Clinical trial NCT05764239
Conditions: Phenylketonuria
Keywords: PKU Inborn error of metabolism Synpheny Synlogic Phe Metabolic
Sponsor: Synlogic
Start date: 2023-07-05; Completion date: 2024-03-15
Enrollment: 35 participants; Allocation: RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
SYNB1934-CP-003 was designed as a 3-part, adaptive study consisting of a dose-escalating, open-label period (DEP; Part 1) of up to 15 weeks, followed by a 4-week, double-blind, placebo-controlled, randomized withdrawal period (RWP; Part 2), and an open-label extension (OLE; Part 3) of up to 36 months

Detailed description:
In the DEP, all enrolled participants maintained a stable diet reflecting their baseline phenylalanine (Phe) intake and received escalating doses of SYNB1934v1 from approximately 3 to 15 weeks to determine an individually titrated dose (iTD), which was defined as the highest dose the participant was able to tolerate. A participant was defined as having reached an iTD if they tolerated 3 weeks at a dose, regardless of whether other doses were tolerated.

Blood Phe level was measured at each dose level after 3 weeks at that level. A responder was defined as a participant who achieved a ≥ 20% reduction in blood Phe level compared to DEP baseline on SYNB1934v1.

Participants who completed at least 3 weeks at their iTD during the DEP entered a 4-week RWP in which they were randomized 1:1 to receive SYNB1934v1 at their iTD determined in the DEP or placebo TID. Randomization was stratified on screening Phe level. Participants remained on their assigned dose (iTD of SYNB1934v1 or matching placebo) for the duration of the RWP, unless they developed intolerance or met other discontinuation criteria, and remained on the same diet they consumed during the DEP. Blood Phe level was measured at Weeks 1, 3, and 4 of the RWP.

Participants who completed the 4-week RWP may have entered the OLE and received SYNB1934v1 for up to 36 months. During the OLE, participants completed a dose ramp to their iTD over time guided by tolerability. The iTD in the OLE may have been different from the iTD in the DEP. The investigator may have escalated the SYNB1934v1 dose up to 1 × 10\^12 live cells based on tolerability. Participants were allowed to modify their standard diet, with guidance from the investigator, if their blood Phe level was \< 240 µmol/L.

Arms: arm 1: Participants received SYNB1934v1 orally immediately after meals on the following dose-ramp regimen:

Dose level 1 (Days 1-9): 3 × 10\^11 live cells partial dose up to 3 times daily (TID); Dose level 2 (Weeks 4-6): 6 × 10\^11 live cells up to TID; Dose level 3 (Weeks 7-9): 1 × 10\^12 live cells up to TID. arm 2: Participants who completed the DEP were randomized 1:1 to receive SYNB1934v1 at their iTD established in the DEP orally immediately after meals. Participants remained on this dose of SYNB1934v1 for the duration of the RWP; doses of SYNB1934v1 were not permitted to be modified during the RWP. arm 3: Participants who completed the DEP were randomized 1:1 to receive placebo orally immediately after meals. Participants remained on the same dose of placebo for the duration of the RWP; doses of placebo were not permitted to be modified during the RWP. arm 4: Participants completed a dose ramp to their iTD guided by tolerability, as described for the DEP, including the full dose-ramp schedule. The iTD in the OLE may have been different from the iTD in the DEP or RWP. The investigator may have escalated the dose of SYNB1934v1 up to 1 × 10\^12 live cells based on tolerability; multiple attempts to escalate to a higher dose level were permitted per investigator discretion.
Interventions: intervention 1: SYNB1934v1 intervention 2: Placebo
Intervention descriptions: intervention 1: SYNB1934v1 consisted of powder for oral suspension packaged in sachets. During dose preparation, the powder was resuspended in water or apple juice prior to administration. intervention 2: Placebo was manufactured using an inactive powder that was color matched to the SYNB1934v1 drug product. In order to maintain study blinding during the RWP, placebo was packaged, labeled, stored, and administered in an identical manner to SYNB1934v1.

Protocol documents:
Prot_000.pdf: 
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CLINICAL STUDY SYNB1934-CP-003 
Protocol Version 4.0_US: 01 July 2023 
 
Study Title: 
A Phase 3, Double-blind, Placebo-controlled, Randomized Withdrawal Study to 
Evaluate the Efficacy and Safety of SYNB1934 in Patients with PKU 
(SYNPHENY-3) 
Study No.: 
SYNB1934-CP-003 
Short Title: 
Synpheny-3 
Study Phase: 
2b/3 
Product Name: 
SYNB1934v1 
IND No. 
IND 27340 
EU Trial No.  
2022-502932-37-00 
HC Control No. 
273002 
Indication: 
Phenylketonuria 
Sponsor: 
Synlogic Operating Company, Inc. 
301 Binney Street, Suite 402 
Cambridge, MA 02142 USA 
Phone: +1 (617) 401-9975 
CONFIDENTIAL INFORMATION: This protocol contains trade secrets and other confidential information. 
Accordingly, this protocol shall be treated as confidential and restricted to its intended use, namely, the guidance of 
the clinical investigator. This material is the sole property of Synlogic Operating Company, Inc.. and shall not be 
disclosed or used by others except as authorized in writing by Synlogic Operating Company, Inc.. This material may 
be disclosed to and used by your staff and associates only to the extent necessary to conduct the clinical study. 
 
 
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Protocol SYNB1934-CP-003 
 
 
 
Version 4.0_US, 01 July 2023 
 
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Revision History 
Version 
Date 
Reason for Change 
1.0 
06 December 2022 
Original version 
2.0_CA 
2.0_EU 
 
2.0_IL 
2.0_TR 
2.0_GE 
21 February 2023; Canada 
21February2023; European 
Union 
21 February 2023; Israel 
21 February 2023; Turkey 
21 February 2023; Georgia 
 
• 
Addition of a minimum number of participants 12-17. 
• 
More complete description of dose ramping in OLE. 
• 
Clarification of statistical measures. 
• 
Removal of optional TCA substudy. 
• 
Simplification of pregnancy testing 
3.0_US 
08 May 2023, United States 
Description of dose ramping in OLE, clarification of statistical 
measures, removal of TCA substudy. Response to regulatory 
guidance: addition of weekly Phe substudy and alteration of 
inclusion age. [Note: Version 2.0 was not released in the US; 
Version 3.0 captures all changes made in Version 2.0] 
4.0_US 
01 July 2023, United States 
Timing of diary card collection, alteration of AESI criteria, 
clarification of responder population. 
 
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Version 4.0_US, 01 July 2023 
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SIGNATURE PAGE (SPONSOR) 
I have read and understand the contents of the clinical protocol for Clinical Study 
SYNB1934-CP-003 Version 4.0_US, dated 01 July 2023, and agree to meet all obligations of 
Synlogic Operating Company, Inc., as detailed in all applicable regulations and guidelines. In 
addition, I will ensure that the lead investigators are informed of all relevant information that 
becomes available during the conduct of the study. 
Synlogic Operating Company, Inc. 
Date 
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Protocol SYNB1934-CP-003 
 
 
 
Version 4.0_US, 01 July 2023 
 
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LEAD INVESTIGATOR’S AGREEMENT 
I have read and understand the contents of the clinical protocol for Clinical Study 
SYNB1934-CP-003 Version 4.0_US, dated 01 July 2023, and will adhere to the study 
requirements as presented, including all statements regarding confidentiality. In addition, I will 
conduct the study in accordance with the requirements of this protocol and also protect the rights, 
safety, privacy and well-being of study participants in accordance with the following: 
• International Council for Harmonisation of Technical Requirements of 
Pharmaceuticals for Human Use (ICH) Harmonised Tripartite Guideline for Good 
Clinical Practice (GCP) E6(R1). 
• All applicable laws and regulations, including, without limitation, data privacy laws 
and regulations. 
• Requirements for reporting serious adverse events as defined in Section 6 of this 
protocol. 
• Terms outlined in the Clinical Study Site Agreement. 
My signature also acknowledges that: 
• Neither my subinvestigators nor I are members of the Institutional Review Board 
reviewing this protocol, or 
• I and/or my subinvestigators are members of the Institutional Review Board, but I/we 
will not participate in the initial review or continuing review of this study. 
 
 
 
 
Printed Name of Investigator 
 
 
 
 
Signature of Investigator 
 
 
 
 
Date 
 
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PROTOCOL SYNOPSIS 
Name of Sponsor/Company: Synlogic Operating Company, Inc.  
Name of Investigational Product: SYNB1934v1 
Title of Study: A Phase 3, Double-blind, Placebo-controlled, Randomized Withdrawal Study to 
Evaluate the Efficacy and Safety of SYNB1934 in Patients with PKU (SYNPHENY-3) 
Protocol Number: SYNB1934-CP-003 
Phase of Development: 2b/3 
Objectives and Endpoints 
This study has 3 parts, and each study part has a distinct set of objectives, endpoints, and analysis 
populations. The primary determination of efficacy will be based on the comparison of SYNB1934v1 
versus placebo in the randomized withdrawal period (Part 2).  
Dose Escalating, Open-Label Period (DEP; Part 1): 
Objective 
Endpoint 
Primary:  
 
• 
To assess the percentage change in blood 
phenylalanine (Phe) level 
• Percent change from baseline in blood Phe level 
at last measurement of the individually titrated 
dose (iTD) of SYNB1934v1  
Key Secondary: 
 
• To assess the absolute change in Phe level 
• Change from baseline in blood Phe level at last 
measurement of the iTD of SYNB1934v1 
• To determine the responder population 
• A ≥ 20% reduction from baseline in blood Phe 
level at any time in the DEP 
Other Secondary: 
 
• To assess proportion of participants achieving 
blood Phe level ≤ 360 µmol/L 
• Blood Phe level ≤ 360 µmol/L at any time of 
the iTD of SYNB1934v1 
• Blood Phe level ≤ 360 µmol/L at any time in the 
DEP 
• To assess the safety and tolerability of 
SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from baseline in clinical laboratory 
parameters 
• Changes from baseline in vital signs 
measurements 
Exploratory: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
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• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Note: baseline and week numbering are specific to study Part 1, unless explicitly noted otherwise in the 
endpoint. 
Randomized Withdrawal Period (RWP; Part 2): 
Objective 
Endpoint 
Primary: 
 
• To evaluate efficacy of SYNB1934v1 versus 
placebo in the responder population 
• Change from baseline to Week 4 in blood Phe 
level 
Key Secondary: 
 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard 
to percent change from DEP baseline in blood 
Phe level 
• Percent change from DEP baseline in blood Phe 
level at Week 4 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard 
to change from DEP baseline in blood Phe level 
• Change from DEP baseline to Week 4 in blood 
Phe level 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard 
to the proportion of participants with a blood 
Phe level ≤ 360 µmol/L 
• Blood Phe level ≤ 360 µmol/L at Week 4 
Other Secondary Objectives: 
 
• To assess the safety and tolerability of 
SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from baseline in clinical laboratory 
parameters 
• Change from baseline in vital signs 
measurements 
• To evaluate efficacy of SYNB1934v1 versus 
placebo in the responder population with regard 
to the proportion of participants with a ≥ 20% 
reduction from DEP baseline in blood Phe level 
at Week 4 
 
 
• A ≥ 20% reduction from DEP baseline in blood 
Phe level at Week 4 
Exploratory: 
 
• 
 
 
 
 
 
  
 
 
 
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• 
 
 
 
 
 
