for suitability in the GUTDM1 study (NL73189.018.20,
METC_2020_105) and invited to participate. At every visit of the study a MMT
will be performed, blood will be drawn for basic biochemistry and patients will
hand in the food diaries, questionnaires, 24-hour urine and faeces. In
addition, we measure height, weight, blood pressure and calculate BMI. As the
faeces is needed to produce the autologous FMT capsules, participants are
instructed collect at least 150 grams of fresh faeces. If the fresh faeces is
insufficient (<150 g), participants are asked to collect more fresh faeces and
bring this to the AMC at a later date (but before the baseline visit).

Visit 2: baseline visit (0 months)
At this visit, a gastroscopy is performed and 6 duodenal biopsy*s are
collected. The MMT will be repeated, blood will be drawn for basic biochemistry
and patients will hand in the food diaries, questionnaires, 24-hour urine and
faeces. In addition, we measure height, weight, blood pressure and calculate
BMI again. Thereafter, the participant will start with the LFMT capsules for 3
months (84 ±6 days). The first capsule is ingested with the investigator
present to ensure the participant tolerates the capsules well. After this visit
the participant is contacted biweekly to ensure adherence.

Visit 3: follow-up visit (3 months)
During this visit, the gastroscopy is repeated and 6 duodenal biopsy*s are
collected. The MMT will be repeated, blood will be drawn for basic biochemistry
and patients will hand in the food diaries, questionnaires, 24-hour urine and
faeces. In addition, we measure height, weight, blood pressure and calculate
BMI again. Participants stop with the LFMT capsules and return any remaining
capsules to the investigator.

Visit 4: follow-up visit (6 months)
During this last visit, the MMT will be repeated, blood will be drawn for basic
biochemistry and patients will hand in the food diaries, questionnaires,
24-hour urine and faeces. In addition, we measure height, weight, blood
pressure and calculate BMI again.

After the 3 month run-in phase of the study, patients will be treated on a
daily basis with capsules containing lyophilized autologous faecal matter for a
period of 3 months. During the run-in phase participants will collect their
stool, which will be processed into the autologous LFMT capsules containing
approximately 1 gram stool per capsule, and stored at -80°C. Participants store
the capsules at home in the freezer and ingest one capsule per day for a period
of 3 months (84 ±6 days). Capsules are advised to be ingested with a glass of
water on an empty stomach in the morning, preferably an hour before breakfast
(while still fasted).

Participant may benefit in terms of helping to further unravel the relation
between the gut microbiome and residual beta cell function. The DIMID study was
very promising, suggesting that manipulation of the gut microbiome may indeed
preserve the residual beta cell mass. Participants may benefit from an
increased residual beta cell function, which is associated with a lower risk of
diabetic complications and hypoglycaemia. In the long-term, the FMT procedure
may be refined to a probiotic formation or a combination of potent microbial
metabolites and antigens that induce immune-tolerance.

For the mixed meal test, morning long- and short acting insulin must be
withheld. This carries a small risk of hypo- and hyperglycaemia. The study
participants will be carefully instructed and no major risk is expected in the
context of this procedure. The placing of the intravenous cannula in our study
can be an unpleasant experience for the subjects and may result in
(self-limiting) bruising. The amount of blood drawn (70 mL) is far below the
maximum 500 mL per day; other study procedures (such as faeces collection or
dietary diary) are considered harmless and will not cause any physical or
physiological discomfort.

Gastroscopy is a procedure associated with discomfort, but when participants
are well fasted it is very safe. The required fasting is associated with a
small risk of hypoglycaemia, for which participants will be adequately
instructed. In the age of continuous glucose monitoring this risk is also very
low.

In our centre, FMT procedures have not been associated with major adverse
events and only minor gastrointestinal symptoms were reported by recipients.
Compared to an allogenic FMT from healthy donors, the autologous FMT approach
used in this study comes with no risk for pathogen transmission. In addition,
the LFMT capsules are not invasive, cause almost no patient discomfort and can
be easily ingested by the patient as compared to the fresh FMT administered.