  
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
• 
 
Note: baseline and week numbering are specific to study Part 2, unless explicitly noted otherwise in the 
endpoint. 
Open-Label Extension (OLE; Part 3): 
Objective 
Endpoint 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
. 
Note: baseline and week numbering are specific to study Part 3, unless explicitly noted otherwise in the 
endpoint. 
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Methodology 
This is a 3-part, adaptive study consisting of a DEP of up to 15 weeks (Part 1), followed by a 4-week, 
double-blind, placebo-controlled RWP (Part 2), and an OLE (Part 3) of up to 36 months.  
DEP (Part 1): 
In the DEP, all enrolled participants will maintain a stable diet reflecting their baseline Phe intake and 
receive escalating doses of SYNB1934v1 from approximately 3 to 15 weeks to determine an iTD, 
which is defined as the highest dose the participant is able to tolerate. A participant will be defined as 
having reached an iTD if they tolerate 3 weeks at a dose, regardless of whether other doses are 
tolerated. 
If a participant tolerates escalation to the initial dose (Dose 1) after 3 weeks, the investigator will 
escalate to Dose 2. If Dose 2 is tolerated, the participant will continue on this dose for a total duration 
of 3 weeks prior to attempting to escalate to Dose 3. A participant will stay on his or her iTD for 
3 weeks. If at any time during a 3-week dosing interval the participant or investigator considers a dose 
level to be intolerable, the dose may be de-escalated after discussion with the medical monitor. In this 
case, the participant will restart 3 weeks at the lower level iTD. After de-escalation, participants cannot 
escalate to a higher dose again during Part 1. In general, a dose is considered intolerable if the 
participant experiences Grade 3 or more severe adverse events (AEs) or Grade 2 gastrointestinal (GI) 
AEs that do not improve with continued dosing at the same dose level over time. If a dose is 
de-escalated due to a reason other than Grade 3 or greater, or Grade 2 GI AEs, or a second 
de-escalation is required, the medical monitor must be consulted. 
Blood Phe level will be measured at each dose level after 3 weeks at that level. A responder will be 
defined as a participant who achieves a ≥ 20% reduction in blood Phe level compared to DEP baseline 
on SYNB1934v1. 
The DEP will contain an adaptive, seamless Phase 2b component. Once the first 20 participants have 
attained an iTD and completed the DEP or the 30th participant has a baseline DEP and has been in the 
study for 105 days, DEP data will be evaluated by an independent data monitoring committee (DMC) 
using a pre-specified set of criteria based on both safety and efficacy to determine whether the dose 
regimen is appropriate or whether any doses should be discontinued as well as whether the study 
should be stopped for futility. 
 
 
 
RWP (Part 2): 
All participants who complete the DEP at their iTD for at least 3 weeks will enter a 4-week RWP. 
Participants will be randomized 1:1 to receive SYNB1934v1 at their iTD determined in the DEP or 
placebo 3 times daily (TID). Randomization will be stratified on screening Phe level. Participants will 
remain on the assigned dose (iTD of SYNB1934v1 or matching placebo) for the duration of the RWP, 
unless they develop intolerance or meet other discontinuation criteria, and will remain on the same diet 
they consumed during the DEP. Blood Phe level will be measured at Week 1, 3 and 4 of the RWP. 
OLE (Part 3): 
Participants who complete the 4-week RWP may enter the OLE and receive SYNB1934v1 for up to 
36 months. (At the sponsor’s discretion, enrollment directly into OLE may be opened.) During the 
OLE all participants will complete a dose ramp to their iTD over time guided by tolerability. The iTD 
in OLE may be different from the iTD in DEP. The investigator may escalate the dose of 
SYNB1934v1 up to a maximum of 1 × 1012 live cells based on tolerability; multiple attempts to 
escalate to a higher dose level are permitted per investigator discretion. Participants will be allowed to 
modify their standard diet if their blood Phe level is < 240 µmol/L, with guidance from the investigator 
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as outlined in the study-specific Diet Manual. The 3-day dietary intake assessments will continue per 
the Schedule of Events. 
Dosing and Dose Escalation 
SYNB1934v1 will be taken with a meal throughout the study. In the DEP, participants will complete a 
dose ramp at the beginning of each dose level.  
The escalating dose levels are as follows: 
• 
Dose 1: 3 × 1011 live cells 
• 
Dose 2: 6 × 1011 live cells  
• 
Dose 3: 1 × 1012 live cells 
During participation in the study, participants will take a proton pump inhibitor (PPI; esomeprazole 
20 mg or equivalent) once per day (QD; recommended before breakfast), starting 7 days before the 
first dose of SYNB1934v1 in Part 1. If they cannot tolerate a PPI, they can use an H2 blocker per 
investigator discretion. If neither the PPI nor H2 blocker can be tolerated, the participant will 
discontinue from the study. The dose of PPI or H2 blocker should remain stable during the DEP and 
RWP; if a dose adjustment to the PPI or H2 blocker is required in OLE, the investigator must discuss 
with the medical monitor (unless the adjustment is required due to an adverse event). 
Diet and Phe Monitoring 
For 3 consecutive days prior to scheduled study visits and blood Phe draws, all participants will have a 
3-day dietary intake assessment. A study dietitian will interview the participant and provide advice on 
maintaining a diet that is consistent with baseline diet throughout the study. Participants will follow 
their usual diet (including Phe, protein, and medical food intake) from baseline in the DEP to the end 
of the RWP.  
During the DEP and RWP, blood Phe and tyrosine (Tyr) levels will be blinded to the sponsor, 
participant, and investigator. The investigator will be notified if Phe is > 1.5 × the DEP baseline value, 
if Phe falls below 30 µmol/L or if Tyr falls below 20 µmol/L to allow for individual safety 
discontinuation. During the OLE, participants will be allowed to modify their standard diet if their 
blood Phe level is < 240 µmol/L, with guidance from the investigator as outlined in the study-specific 
Diet Manual. The 3-day dietary intake assessments will continue per the Schedule of Events. 
Number of Participants (planned): 
 are planned to be enrolled. 
Study Inclusion and Exclusion Criteria 
Male and female participants will be enrolled. 
Inclusion Criteria: 
1. Age ≥ 18 years.  
2. Able and willing to voluntarily complete the informed consent process  
3. Diagnosis of phenylketonuria (PKU) and failure to maintain recommended blood Phe levels 
on existing management (sapropterin, sepiapterin and/or Phe-restricted diet), demonstrated by 
uncontrolled blood Phe level > 360 μmol/L on current therapy any time during screening and 
uncontrolled blood Phe level > 360 μmol/L on current therapy when taking the average of the 
3 most recent Phe levels from the participant’s medical history (inclusive of any screening 
values). All screening values must be obtained more than 7 days apart, as determined by 
central or local laboratory.  
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4. Females of childbearing potential must have a negative pregnancy test at screening and the end 
of DEP (in order to enter the RWP) and RWP (in order to enter the OLE) and be willing to 
have additional pregnancy tests during the study. 
5. Sexually active female participants of childbearing potential must be willing to use an 
acceptable method of contraception while participating in the study and for 2 weeks after the 
last dose.  
6. Stable diet including stable medical formula regimen (if used) for at least 1 month prior to 
screening.  
7. If using sapropterin or sepiapterin, must be on a stable dose for at least 3 months. 
8. Willing and able to continue current diet, sapropterin, sepiapterin and large neutral amino 
acids unchanged during screening, DEP and RWP and to engage in all study activities. 
Exclusion Criteria: 
1. Currently taking Palynziq® (pegvaliase-pqpz) (within 1 month of screening). 
2. Acute or chronic medical, surgical, psychiatric, or social condition or laboratory abnormality 
that may increase participant risk associated with study participation, compromise adherence 
to study procedures and requirements, and, in the judgment of the investigator, would make 
the participant inappropriate for enrollment. 
3. A known or suspected diagnosis of DNAJC12 deficiency, biopterin synthesis deficiency, or 
irritable bowel syndrome. 
4. Intolerance to or allergic reaction to E. coli Nissle or any of the ingredients in SYNB1934v1 
formulation, or an allergy to cinnamon. Known intolerance to proton pump inhibitors and H2 
blockers, since one or the other must be used. 
5. Currently taking or plans to take any type of systemic (e.g., oral or intravenous) antibiotic 
within 28 days prior to the first dose of SYNB1934v1 through final safety assessment in RWP, 
including planned surgery, hospitalizations, dental procedures, or interventional studies that 
are expected to require antibiotics. Exception: topical antibiotics are allowed. 
6. Pregnant, planning to become pregnant, or breastfeeding. 
7. Current participation in any other investigational drug study or use of any investigational agent 
within 30 days or 5 half-lives (whichever is longer) prior to screening.  
8. Ever received gene therapy for treatment of PKU. 
Study Drug: SYNB1934v1 at 3 × 1011, 6 × 1011, or 1 × 1012 live cells or matching placebo 
Duration of Treatment 
• 
Screening period: up to 45 days 
• 
Part 1: DEP: up to 15 weeks 
• 
Part 2: RWP: 4 weeks 
• 
Part 3: OLE: up to 36 months  
Reference Therapy, Dosage, and Mode of Administration: Placebo to match SYNB1934v1 
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Removal of Individual Participants from Investigational Medicinal Product and Withdrawal of 
Consent 
Reasons for discontinuation of investigational medicinal product (IMP; i.e., SYNB1934v1 or placebo) 
may include the following:  
• 
The participant withdraws consent. 
• 
The investigator or sponsor notes a significant noncompliance with protocol procedures. 
• 
The participant develops an intolerable toxicity including but not limited to a Grade 3 or 
more severe adverse event or serious adverse event (SAE) assessed as related to IMP by 
the investigator.  
• 
The investigator determines that the participant must discontinue further study dosing for 
medical reasons (including pregnancy). 
• 
The participant has low levels of Phe (< 30 µmol/L) or Tyr (< 20 µmol/L) or has profound 
elevations in Phe (> 1.5 × the DEP baseline value during DEP and RWP and > 2200µM 
during OLE). 
If possible, the investigator will confer with the sponsor or medical monitor before discontinuing 
dosing. Participants who discontinue from IMP in RWP will be encouraged to remain on study and 
complete assessments (particularly safety assessments) through the remainder of RWP of the study. 
Participants who discontinue from DEP and OLE will be asked to have an early termination visit 
30 days after the last dose of IMP. 
Participants may withdraw their consent at any time for any reason without prejudice to their future 
medical care by the physician or at the institution. If a participant withdraws consent, the date and 
stated reason for consent withdrawal should be documented. Participant data collected up to the date of 
consent withdrawal will be included in the analyses. 
Study Stopping Rules 
If any of the following criteria are met, enrollment will be halted. Participants already enrolled in the 
study will continue dosing until review by the Data Monitoring Committee (DMC). 
1. Two or more participants experience an AE ≥ Grade 3, per protocol severity grading criteria, 
related to SYNB1934v1 as assessed by the investigator.  
2. Clinical infection is detected in a sterile space with a gram-negative aerobic bacillus confirmed 
by qPCR to be SYNB1934v1.  
If any of the above criteria are met, the DMC will review all relevant data and determine whether 
enrollment in the study should resume and whether any modifications to the study are warranted. If the 
DMC decides that further enrollment should be permanently suspended, subjects already enrolled in 
the study will continue to be followed for the duration of the study.  
If a death occurs at any time during the study that is considered by the investigator to be related to 
SYNB1934v1, enrollment will be halted and dosing for subjects already enrolled will be stopped until 
the DMC reviews all relevant data and makes further recommendations.  
Statistical Methods 
General: 
All evaluations and tabulations will be carried out as described in detail in a statistical analysis plan 
(SAP), which will be finalized and approved prior to the DEP interim analysis and database lock and 
unblinding.  
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A disposition of participants will be provided for each of the 3 parts of the study. 
Continuous variables will be summarized using the number of observations, mean, standard deviation 
(SD), median, minimum, and maximum. Categorical variables will be summarized using frequency 
counts and percentages. 
Baseline for blood Phe level in each of the 3 parts of the study will be defined as the mean of the 
duplicate blood Phe level measurements obtained immediately prior to administration of the first dose 
of each part. If only one blood Phe level measurement is available, then that measure will be used as 
baseline. Similarly, for the determination of responders and participants who achieve a specific 
reduction in Phe (e.g., Phe level ≤ 360 µmol/L at any time in the DEP, or a ≥ 20% reduction from 
baseline) the mean of the duplicate blood Phe level measurements at the applicable post-baseline 
timepoint (e.g., Week 3) will be used. If only one of the duplicate blood Phe levels is available at a 
particular post-baseline timepoint, then that measurement will be used.  
In describing the endpoints, the baseline and week numbering are specific to the corresponding study 
part, unless explicitly noted otherwise. 
Analysis Populations: 
Analysis populations will be defined for each of the 3 parts of the study (DEP, RWP, and OLE). 
Full analysis set (FAS): The full analysis sets are based on the intention-to-treat principle. The DEP 
FAS includes all participants enrolled in the DEP. 
The RWP FAS is defined as all participants randomized into the RWP. Additionally: 
• 
The responder RWP FAS is defined as participants in the RWP FAS who are responders 
• 
The nonresponder RWP FAS is defined as participants in the RWP FAS who are 
nonresponders 
A responder is defined as a participant who achieves a ≥ 20% reduction in blood Phe level compared 
to DEP baseline on SYNB1934v1. 
The responder RWP FAS will be the primary efficacy analysis population for the RWP. Analyses on 
the RWP FAS will be performed according to the randomized treatment group. 
In the event that, based on the interim analysis results utilizing the protocol-defined DMC criteria for 
dose changes and futility, the DMC recommends dropping either the 1 × 1012 or the 1× 1012 and 
6 × 1011 doses, the DEP FAS will exclude all participants who were enrolled and treated at a dropped 
dose in the DEP. Additionally, the RWP FAS will exclude all participants who have been randomized 
in the RWP up to this time. The participants in the DEP who have an iTD corresponding to a dropped 
dose will go directly into the OLE part of the study (i.e., as opposed to being randomized into the 
RWP).  
These participants who are excluded from the DEP FAS and RWP FAS will be summarized 
separately.  
Safety analysis set (SAS): For each of the 3 study parts, the SAS will include all participants who 
received any amount of IMP in the corresponding study part with treatment assignment based on the 
treatment received. 
Per-protocol analysis set (PPS): The DEP PPS is defined as all DEP FAS participants, who had no 
major protocol deviations in DEP that would affect efficacy, completed their final 3-week iTD dosing 
period, whose dietary Phe remained within 20% of their DEP baseline value, and who did not require 
systemic antibiotic treatment during DEP. The DEP PPS will be used as a sensitivity analysis for the 
DEP primary efficacy endpoint. 
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RWP baseline blood Phe level, iTD dose level, visit, and visit × treatment group (interaction effect), 
visit × iTD dose level (interaction effect), and visit × RWP baseline blood Phe level (interaction effect) 
as fixed effects. The MMRM will incorporate the participant’s repeated measures at visit Weeks 1, 3, 
and 4, with visit being treated as a categorical variable. Least-squares means for each treatment group 
and the SYNB1934v1 treatment group difference from placebo, along with the corresponding
 
 and p-values will be calculated for Weeks 1, 3, and 4. The primary efficacy 
analysis will compare the least-squares means treatment difference between SYNB1934v1 and placebo 
in the change from baseline in the blood Phe level at Week 4. 
At each of the scheduled assessments, blood Phe will be drawn in duplicate. The MMRM will 
incorporate these participant repeated measures. For the MMRM analysis, the results of each of the 
post-baseline duplicate samples from a visit will be included in the statistical model (i.e, without 
averaging the results within a given visit before inclusion into the MMRM). The responder RWP FAS 
will be the primary efficacy analysis population. 
Key Secondary Efficacy Endpoint Analyses: 
DEP (Part 1): The key secondary efficacy endpoints for the DEP are listed in the objectives and 
endpoints table above. The key secondary endpoint, change from baseline in blood Phe level at last 
measurement of the iTD of SYNB1934v1, will be analyzed using an MMRM analysis similar to the 
model described for the DEP primary efficacy analysis, with the dependent variable being the change 
in blood Phe level from DEP baseline. 
For the secondary endpoint, a ≥ 20% reduction from baseline in blood Phe level at any time in the 
DEP, the proportion of participants meeting these criteria will be summarized along with 95% 
  
These analyses will be performed on the DEP FAS. 
RWP (Part 2): The key secondary efficacy endpoints for the RWP are listed in the objectives and 
endpoints table above. The proportion of participants meeting the key secondary endpoint, a blood Phe 
level ≤ 360 µmol/L at Week 4, will be tested between the placebo and SYNB1934v1 dose groups 
using a Fisher’s Exact test. The key secondary endpoint, change from DEP baseline to Week 4 in 
blood Phe level, will be analyzed using an MMRM analysis similar to the model described for the 
RWP primary efficacy analysis, with the dependent variable being the change in blood Phe level from 
DEP baseline, and the DEP baseline blood Phe level as the baseline blood Phe level covariate. The key 
secondary endpoint, percent change from DEP baseline in blood Phe level at Week 4, will be analyzed 
using an MMRM analysis similar to the model described for the RWP primary efficacy analysis, with 
the dependent variable being the percent change in blood Phe level from DEP baseline, and the DEP 
baseline blood Phe level as the baseline blood Phe level covariate.  
These analyses will be performed on the responder RWP FAS. 
Analyses of other secondary endpoints are described in the full protocol and will be included in the 
SAP. Analyses of exploratory endpoints will be described in the SAP. 
Interim Analysis: 
Once the first 
 have attained an iTD and completed the DEP or the 
 participant has a 
baseline DEP and has been in the study for
, whichever occurs earlier, the DEP data will be 
evaluated by an independent DMC. The DMC will use the below pre-specified set of criteria based on 
both safety and efficacy to determine whether the dose regimen is appropriate or whether any doses 
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should be discontinued as well as whether the study should be stopped for futility. Enrollment will not 
be paused during the interim analysis. 
If a dose level is dropped, participants who are in the DEP and have an iTD corresponding to a 
dropped dose will not be randomized into the RWP but will instead enter the OLE directly. In addition, 
participants who are in the OLE at a dose level that has been dropped will be transitioned to the highest 
dose level retained. 
The size of the DEP interim analysis cohort and corresponding criteria for dropping a dose (see below) 
provide reasonable operating characteristics for proper determination if one or more doses should be 
dropped. 
DMC Criteria for Dose 
Changes and Futility 
Evaluation by DMC 
Criteria to Drop the Dose 
1 × 1012 dose 
< 20% of participants who attain an iTD are at the 1 × 1012 dose 
OR 
> 75% of participants who have 1 × 1012 as their iTD had a greater 
Phe reduction at their 6 × 1011 dose versus their 1 × 1012 dose 
6 × 1011 dose 
 
Note: considered only 
if the Drop 1 × 1012 Criteria 
has been met 
< 30% of participants who attain an iTD have 6 × 1011 or 1 × 1012 as 
their iTD 
OR 
> 75% of participants who have 6 × 1011 or 1 × 1012 as their iTD had a 
greater Phe reduction at their 3 × 1011 dose versus their 6 × 1011 dose 
Stop for futility 
< 30% of participants enrolled into the DEP attain an iTD and 
complete the DEP 
OR 
< 25% of participants who attain an iTD and complete the DEP are 
responders 
DEP = dose-escalating, open-label period; DMC = data monitoring committee; iTD = individually titrated dose; 
Phe = phenylalanine 
In addition, the DMC will review interim data at defined intervals in the study. The Sponsor will 
remain blinded to the subject-level blood Phe levels during the DEP and RWP but will have access to 
the DEP interim analysis results.  
Multiplicity:  
The overall Type I error rate for the DEP will be controlled at the
 
 
 
 
 
 
  
 
 
 
 
 
  
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1) Blood Phe level ≤ 360 μmol/mL at Week 4  
2) Change from DEP baseline to Week 4 in blood Phe level 
3) Percent change from DEP baseline to Week 4 in blood Phe level.  
These key secondary endpoints will be tested following this prespecified order only if the RWP 
primary efficacy endpoint null hypothesis is rejected.  
Safety Analysis: 
Safety will be evaluated by scheduled monitoring of AEs, vital signs, and clinical laboratory 
measurements. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities, and severity of 
AEs and laboratory abnormalities will be graded using the National Cancer Institute Common 
Terminology Criteria for Adverse Events (CTCAE), v5.0, with certain exceptions from CTCAE 
grading for diarrhea and nausea. Adverse events will be tabulated by study period, system organ class, 
and preferred term. Incidence tables of participants with AEs will be presented for all AEs by 
maximum severity, AESIs, SAEs, AEs assessed as related to IMP, and AEs resulting in 
discontinuation of study dosing. 
For the DEP, safety analyses will be performed on the DEP SAS. For the RWP and OLE study parts, 
safety analyses will be performed on the corresponding SAS, with results being presented for the 
responder and nonresponder populations as well as overall population. 
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TABLE OF CONTENTS 
SIGNATURE PAGE (SPONSOR) ..................................................................................................3 
LEAD INVESTIGATOR’S AGREEMENT ...................................................................................4 
PROTOCOL SYNOPSIS ................................................................................................................5 
TABLE OF CONTENTS ...............................................................................................................17 
LIST OF TABLES .........................................................................................................................21 
LIST OF FIGURES .......................................................................................................................21 
LIST OF ABBREVIATIONS ........................................................................................................22 
1. 
INTRODUCTION ......................................................................................................25 
1.1. 
Background of Phenylketonuria .................................................................................25 
1.2. 
Unmet Medical Need in Phenylketonuria ...................................................................26 
1.3. 
Description of SYNB1934 and SYNB1934v1 ...........................................................27 
1.4. 
Nonclinical Studies .....................................................................................................28 
1.5. 
Clinical Experience .....................................................................................................28 
1.5.1. 
Study SYNB1934-CP-001 ..........................................................................................28 
1.5.2. 
Studies of SYNB1618.................................................................................................29 
1.6. 
Study Rationale ...........................................................................................................30 
1.6.1. 
Enrichment Design .....................................................................................................31 
1.7. 
Benefit-Risk Assessment ............................................................................................32 
2. 
STUDY OBJECTIVES AND ENDPOINTS ..............................................................33 
3. 
INVESTIGATIONAL PLAN .....................................................................................35 
3.1. 
Overall Study Design ..................................................................................................35 
3.2. 
Dosing and Dose Escalation .......................................................................................36 
3.2.1. 
Part 1: Dose-Escalating Open-Label ..........................................................................37 
3.2.1.1. 
Intolerance ..................................................................................................................37 
3.2.2. 
Part 2: Randomized Withdrawal .................................................................................38 
3.2.3. 
Part 3: Open-Label Extension .....................................................................................38 
3.3. 
Diet and Phenylalanine Monitoring ............................................................................38 
3.4. 
Study Stopping Rules .................................................................................................39 
3.5. 
Study Discontinuation for Individual Participants .....................................................39 
3.6. 
Duration of Study Participation ..................................................................................40 
4. 
STUDY POPULATION .............................................................................................40 
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4.1. 
Number of Participants ...............................................................................................40 
4.2. 
Selection of Participants .............................................................................................40 
4.2.1. 
Participant Inclusion Criteria ......................................................................................40 
4.2.2. 
Participant Exclusion Criteria .....................................................................................41 
4.3. 
Treatment of Participants ............................................................................................41 
4.3.1. 
SYNB1934 ..................................................................................................................41 
4.3.2. 
Placebo ........................................................................................................................42 
4.3.3. 
Treatment Compliance ................................................................................................42 
4.3.4. 
Concomitant Medications ...........................................................................................42 
4.3.4.1. 
Proton Pump Inhibitor ................................................................................................42 
4.3.4.2. 
43 
4.3.4.3. 
Antibiotics ...................................................................................................................43 
4.3.4.4. 
Prohibited Therapies ...................................................................................................43 
5. 
STUDY PROCEDURES AND ASSESSMENTS......................................................43 
5.1. 
Schedule of Events .....................................................................................................43 
5.2. 
Screening and Baseline Periods ..................................................................................46 
5.3. 
Randomization ............................................................................................................47 
5.4. 
Pharmacodynamic Assessments .................................................................................47 
5.4.1. 
Blood Phenylalanine and Tyrosine .............................................................................47 
5.4.2. 
DEP Weekly Substudy ...............................................................................................47 
5.5. 
Safety Assessments .....................................................................................................47 
5.5.1. 
Adverse Events ...........................................................................................................47 
5.5.2. 
Vital Signs, Weight, Height, and Electrocardiograms ................................................48 
5.5.3. 
Clinical Laboratory Measurements.............................................................................48 
5.6. 
Early Termination Assessments .................................................................................48 
5.7. 
End of Study Assessment ...........................................................................................48 
6. 
ADVERSE EVENTS..................................................................................................48 
6.1. 
Adverse Event Definition ...........................................................................................48 
6.1.1. 
Assessment of Severity ...............................................................................................49 
6.1.1.1. 
Gastrointestinal-related Events ...................................................................................49 
6.1.2. 
Assessment of Causality .............................................................................................49 
6.2. 
Serious Adverse Events ..............................................................................................50 
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6.2.1. 
Clarification of Serious Adverse Event Definition .....................................................51 
6.2.2. 
Serious, Unexpected, Suspected Adverse Reactions ..................................................51 
6.3. 
Adverse Events of Special Interest .............................................................................52 
6.4. 
Reporting of Adverse Events ......................................................................................52 
6.5. 
Pregnancy ...................................................................................................................52 
6.6. 
Clinical Laboratory Abnormalities .............................................................................53 
6.7. 
Review of Safety Data ................................................................................................53 
7. 
ESTIMANDS .............................................................................................................53 
7.1. 
Part 1: Primary and Key Secondary Efficacy Objective Estimands ...........................53 
7.2. 
Part 2: Primary and Key Secondary Efficacy Objectives Estimands .........................55 
8. 
STATISTICAL METHODS AND DETERMINATION OF SAMPLE SIZE ...........57 
8.1. 
General Statistical Methods ........................................................................................57 
8.2. 
Populations for Analysis .............................................................................................57 
8.3. 
Determination of Sample Size ....................................................................................58 
8.4. 
Primary Efficacy Endpoint Analyses ..........................................................................59 
8.4.1. 
Part 1: DEP .................................................................................................................59 
8.4.2. 
Part 2: RWP ................................................................................................................59 
8.5. 
Key Secondary Efficacy Endpoint Analyses ..............................................................60 
8.5.1. 
Part 1: DEP .................................................................................................................60 
8.5.2. 
Part 2: RWP ................................................................................................................60 
8.6. 
Other Secondary Efficacy Endpoint Analyses ...........................................................61 
8.6.1. 
Part 1: DEP .................................................................................................................61 
8.6.2. 
Part 2: RWP ................................................................................................................61 
8.7. 
Exploratory Endpoint Analyses ..................................................................................61 
8.8. 
Missing Data ...............................................................................................................61 
8.9. 
Multiplicity .................................................................................................................62 
8.10. 
Safety Analysis ...........................................................................................................63 
8.11. 
Randomization and Blinding ......................................................................................63 
8.12. 
Interim Analysis ..........................................................................................................63 
9. 
CHANGES IN THE CONDUCT OF THE STUDY OR PLANNED 
ANALYSIS.................................................................................................................64 
10. 
DATA RECORDING, RETENTION, AND MONITORING ...................................65 
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10.1. 
Case Report Forms .....................................................................................................65 
10.2. 
Data Retention ............................................................................................................65 
10.3. 
Data Monitoring ..........................................................................................................65 
10.4. 
Quality Control and Quality Assurance ......................................................................66 
11. 
REGULATORY, ETHICAL, AND LEGAL OBLIGATIONS .................................66 
11.1. 
Good Clinical Practice ................................................................................................66 
11.2. 
Institutional Review Board/Independent Ethics Committee/Research Ethics 
Board Approval ..........................................................................................................66 
11.3. 
Regulatory Authority Approval ..................................................................................66 
11.4. 
Other Required Approvals ..........................................................................................66 
11.5. 
Informed Consent .......................................................................................................67 
11.6. 
Participant Confidentiality ..........................................................................................67 
11.7. 
Study Confidentiality and Disclosure of Information ................................................68 
11.8. 
Publication of Study Data ...........................................................................................68 
11.9. 
Ethical Standards ........................................................................................................68 
12. 
REFERENCES ...........................................................................................................69 
APPENDIX 1. CLINICAL LABORATORY TESTS ................................................................71 
 
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LIST OF TABLES 
Table 1: 
Objectives and Endpoints: Part 1, Dose Escalating, Open-Label Period 
(DEP) ..........................................................................................................................33 
Table 2: 
Objectives and Endpoints: Part 2, Randomized Withdrawal Period (RWP) ..............34 
Table 3: 
Objectives and Endpoints: Part 3, Open-label Extension (OLE) ...............................35 
Table 4: 
Dose Ramp Schedule ..................................................................................................37 
Table 5: 
Schedule of Events: Dose-escalation (Part 1) and Randomized Withdrawal 
(Part 2) ........................................................................................................................44 
Table 6: 
Schedule of Events: Part 3, Open-Label Extension Period and Follow-up, 
End of Study and Early Termination Visits ................................................................46 
Table 7: 
Severity Assessment of Diarrhea and Nausea ............................................................49 
Table 8: 
Estimand for the Part 1 Primary Objective .................................................................53 
Table 9: 
Estimands for the Part 1 Key Secondary Objectives ..................................................54 
Table 10: Estimand for the Part 2 Primary Objective .................................................................55 
Table 11: Estimands for the Part 2 Key Secondary Objectives ..................................................56 
Table 12: DMC Criteria for Dose Changes and Futility .............................................................64 
 
LIST OF FIGURES 
Figure 1: 
Schematic of SYNB1934v1 ........................................................................................28 
Figure 2: 
Study SYNB1934-CP-003 Schematic ........................................................................36 
 
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LIST OF ABBREVIATIONS 
Abbreviation or Specialist Term 
Explanation 
AE 
Adverse event 
AraC  
Arabinose-responsive transcriptional regulator 
AUC 
Area under the curve 
BH4 
Tetrahydrobiopterin 
BID 
Twice per day 
CBC 
Complete blood count 
CFU 
Colony forming units 
CRO 
Contract research organization 
CRP 
C-reactive protein 
CTCAE 
Common Terminology Criteria for Adverse Events 
DAP 
Diaminopimelate 
DEP 
Dose-escalating, open-label period 
DMC 
Data Monitoring Committee 
ECG 
Electrocardiogram 
EcN 
Escherichia coli Nissle 1917 
eCRF 
Electronic case report form 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
GCP 
Good Clinical Practice 
GI 
Gastrointestinal 
GLP 
Good Laboratory Practice 
IPTG 
Isopropyl ß-D-1-thiogalactopyranoside 
HA 
Hippuric acid 
HPA 
Hyperphenylalaninemia 
HV 
Healthy volunteer 
ICF 
Informed consent form 
ICH 
International Council for Harmonisation of Technical 
Requirements of Pharmaceuticals for Human Use 
IEC 
Independent Ethics Committee 
IMP 
Investigational medicinal product 
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Abbreviation or Specialist Term 
Explanation 
IRB 
Institutional Review Board 
iTD 
Individually titrated dose 
IXRS 
Interactive response system 
LAAD 
L-amino acid deaminase 
MAD 
Multiple ascending dose 
MMRM 
Mixed-model with repeated measures 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
PAH 
Phenylalanine hydroxylase 
(m)PAL 
(Modified) phenylalanine ammonia lyase 
PD 
Pharmacodynamics 
Phe 
Phenylalanine 
PheP  
High-affinity phenylalanine transporter 
PK 
Pharmacokinetics 
PKU 
Phenylketonuria 
PP 
Phenylpyruvate 
PPI 
Proton pump inhibitor 
PPS 
Per-protocol analysis set 
QD 
Once per day 
qPCR 
Quantitative polymerase chain reaction 
REB 
Research Ethics Board 
RWP 
Randomized withdrawal period 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety analysis set 
SD 
Standard deviation 
SST 
Stop signal task 
t½  
Half-life 
TCA 
Trans-cinnamic acid 
TEAE 
Treatment-emergent adverse event 
TID 
Three times daily 
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Abbreviation or Specialist Term 
Explanation 
Tyr 
Tyrosine 
WOCBP 
Women of childbearing potential 
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1. 
INTRODUCTION 
The target indication for clinical development of SYNB1934v1 is for the treatment of 
phenylketonuria (PKU) in adult patients. 
1.1. 
Background of Phenylketonuria 
Phenylketonuria is a rare inherited metabolic disorder characterized by an inability to utilize the 
amino acid phenylalanine (Phe). The incidence of PKU varies widely among different 
populations with an incidence in the United States of 1:13,500 to 1:21,000.1 There are pockets of 
high incidence, for example, in Ireland, where the incidence is 1:4,500 births. In the United 
States, newborn screening for PKU was initiated in the 1960s, which changed the natural history 
of the disease and resulted in improved outcomes for patients who were diagnosed in the 
neonatal period.2  
PKU is caused by a mutation in the gene encoding phenylalanine hydroxylase (PAH), which is 
inherited in an autosomal recessive pattern and affects males and females equally. The PAH gene 
is located on chromosome 12q23.1. Tetrahydrobiopterin (BH4) is a cofactor required for the 
activity of the PAH enzyme, and hyperphenylalaninemia (HPA) can also result from defects in 
the biosynthesis or recycling of BH4. Phe, an essential amino acid, which is found in all protein-
containing foods, is converted to tyrosine (Tyr) by PAH in the healthy population. PAH is either 
lacking or its activity is impaired in patients with PKU, leading to accumulation of Phe in the 
blood (a condition designated as HPA) and central nervous system and causing detrimental 
effects on brain development and function. Untreated, the disease results in severe neurological 
complications, including irreversible loss of cognitive capacity and parkinsonism.3,4  
Phenylketonuria is diagnosed based on newborn screening. PKU may be classified by the degree 
of HPA, and patients presenting with plasma Phe levels > 1200 μmol/L have been described as 
having classical PKU.6 Patients with mild to moderate PKU present with Phe serum 
concentrations ranging from 360 to 1200 μmol/L, and benign HPA is characterized by the ability 
to maintain plasma Phe below the range required for treatment.6 A widely accepted treatment 
goal is to achieve a blood Phe level of 120 to 360 μmol/L.7 A majority of specialized centers 
initiate treatment at a baseline plasma Phe level of > 360 μmol/L, although other centers initiate 
treatment at 600 μmol/L. 
Blood Phe lowering is the goal of therapy. If PKU is detected early and treatment is started 
within first weeks of life, intellectual disability can be prevented.8 Current recommendations 
require lifelong dietary support, with intensification during preconception and pregnancy for 
women.7 According to US-based treatment guidelines, the goal of PKU treatment is to maintain 
the blood concentrations of Phe between 120 and 360 μmol/L for all patients regardless of age.7  
Allelic variation in the PAH locus leads to a wide range of phenotypes and variation in Phe 
level.10 Treatment and maintenance of metabolic control throughout life is essential to optimal 
functioning of PKU patients. While intellectual disability does not occur in patients who are well 
controlled in infancy and childhood, a variety of adverse neurocognitive and psychiatric 
outcomes, including deficits in executive functioning and psychiatric symptoms such as anxiety, 
depression, and phobias can develop later in life when Phe control is relaxed. High Phe is 
associated with an increased prevalence of neuropsychiatric symptoms and executive functioning 
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deficits, whereas low Phe is associated with improved neurological performance.11,12 Even in 
well-controlled patients with normal IQ, some impairment of intellectual abilities has been 
observed.13 During childhood, each 100 μmol/L increase in blood Phe predicted a 1.3- to 
3.1-point reduction in IQ over a range of Phe levels of 423 to 750 μmol/L. When imputed over 
lifetime Phe levels, the 100 μmol/L increase in Phe predicted a 1.9 to 4.1 reduction in IQ.14 Even 
small, short term (~200 μmol/L, 1–2 weeks) shifts in blood Phe levels can impact outcomes of 
working memory and sustained attention.15 
1.2. 
Unmet Medical Need in Phenylketonuria 
Current standard treatment for patients with PKU is a stringent Phe-restricted diet, combined 
with amino acid mixtures supplemented with trace elements to prevent nutritional 
deficiencies.16,17 This is achieved by excluding or severely curtailing protein-containing foods 
and providing a protein supplement that has other amino acids, but not Phe (medical food, 
“formula”), along with frequent monitoring of blood Phe levels.16 Most patients with classic 
PKU tolerate < 500 mg Phe per day (10 g natural protein), while patients with mild to moderate 
PKU tolerate < 1000 mg Phe per day (20 g natural protein).16 While in the past it was thought 
that strict dietary control was only required in early childhood, current recommendations require 
lifelong dietary support with intensification during preconception and pregnancy for women, 
given the risk of teratogenic effects of elevated blood Phe on the developing fetus.18 
Two therapeutics for PKU have been approved by the Food and Drug Administration (FDA), 
sapropterin dihydrochloride (Kuvan®) and pegvaliase (Palynziq®), which are described below.  
Kuvan is a synthetic form of the BH4 cofactor of the PAH. The BH4 cofactor increases the 
activity level of the PAH enzyme and increases the amount of Phe that can be converted to Tyr. 
Kuvan is indicated for the treatment of HPA in adults and pediatric patients of all ages with PKU 
who have been shown to be responsive to such treatment and also for the treatment of HPA in 
adults and pediatric patients of all ages with BH4 deficiency who have been shown to be 
responsive to such treatment (Kuvan, Union Register EU/1/08/481, last updated 31 March 
2021).19 Approximately 25% to 50% of PKU patients are sapropterin-responsive.20  
Palynziq is a Phe-metabolizing enzyme that is composed of recombinant phenylalanine ammonia 
lyase (PAL) (rAvPAL) conjugated to N-hydroxysuccinimide -methoxypolyethylene glycol. It is 
indicated for the treatment of patients with PKU aged 16 years and older who have inadequate 
blood Phe control (blood Phe levels greater than 600 µmol/L) despite prior management with 
available treatment options (Palynziq, Union Register, EU/1/19/1362, last updated 31 May 
2021).21 Palynziq is dosed as daily subcutaneous injections. Anaphylaxis has been reported after 
administration of Palynziq and may occur at any time during treatment.  
There remains a significant unmet medical need despite existing therapies. Strict blood Phe 
control by dietary management is challenging and poses a significant burden on the patients.22 
Despite recommendations supporting life-long control of Phe levels, some children and most 
adults cannot comply due to the highly restrictive nature of the diet.23 This puts these patients at 
risk of cognitive and psychiatric disease and supports the need for novel treatment approaches. 
Adult patients who are no longer on a Phe-restricted diet can experience neurological 
complications that can improve or even reverse after reinstitution of treatment.7 Based on clinical 
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experience and research data, adults and older adolescents are less adherent to a strict 
Phe-restricted diet than younger children, who are under their parents’ control.8  
1.3. 
Description of SYNB1934 and SYNB1934v1 
SYNB1934 was derived from Escherichia coli Nissle 1917 (EcN) in a series of genetic 
manipulations designed to allow enhanced degradation of Phe within the human gut. The 
degradation of Phe is carried out by the activity of engineered genes encoding phenylalanine 
ammonia lyase (PAL), which catalyzes the conversion of Phe to trans-cinnamic acid (TCA), and 
L-amino acid deaminase (LAAD), which catalyzes the conversion of Phe to phenylpyruvate 
(PP). The PAL expressed in SYNB1934 was derived from Photorhabdus luminescens (recently 
reclassified as Photorhabdus laumondii subsp. laumondii), the same PAL utilized in the closely 
related strain SYNB1618. However, in SYNB1934, this PAL differs from the wild-type PAL 
sequence at 5 amino acid positions. This modified PAL (mPAL) was identified in a high 
throughput screening campaign that selected for PAL enzyme variants with higher rates of TCA 
production compared to the wild-type PAL. Additionally, SYNB1934 contains the following 
modifications within the genome of EcN that are intended to enhance Phe degradation while 
augmenting biologic containment through diaminopimelate (DAP) auxotrophy: 
a. Insertion of 1 additional copy of an endogenous Nissle gene encoding a high-affinity 
phenylalanine transporter (PheP) under the regulatory control of an isopropyl ß-D-1-
thiogalactopyranoside (IPTG)-inducible promoter (Ptac) and transcriptional repressor 
LacI  
b. Insertion of 4 copies of a gene encoding mPAL under the regulatory control of Ptac 
and LacI 
c. Insertion of the gene encoding L-amino acid deaminase (LAAD) derived from 
Proteus mirabilis under the regulatory control of the arabinose-inducible promoter 
PBAD and the arabinose-responsive transcriptional activator (AraC) 
d. Deletion of the dapA gene that encodes 4-hydroxy-tetrahydropicolinate synthase to 
create an auxotrophy for the essential cell wall component DAP 
e. Inactivation of the endogenous Nissle prophage 
f. Deletion of the polyketide synthase (pks) island containing the colibactin-production 
genes  
The deletion of the pks island resulted in a modified strain of SYNB1934, which is referred to as 
SYNB1934v1. 
A schematic of SYNB1934v1 is shown in Figure 1.  
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Figure 1: 
Schematic of SYNB1934v1 
 
AraC = arabinose-responsive transcriptional regulator; LAAD = L-amino acid deaminase from Proteus mirabilis; 
mPAL = gene encoding phenylalanine ammonia lyase from Photorhabdus luminescens with the following 
engineered mutations: S92G, H133M, I167K, L432I, V470A; pheP = high affinity phenylalanine transporter; 
Ptac = synthetic promoter controlled by LacI; ΔdapA = deletion of dapA gene leading to diaminopimelate 
auxotrophy; ΔΦ = partial deletion of endogenous Nissle prophage; Δpks = deletion of the genetic island responsible 
for the production of colibactin 
1.4. 
Nonclinical Studies 
The pharmacologic activity of SYNB1934v1 and its closely related strain, SYNB1618, has been 
characterized in a series of non-Good Laboratory Practices (GLP) studies in mice and nonhuman 
primates that demonstrate the in vitro and in vivo Phe-consuming activity of the strain, as well as 
dose response, biodistribution, excretion, and pH sensitivity. The safety of SYNB1618 has been 
evaluated in a 28-day GLP toxicology study in mice. Further details on the nonclinical studies 
can be found in the Investigator’s Brochure.  
1.5. 
Clinical Experience  
SYNB1934 has been investigated in 2 clinical studies, SYNB1934-CP-001 and 
SYNB1618-CP-003. The related strain SYNB1618 has been investigated in 3 completed studies, 
SYNB1618-CP-001, SYNB1618-CP-002, and SYNB1618-CP-003. 
1.5.1. 
Study SYNB1934-CP-001 
SYNB1934-CP-001, a Phase 1, dose-escalation, placebo- and active-controlled crossover study, 
assessed the safety, tolerability, and pharmacodynamics of SYNB1934 (an engineered EcN 
closely related to SYNB1618) and 
 in healthy 
volunteers. Part 1 was a multiple-ascending dose design in which participants were dosed with 
either SYNB1934, SYNB1618, or placebo. In Cohort 2 of Part 1, participants received 
SYNB1934 or SYNB1618 or placebo in the first treatment period. After a ≥ 7-day washout 
period after the first treatment period, they crossed over to the alternate EcN strain in the second 
treatment period (or remained on placebo). 
 
 Forty subjects were dosed in Part 1. 
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Part 2 was an open-label, randomized sequence, crossover study of SYNB1934 
 
 Twenty-two subjects were dosed in Part 2. 
The maximum tolerated dose was not reached. The highest dose administered was 
2 × 1012 SYNB1934. Dose-related decreases in labeled Phe area under the curve (AUC), as well 
as increases in the Phe metabolites, TCA and HA, were observed. 
No deaths, serious adverse reactions, or SAEs occurred. The most commonly reported AEs were 
nonserious gastrointestinal (GI)-related AEs, which are a known potential risk of EcN. A 
dose-related increase in the number and severity of GI-related AEs was observed. The majority 
of AEs were mild or moderate. Four participants discontinued IMP due to an AE. No clinically 
significant changes in laboratory values or vital signs have been observed. 
Data on clearance of SYNB1934 from this study have confirmed the expected time to clearance 
from the GI tract, with no participant with a positive fecal quantitative polymerase chain reaction 
result more than 1 week after the last dose. There were no signs of colonization, and no 
participants have required antibiotics for clearance.  
1.5.2. 
Studies of SYNB1618 
Study SYNB1618-CP-001 was a Phase 1/2a, first-in-human, oral, single and multiple 
dose-escalation, randomized, double-blinded, placebo-controlled study of SYNB1618 in healthy 
volunteers (HVs) and adult participants with PKU to evaluate the safety, tolerability, kinetics and 
pharmacodynamics (PD) of the frozen liquid formulation. The study consisted of 2 parts: a 
single-ascending dose (SAD) study and a multiple-ascending dose (MAD) study. 
The study enrolled 56 HVs and 14 PKU participants, all of whom received at least one dose of 
SYNB1618 or placebo. Dose-related increase in strain-specific biomarkers of Phe metabolism 
(D5-labeled and unlabeled plasma TCA and urine hippuric acid [HA]) were observed in both HV 
and PKU active treatment groups compared to placebo in both SAD and MAD study parts. 
No treatment-related serious adverse events (SAEs) or systemic toxicity or infections were 
observed. The majority of treatment-emergent adverse events (TEAEs) were either mild or 
moderate in severity and reversible. Most TEAEs were GI-related and similar to those described 
for the parent strain EcN (i.e., abdominal pain, gut noises, loose stools/diarrhea, nausea, 
vomiting, and headache). At increasing doses, GI related TEAEs, mainly nausea and vomiting, 
were dose limiting. One HV in the highest MAD cohort (1 × 1011 colony forming units [CFU]) 
discontinued dosing due to Grade 2 nausea that was considered related to treatment. 
All participants cleared the bacteria following discontinuation of dosing. The single-dose MTD 
was defined as 2 × 1011 CFU. 
SYNB1618-CP-002 was conducted in adult male and female HVs to bridge the safety, 
tolerability, and PD of SYNB1618, using the lyophilized powder for oral suspension. This study 
included a tracer study and a biomarker study to assess levels of Phe and its metabolites. In total, 
88 subjects were enrolled.  
Dose-dependent reductions in D5-Phe AUC were observed in subjects administered SYNB1618 
compared with placebo subjects after the protein load in the tracer study. In addition, 
dose-related increase in strain-specific biomarkers of Phe metabolism (D5-labeled and unlabeled 
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plasma TCA and urine HA) were observed. In the biomarker study, the production of TCA in the 
fasted state was similar in magnitude to the production after a protein load in the 
SYNB1618-treated subjects. No TCA production was observed in the placebo group. This 
supports strain activity in the fasted state, regardless of dietary Phe intake. 
No treatment-related SAEs were reported. As in the SYNB1618-CP-001 study, mild to moderate 
GI AEs were observed in a dose-related fashion. Nausea and vomiting were the dose-limiting 
symptoms. The MTD was determined to be 2 × 1012 live cells TID. A small, dose-related 
increase in C-reactive protein (CRP) was observed in some participants; the clinical significance 
of this finding is unclear, and no AEs appeared to be related to CRP increases. The CRP level 
returned to normal after the end of dosing in those participants who had a follow-up sample 
available. 
SYNB1618-CP-003 (SynPheny-1) was a Phase 2, open-label study of the efficacy and safety of 
SYNB1618 and SYNB1934 in participants with PKU, designed as a proof-of-concept study to 
show Phe lowering, relative to baseline. This 2-arm study used a dose-ramp regimen consisting 
of 4 dose levels of SYNB1618 (1 × 1011, 3 × 1011, 1 × 1012, and 2 × 1012 live cells) over 15 days 
of treatment in Arm 1, and 4 dose levels of SYNB1934 (3 × 1011, 6 × 1011, 1 × 1012, and 2 × 1012 
live cells) over 15 days of treatment in Arm 2. The treatment period was preceded by a 6-day 
diet run-in period, with concomitant PPI administration throughout the treatment period. A tracer 
study, using D5-labeled Phe, and a biomarker study were conducted. 
Interim data from Arm 1 of the study and preliminary data from Arm 2 of the study support the 
Phe-lowering capacity of SYNB1618 and SYNB1934, respectively. Nine out of 15 PKU 
participants (60%) had at least a 20% reduction in fasting Phe. Of these 9 participants, 
7 participants demonstrated Phe lowering below the guidance threshold for adults with PKU of 
600 μmol/L, and 2 participants had levels ≤ 360 μmol/L, including a single participant reaching a 
normal physiological Phe level. Evaluation of labeled and unlabeled plasma TCA and urinary 
HA supported findings of the primary and secondary efficacy analysis: Levels of these 
metabolites increased in a dose-related manner during the treatment period, demonstrating 
breakdown of Phe in the GI tract. 
No deaths, serious adverse reactions, or treatment-related SAEs have been reported. Two 
participants in Arm 1 experienced increased levels of CRP on Day 14; these levels had returned 
to normal at follow-up on Day 29. One participant in Arm 2 experienced increased CRP on an 
unscheduled visit; these levels had returned to normal at follow-up on Day 29. The clinical 
significance of this finding, if any, is unclear. No other potentially clinically significant 
laboratory values or clinically significant changes in vital signs measurements have been 
reported.  
1.6. 
Study Rationale 
Due to the restrictive nature of the diet and limitations of current treatment, there remains an 
unmet medical need for new treatments for people with PKU. Given the favorable safety profile 
of SYNB1934 and the closely related strain SYNB1618 to date, gut-restricted mechanism of 
action, and demonstrated potential for clinical benefit as shown by blood Phe lowering in PKU 
participants in study SYNB1618-CP-003, further clinical development of SYNB1934v1 as a 
potential treatment for PKU is warranted. 
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PKU participants on sapropterin who do not reach the treatment target of blood Phe 
≤ 360 µmol/L could benefit from addition of SYNB1934v1 in the management of blood Phe. 
Given the differing mechanisms of action of SYNB1934v1 and sapropterin, and different sites of 
action (sapropterin acting intracellularly in the liver on PAH activity, and SYNB1934v1 acting 
locally in the GI tract on Phe degradation), interaction between these 2 agents should not be 
anticipated. Participants on sapropterin enrolled in prior studies have had similar safety and PD 
compared to the overall population. Participants on a stable dose of sapropterin and stable diet, 
with blood Phe > 360 µmol/L are eligible for this study; therefore, any additional blood Phe 
lowering in SYNB1934v1-treated participants could be attributed to the IMP. 
Based on the mechanism of action of the strain and data in prior clinical studies, there is a linear 
relationship between the dose and Phe metabolism, which has not reached a plateau at the 
maximum dose studied (2 × 1012 live cells). Dosing is limited by GI tolerability which appears to 
be variable across individuals. Titration of dose over time has led to improved tolerability. Blood 
Phe lowering has been demonstrated at a dose of 3 × 1011 live cells with the closely related strain 
SYNB1618, and this dose will be the lowest potential iTD for the study. Data from study 
SYNB1618-CP-003 show that a higher dose leads to increased Phe lowering potential and thus, 
improved clinical benefit. Therefore, in dose-escalating, open-label period (DEP) participants 
will titrate to their individual titrated dose (iTD) over time guided by AEs in order to achieve the 
maximum clinical benefit  
Part 2, the randomized withdrawal period (RWP), will allow blinded comparison to placebo 
while participants maintain a stable diet. Phe half-life (t1/2) in PKU is 24 hours, and a steady state 
is reached within 5 days. The 4-week duration in RWP will minimize time off active treatment 
while allowing blinded comparison to placebo. 
Response to SYNB1934v1 varies based on individual factors related to GI physiology 
(e.g., gastric pH, GI transit time) as well as total intake of dietary Phe. Subjects achieving 
≥ 20% blood Phe lowering in DEP will be considered responders and will be included in the 
primary efficacy analysis population. A 20% change in biomarker level exceeds assay variability 
and can generally be considered biologically meaningful and was therefore selected as the 
threshold for responder definition.  
The OLE will enable collection of long-term safety and efficacy data for up to 36 months. 
Dietary intake of Phe may be modified during the OLE based on measured blood Phe lowering if 
the recommended level of Phe < 240 µmol/L has been achieved. Dietary liberalization of natural 
protein while maintaining controlled Phe levels is of benefit to the participants and facilitates 
dietary compliance.  
1.6.1. 
Enrichment Design 
The trial will employ an enrichment strategy of having PKU participants who have a 
≥ 20% reduction from baseline in blood Phe during DEP (“responders”) be the primary analysis 
population in the RWP portion of the study. This strategy is consistent with the predictive 
enrichment strategy discussed in the FDA guidance on enrichment strategies.24 As discussed in 
the guidance, this enrichment strategy chooses participants who are more likely to respond to the 
drug treatment than other participants with the condition being treated. The objective of 
predictive enrichment is to increase study efficiency/feasibility by having a larger effect size and 
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use of a smaller study population and provide an enhanced benefit-risk relationship for the 
responder participants compared to the overall population. 
The criteria for the enriched population, a ≥ 20% reduction from baseline in blood Phe, is 
justified in Section 1.6. This enrichment approach is consistent with similar approved therapies 
for PKU (Kuvan and Palynziq). 
As the final blood Phe assessments from the DEP will not be available at the time of 
randomization, all participants who complete the DEP will be randomized into the RWP. 
Although responder participants will be the primary analysis population, the nonresponders will 
also be included in the RWP portion of the study as well as the OLE of the study. This will allow 
for the assessment of the treatment effect and safety profile in the nonresponder population. 
1.7. 
Benefit-Risk Assessment 
SYNB1934 and its closely related strain SYNB1618 have been studied in more than 230 HVs 
and PKU participants. The safety profile has been consistent with the probiotic chassis EcN, 
which has been used in participants for decades in the European Union and Canada without 
requirement for prescription. There have been no treatment-related SAEs, no deaths, no systemic 
toxicity, and no infections. Interindividual variability in tolerability has been observed. The 
majority of TEAEs have been either mild or moderate in severity and reversible. Most AEs have 
been GI-related and included GI discomfort, nausea, and vomiting. At increasing doses, 
GI-related AEs, mainly nausea and vomiting, have been dose limiting. A small dose-related and 
reversible increase in CRP has been observed in some participants; the clinical significance of 
this finding is unclear. The CRP level has returned to normal after end of dosing. 
Consistent with the design of SYNB1934, clearance is rapid following discontinuation of dosing. 
In study SYNB1618-CP-001, SYNB1618 was cleared within 4 days following the last dose, as 
indicated by fecal quantitative polymerase chain reaction (qPCR) analyses. Data from study 
SYNB1618-CP-002 are consistent. Across all study parts, following up to 7 days of repeat 
dosing with SYNB1618, no fecal sample was above the limit of quantification at 7 days after last 
dose. In neither study was there any evidence of colonization, nor did any participant require 
antibiotic treatment to achieve clearance. Clearance data for SYNB1934 are consistent with that 
for SYNB1618 with no subject having a positive qPCR sample more than 1 week after the last 
dose. 
Data from completed studies demonstrates that SYNB1934 can access Phe from within the GI 
tract, as demonstrated by the D5-Phe meal challenge. A dose-related decrease in plasma D5-Phe 
AUC has been observed, with corresponding increase in strain-specific biomarkers plasma 
D5-TCA and urinary D5-HA. In the Phase 2 study SYNB1618-CP-003, SYNB1934 led to a 
mean 34% decrease in plasma Phe in PKU participants, thus demonstrating potential for clinical 
benefit in the treatment of PKU. Individual variability in Phe lowering was observed. Data from 
HVs in SYNB1934-CP-001 demonstrate increased Phe conversion capacity of SYNB1934 
compared to SYNB1618 based on strain-specific biomarkers. 
The risk of unintended effects of using genetically engineered EcN is considered low. The 
products of the 2 engineered Phe degradation pathways are TCA, produced via the PAL 
pathway, and PP, produced by the deamination of Phe by LAAD. TCA is a naturally occurring 
compound and is found in several plants that are consumed as food. It is frequently added to food 
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as a flavor ingredient and is generally regarded as safe.25 Phenylpyruvate is normally present in 
low amounts in human blood and urine but in patients with PKU who lack PAH enzyme activity, 
large quantities can be excreted in the urine. It is not associated with any known toxicity. An 
additional on-target toxicity risk is related to lowering of blood Phe levels below the normal 
range. However, low blood Phe levels could be tolerated without significant sequelae for at least 
2 months based on clinical observations in prior PKU trials.23 Blood Phe levels will be monitored 
throughout the study and measures are implemented to correct Phe levels if they fall below the 
acceptable range. 
Overall, the benefit–risk assessment for SYNB1934 supports initiation of this Phase 3 trial to 
evaluate the safety and efficacy of SYNB1934v1 in a larger cohort of PKU participants.  
2. 
STUDY OBJECTIVES AND ENDPOINTS 
This study has 3 parts, and each part has a distinct set of objectives, endpoints, and analysis 
populations. The determination of the primary evidence of efficacy will be the comparison of 
SYNB1934v1 versus placebo in the randomized withdrawal period (Part 2).  
Objectives and endpoints are presented in Table 1 to Table 3 for the respective parts of the study.  
Table 1: 
Objectives and Endpoints: Part 1, Dose Escalating, Open-Label Period (DEP) 
Objective 
Endpoint 
Primary: To assess the percent change in blood Phe level 
• Percent change from baseline in blood Phe 
level at last measurement of the iTD of 
SYNB1934v1 
Key Secondary: 
 
•  To assess the absolute change in Phe level 
• Change from baseline in blood Phe level at 
last measurement of the iTD of 
SYNB1934v1 
• To determine the responder population 
• A ≥ 20% reduction from baseline in blood 
Phe level at any time in the DEP  
Other Secondary: 
 
• To assess proportion of participants achieving blood 
Phe level ≤ 360 µmol/L 
• Blood Phe level ≤ 360 µmol/L at any time 
of the iTD of SYNB1934v1 
• Blood Phe level ≤ 360 µmol/L at any time in 
the DEP 
• To assess the safety and tolerability of SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from baseline in clinical laboratory 
parameters 
• Changes from baseline in vital signs 
measurements 
Exploratory: 
 
• 
 
 
 
 
 
 
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Objective 
Endpoint 
• 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Note: baseline and week numbering are specific to study Part 1, unless explicitly noted otherwise in the endpoint. 
Table 2: 
Objectives and Endpoints: Part 2, Randomized Withdrawal Period (RWP) 
Objective 
Endpoint 
Primary: To evaluate efficacy of SYNB1934v1 versus 
placebo in the responder population 
• Change from baseline to Week 4 in blood Phe 
level 
Key Secondary: 
 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard to 
percent change from DEP baseline in blood Phe level 
• Percent change from DEP baseline in blood 
Phe level at Week 4 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard to 
change from DEP baseline in blood Phe level 
• Change from DEP baseline to Week 4 in blood 
Phe level 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard to the 
proportion of participants with a blood Phe level 
≤ 360 µmol/L 
• Blood Phe level ≤ 360 µmol/L at Week 4 
Other Secondary Objectives: 
 
• To assess the safety and tolerability of SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from baseline in clinical laboratory 
parameters 
• Change from baseline in vital signs 
measurements 
• To evaluate efficacy of SYNB1934v1 versus placebo 
in the responder population with regard to the 
proportion of participants with a ≥ 20% reduction 
from DEP baseline in blood Phe level at Week 4 
• A ≥ 20% reduction from DEP baseline in 
blood Phe level at Week 4 
Exploratory: 
 
• 
 
 
 
 
 
  
 
 
 
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• 
 
 
 
 
 
  
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Note: baseline and week numbering are specific to study Part 2, unless explicitly noted otherwise in the endpoint. 
Table 3: 
Objectives and Endpoints: Part 3, Open-label Extension (OLE) 
Objective 
Endpoint 
• To assess the safety and tolerability of SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from DEP baseline in clinical 
laboratory parameters 
• Changes from DEP baseline in vital signs 
measurements 
• To assess absolute and relative change in blood Phe 
level in the responder and nonresponder populations 
• Change from DEP baseline in blood Phe levels 
• Blood Phe level ≤ 360 μmol/L  
• To assess impact of SYNB1934v1 and dietary 
protein intake on Phe levels 
• Change from DEP baseline in blood Phe levels 
over time 
• Change from DEP baseline in blood Phe level 
over time until first protocol-specified increase 
in dietary protein intake 
• To assess impact of SYNB1934v1 on dietary Phe 
intake 
• Change from DEP baseline in dietary Phe 
intake 
Note: baseline and week numbering are specific to study Part 3, unless explicitly noted otherwise in the endpoint. 
3. 
INVESTIGATIONAL PLAN 
3.1. 
Overall Study Design 
This is a 3-part, adaptive study consisting of a dose-escalating, open-label period (DEP; Part 1) 
of up to 15 weeks, followed by a 4-week, double-blind, placebo-controlled, randomized 
withdrawal period (RWP; Part 2), and an open-label extension (OLE; Part 3) of up to 36 months. 
Figure 2 presents a study schematic. 
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In the DEP, all enrolled participants will maintain a stable diet reflecting their baseline Phe 
intake and receive escalating doses of SYNB1934v1 from approximately 3 to 15 weeks to 
determine an iTD, which is defined as the highest dose the participant is able to tolerate. A 
participant will be defined as having reached an iTD if they tolerate 3 weeks at a dose, regardless 
of whether other doses are tolerated. 
Blood Phe level will be measured at each dose level after 3 weeks at that level. A responder will 
be defined as a participant who achieves a ≥ 20% reduction in blood Phe level compared to DEP 
baseline on SYNB1934v1.  
The DEP will contain an optional substudy (DEP weekly substudy) 
 
 
 Additional values obtained as 
part of the substudy will not be used to determine responder status. 
Participants who complete at least 3 weeks at their iTD during the DEP will enter a 4-week 
RWP. Participants will be randomized 1:1 to receive SYNB1934v1 at their iTD determined in 
the DEP or placebo TID. Randomization will be stratified on screening Phe level. Blood Phe 
level will be measured at Weeks 1, 3, and 4 of the RWP. Participants will remain on the same 
diet they consumed during the DEP. 
Participants who complete the 4-week RWP may enter the OLE and receive SYNB1934v1 for up 
to 36 months at their iTD. (At the sponsor’s discretion, enrollment of new participants directly 
into OLE may be opened.) The iTD in Parts 1 and 3 can differ. Participants will be allowed to 
modify their standard diet if their blood Phe level is < 240 µmol/L, as determined by either local 
or central laboratory testing, with guidance from the investigator as outlined in the study-specific 
Diet Manual. The 3-day dietary intake assessments will be conducted per the Schedule of Events 
(Table 5). 
Figure 2: 
Study SYNB1934-CP-003 Schematic 
 
iTD = individually titrated dose. Should the DMC remove doses from the study after completion of the interim 
analysis, the dose ramp will be altered to reflect these changes. 
3.2. 
Dosing and Dose Escalation  
SYNB1934v1 will be taken with a meal throughout the study. Participants will take a proton 
pump inhibitor (PPI) once per day (QD), recommended to occur before breakfast, during all parts 
of the study (see Section 4.3.4), starting at Day –7 of the DEP. If participants are unable to 
tolerate the PPI, they may attempt to use an H2 blocker per investigator discretion. If neither the 
PPI nor H2 blocker can be tolerated, the participant will discontinue from the study. 
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3.2.1. 
Part 1: Dose-Escalating Open-Label 
In the DEP, participants will complete a dose ramp at the beginning of each dose level. During 
the dose ramp, SYNB1934v1 will be administered as shown in Table 4. Dosing will be with 
meals. On days with QD dosing, SYNB1934v1 should be taken with the mid-day meal. On days 
with twice per day (BID) dosing, SYNB1934v1 should be taken with the morning and evening 
meal. 
Table 4: Dose Ramp Schedule 
 
Dose Level 1 
Dose Level 2 
Dose Level 3 
Timing 
D1-9 
D10-14 
D15-21 Wk 4 
Wk 5 
Wk 6 
Wk 7 
Wk 8 
Wk 9 
Dose (live 
cells) 
  
3 × 1011*  
3 × 1011 
3 × 1011 6 × 1011 
6 × 1011 6 × 1011 1 × 1012 
1 × 1012 1 × 1012 
Frequency QD, D1-3 QD, D10-11 TID 
QD × 3 days TID 
TID 
QD × 3 days 
TID 
TID 
 
BID, D4-6  BID, D12-14  
BID × 4 days  
 
BID × 4 days  
 
 
TID, D7-9  
 
 
 
 
 
 
 
BID = twice per day; D = day; QD = once per day; TID = 3 times per day. 
*See Section 4.3.1 
 
 
 
 
 
Participants will dose escalate to their iTD based on tolerability. If the participant tolerates the 
3 × 1011 dose after 3 weeks, and the investigator believes that further tolerance is likely, the 
investigator will escalate to the 6 × 1011 dose. If the 6 × 1011 is tolerated, the participant will 
continue with this dose for a total duration of 3 weeks. If the participant tolerates the 6 × 1011 
after 3 weeks, and the investigator believes that further tolerance is likely, the investigator will 
escalate to the 1 × 1012 dose. The investigator can, with the medical monitor’s assent, declare 
that the participant has reached iTD at any point during dose escalation, but the participant must 
stay on his or her iTD for 3 weeks prior to entering RWP. 
A subset of the population will participate in the DEP weekly substudy, intended to enroll 20 
 
Participants in this substudy will have 
. 
Additional values obtained as part of the substudy will not be used to determine responder status. 
3.2.1.1. 
Intolerance 
Dose-related GI TEAEs have been seen with SYNB1934 in prior studies. These events have 
been most likely to occur with an escalation of dose and can often be addressed by treatment 
with concomitant medications (e.g., ondansetron) and/or dose reduction. If at any time during a 
3-week dosing interval the participant or investigator considers a dose level to be intolerable, the 
following actions may be taken: 
• Start 
 the investigator’s discretion 
 
as needed). 
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• Decrease the frequency of dosing for 1 to 2 days. This can be initiated without the 
approval of the medical monitor. 
• Skip dosing 1 to 2 days. This can be initiated without the approval of the medical 
monitor. 
• Skip dosing ≥ 3 days. This must be discussed with the medical monitor. 
• De-escalate to a previous level after discussion with the medical monitor. In this case, 
the participant will restart 3 weeks at a previously tolerated dose including its ramp, 
and this previously tolerated dose will be the iTD. After de-escalation, participants 
cannot escalate to a higher dose again during Part 1.  
In general, a dose is considered intolerable if the participant experiences Grade 3 or more severe 
TEAEs or Grade 2 GI AEs that do not improve with continued dosing at the same dose level 
over time. If a dose is de-escalated due to a reason other than Grade 3 or greater or Grade 2 GI 
TEAEs, or a second de-escalation is required, the medical monitor must be consulted. 
Participants must take at least 8 of the 9 expected doses on the final 3 days of their iTD period. If 
not, the medical monitor must be consulted before the participant enters Part 2. 
3.2.2. 
Part 2: Randomized Withdrawal 
In the RWP, participants who completed DEP will be randomized 1:1 to receive either 
SYNB1934v1 at their iTD established in the DEP or placebo. Participants will remain on this 
dose of investigational medicinal product (IMP; i.e., SYNB1934v1 or placebo) for the duration 
of the RWP, unless they develop intolerance or meet other discontinuation criteria (see 
Section 3.5). Doses of SYNB1934v1 cannot be modified during the RWP. 
3.2.3. 
Part 3: Open-Label Extension 
During the OLE, all participants will complete a dose ramp to their iTD over time guided by 
tolerability, as described for the DEP, including the full dose ramp schedule (Table 4). The iTD 
in OLE may be different from the iTD in DEP or RWP. The investigator may escalate the dose 
of SYNB1934v1 up to a maximum of 1 × 1012 live cells based on tolerability; multiple attempts 
to escalate to a higher dose level are permitted per investigator discretion.  
3.3. 
Diet and Phenylalanine Monitoring 
For 3 consecutive days prior to scheduled study visits and blood Phe draws, all participants will 
have a 3-day dietary intake assessment (see Table 5). A study dietitian will interview the 
participant and provide advice on maintaining a diet that is consistent with baseline diet 
throughout the study. Participants will follow their usual diet (including Phe, protein, and 
medical food intake) from baseline in the DEP to the end of the RWP.  
During the DEP and RWP, blood Phe levels will be blinded to the sponsor, participant and 
investigator. The investigator will be notified by the central laboratory if Phe is > 1.5 × the DEP 
baseline value, if Phe falls below 30 μmol/L or if Tyr falls below 20 μmol/L, based on a defined 
monitoring plan (to be provided in the Study Manual; see also Section 8.11). During the OLE, 
participants will be allowed to modify their standard diet if their blood Phe level is < 240 
µmol/L, as determined by either local or central lab, with guidance from the investigator as 
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outlined in the study-specific Diet Manual. The 3-day dietary intake assessments will continue 
per the Schedule of Events (Table 6). 
3.4. 
Study Stopping Rules 
If any of the following criteria are met, enrollment will be halted. Participants already enrolled in 
the study will continue dosing until review by the Data Monitoring Committee (DMC). 
1. Two or more participants experience an AE ≥ Grade 3, per protocol severity grading 
criteria, related to SYNB1934v1 as assessed by the investigator.  
2. Clinical infection is detected in a sterile space with a gram-negative aerobic bacillus 
confirmed by qPCR to be SYNB1934v1.  
If any of the above criteria are met, the DMC will review all relevant data and determine whether 
enrollment in the study should resume and whether any modifications to the study are warranted. 
If the DMC decides that further enrollment should be permanently suspended, subjects already 
enrolled in the study will continue to be followed for the duration of the study.  
If a death occurs at any time during the study that is considered by the investigator to be related 
to SYNB1934v1, enrollment will be halted and dosing for subjects already enrolled will be 
stopped until the DMC reviews all relevant data and makes further recommendations.  
This study may also be discontinued at any time due to safety concerns (including, but not 
limited to, the stopping rules described below), failure to meet expected enrollment goals, 
administrative reasons, or at the discretion of the sponsor. Should the study be terminated 
prematurely, the sponsor will provide written notification to the investigator and regulatory 
authorities and will specify the reasons for early termination. The investigator must inform the 
Institutional Review Board (IRB), Independent Ethics Committee (IEC), or Research Ethics 
Board (REB) promptly and provide the reasons for the termination. 
3.5. 
Study Discontinuation for Individual Participants 
Reasons for discontinuation of IMP may include the following:  
• The participant withdraws consent. 
• The investigator or sponsor notes a significant noncompliance with protocol 
procedures (e.g., use of a prohibited concomitant medication). 
• The participant develops an intolerable toxicity including but not limited to a Grade 3 
or more severe AE or SAE assessed as related to IMP by the investigator.  
• The investigator determines that the participant must discontinue further study dosing 
for medical reasons (including pregnancy). 
• The participant has low levels of Phe (< 30 µmol/L) or Tyr (< 20 µmol/L) or has 
profound elevations in Phe (>1.5 × the DEP baseline value during DEP and RWP and 
> 2200µM during OLE). 
If possible, the investigator will confer with the sponsor or medical monitor before discontinuing 
dosing. Participants who discontinue from IMP in RWP will be encouraged to remain on study 
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and complete assessments (particularly safety assessments) through the remainder of RWP. 
Participants who discontinue from DEP and OLE will be asked to have an early termination visit 
30 days after the last dose of IMP (see Section 5.6).  
Participants may withdraw their consent at any time for any reason without prejudice to their 
future medical care by the physician or at the institution. If a participant withdraws consent, the 
date and stated reason for consent withdrawal should be documented. Participant data collected 
up to the date of consent withdrawal will be included in the analyses. 
3.6. 
Duration of Study Participation 
The maximum time of study participation for a participant is planned to be approximately 
6 months for screening through RWP and up to 36 months for Part 3: 
• Screening: up to 45 days 
• Part 1 (DEP): up to 15 weeks 
• Part 2 (RWP): 4 weeks 
• Part 3 (OLE): up to 36 months  
The clinical trial concludes with the final visit of the OLE (36 months). No additional 
arrangements are made for the care of subjects following the completion of the trial, as they will 
be returned to the standard of care for PKU. 
4. 
STUDY POPULATION 
4.1. 
Number of Participants 
This study plans to enroll 
. More participants 
may be enrolled to achieve 
 randomized in Part 2. 
4.2. 
Selection of Participants 
This study will enroll male and female participants ≥ 18 years of age. Participants will be eligible 
for enrollment in this study regardless of race/ethnicity.  
4.2.1. 
Participant Inclusion Criteria 
1. Age ≥ 18 years.  
2. Able and willing to voluntarily complete the informed consent process  
3. Diagnosis of PKU and failure to maintain recommended blood Phe levels on existing 
management (sapropterin, sepiapterin and/or Phe-restricted diet), demonstrated by uncontrolled 
blood Phe level > 360 μmol/L on current therapy any time during screening and uncontrolled 
blood Phe level > 360 μmol/L on current therapy when taking the average of the 3 most recent 
Phe levels from the participant’s medical history (inclusive of any screening values). All 
screening values must be obtained more than 7 days apart, as determined by central or local 
laboratory.  
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4. Females of childbearing potential must have a negative pregnancy test at screening and the 
end of DEP (in order to enter Part 2) and RWP (in order to enter Part 3) and be willing to have 
additional pregnancy tests during the study. 
5. Sexually active female participants of childbearing potential must be willing to use an 
acceptable method of contraception while participating in the study and for 2 weeks after the last 
dose.  
6. Stable diet including stable medical formula regimen (if used) for at least 1 month prior to 
screening.  
7. If using sapropterin or sepiapterin, must be on a stable dose for at least 3 months. 
8. Willing and able to continue current diet, sapropterin, sepiapterin and large neutral amino 
acids unchanged during screening, DEP, and RWP and to engage in all study activities. 
4.2.2. 
Participant Exclusion Criteria 
1. 
Currently taking Palynziq® (pegvaliase-pqpz) (within 1 month of screening). 
2. 
Acute or chronic medical, surgical, psychiatric, or social condition or laboratory 
abnormality that may increase participant risk associated with study participation, 
compromise adherence to study procedures and requirements, and, in the judgment of the 
investigator, would make the participant inappropriate for enrollment. 
3. 
A known or suspected diagnosis of DNAJC12 deficiency, biopterin synthesis deficiency, or 
irritable bowel syndrome. 
4. 
Intolerance of or allergic reaction to E. coli Nissle or any of the ingredients in 
SYNB1934v1 or placebo formulations, or an allergy to cinnamon. Known intolerance to 
proton pump inhibitors and H2 blockers, since one or the other must be used. 
5. 
Currently taking or plans to take any type of systemic (e.g., oral or intravenous) antibiotic 
within 28 days prior to the first dose of IMP through final safety assessment in RWP, 
including planned surgery, hospitalizations, dental procedures, or interventional studies that 
are expected to require antibiotics. Exception: topical antibiotics are allowed. 
6. 
Pregnant, planning to become pregnant, or breastfeeding. 
7. 
Current participation in any other investigational drug study or use of any investigational 
agent within 30 days or 5 half-lives (whichever is longer) prior to screening.  
8. 
Ever received gene therapy for treatment of PKU. 
4.3. 
Treatment of Participants 
Within this protocol, IMP refers to SYNB1934v1 or placebo. 
4.3.1. 
SYNB1934 
SYNB1934v1 is an engineered bacterium derived from EcN that has been designed to treat PKU 
by consuming and converting Phe to the metabolites TCA and phenylpyruvate. 
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SYNB1934v1 IMP consists of powder for oral suspension packaged in sachets. SYNB1934v1 is 
administered orally so that it acts upon Phe liberated from the diet in the gastrointestinal tract. It 
is provided 3 times daily with meals so that it is effective in covering protein intake during the 
course of the day. During dose preparation, the powder will be resuspended in 100 mL of water 
or apple juice. The prepared dose is to be administered within 15 minutes of resuspension. 
During the first 9 days of the DEP, participants will 
 
 
 
 before using the full dose of 
3 × 1011 cells (see Table 4). This partial amount, only used during ramping, is prepared by 
making a 
 
 
 
. 
The study pharmacist or their designee is responsible for tracing the medication. Drug 
accountability for this study will be maintained through the electronic data capture system on an 
ongoing basis. All unused IMP must be returned by participants to the site for destruction per 
local requirements. Detailed instructions for the storage, handling, administration, and disposal 
of IMP will be provided in the study-specific pharmacy manual and in a participant instruction 
pamphlet.  
4.3.2. 
Placebo 
A placebo will be manufactured using an inactive powder that is color matched to the 
SYNB1934v1 drug product. In order to maintain study blinding during the RWP, placebo will be 
packaged, labeled, stored, and administered in an identical manner to SYNB1934v1. 
4.3.3. 
Treatment Compliance 
Study participants will prepare the individual doses at home. They will be instructed on the 
required procedures by trained study personnel and will receive written instructions on IMP 
preparation and storage. Compliance will be assessed by recording the number of sachets 
received and used by each participant. Participants who take ≥ 75% of doses will be considered 
compliant. 
4.3.4. 
Concomitant Medications 
Concomitant medications can be administered at the investigator’s discretion to conform to 
standard practice. Given the gut-restricted mechanism of action, SYNB1934v1 has a low 
likelihood for eliciting clinically relevant drug–drug interactions; however, no traditional drug–
drug interaction studies have been conducted. Investigators should use caution when prescribing 
co-medications and should contact the medical monitor if they are unsure whether a drug should 
be prescribed to a participant. All concomitant medications and dietary supplements will be 
recorded in the electronic case report forms (eCRFs), using generic drug names when possible. 
4.3.4.1. 
Proton Pump Inhibitor 
During participation in the study, participants will take a PPI 
 
QD (recommended to occur before breakfast) starting 7 days before first dose of IMP 
in Part 1. The PPI should be taken at the same time each day, even if no meal is consumed. If 
participants are already on a PPI regimen consistent with instructions in the package insert, they 
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may continue on that and not 
. If participants cannot tolerate a PPI, they 
can use an H2 blocker per investigator discretion. If neither a PPI nor H2 blocker are tolerated 
during the screening, the participant will discontinue the study. The dose of PPI or H2 blocker 
should remain stable for Parts 1 and 2 of the study; if a dose adjustment to the PPI or H2 blocker 
is required in Part 3, the investigator must discuss with the medical monitor (unless the 
adjustment is required due to an adverse event). If a PPI or H2 dose is missed, it should be taken 
as soon as realized and then resume the normal before-breakfast QD or BID schedule, according 
to the locally approved PPI or H2 label. 
4.3.4.2. 
 
During participation in the study, participants will be provided with o
, an approved 
medication for 
 
The use of this medication in the study is optional and at the discretion of the 
investigator (see Section 3.2.1.1). 
4.3.4.3. 
Antibiotics  
Participants who anticipate requiring systemic (e.g., oral or intravenous) antibiotics are not 
eligible; however, unanticipated antibiotic administration may occur during the study. Topical 
antibiotics are permitted at any time. 
Participants who require systemic antibiotics in Parts 1 and 2 will continue IMP dosing but will 
not be included in the per-protocol analysis set (see Section 8.2).  
Participants who require systemic antibiotics in OLE will continue IMP dosing. Any planned Phe 
assessments taken during antibiotic treatment or within 14 days of the last dose of antibiotics in 
the OLE should be obtained > 14 days after the completion of antibiotic treatment. 
4.3.4.4. 
Prohibited Therapies 
Palynziq® (pegvaliase-pqpz) is prohibited within 1 month of screening through the duration of 
the study, as well as any investigational product from screening through the duration of the 
study. 
Participants who have ever received gene therapy for PKU are not eligible for the study. 
5. 
STUDY PROCEDURES AND ASSESSMENTS 
5.1. 
Schedule of Events 
Table 5 and Table 6 presents the schedules of events for the main study. A description of the 
study procedures is provided in the following sections.
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Table 5: 
Schedule of Events: Dose-escalation (Part 1) and Randomized Withdrawal (Part 2) 
 
 
DEP (Part 1)  
RWP (Part 2) 
Description/Weeka 
Screenb 
Study/DEP 
Baseline 
iTD-Finding 
Period 
End of DEPb 
(RWP 
Baseline)c 
Wk 1 
Wk 3 
Wk 4 (OLE 
Baseline)c 
Study Day 
-45 to -1 
1 
Up to 105 
daysd 
21 days after 
iTD 
7 
21 
28 
Assessmentb (Protocol Section) 
 
 
 
 
 
 
 
Informed consent (11.5) 
● 
 
 
 
 
 
 
Medical history and prior medications (5.2) 
● 
 
 
 
 
 
 
Vital signs (SBP/DBP, pulse, body temperature, height, 
weighte) (5.5.2) 
● 
● 
 
● 
 
 
● 
Physical examination (5.2) 
● 
 
 
 
 
 
 
FSH test (postmenopausal women only) (5.5.3) 
● 
 
 
 
 
 
 
Pregnancy test (WOCBP only)f (5.5.3) 
● 
● 
 
● 
 
 
● 
Record concomitant medications (4.3.4) 
● 
● 
● 
● 
● 
● 
● 
Adverse event reporting (5.5.1) 
● 
● 
● 
● 
● 
● 
● 
Collection of adverse event diary cards (5.5.1) 
 
● 
● 
● 
● 
● 
● 
Weekly contact by site personnel (telephone, text, email) 
 
 
● 
● 
● 
● 
● 
Randomization (8.11) 
 
 
 
● 
 
 
 
Blood Pheg and Tyr (5.4.1) 
● 
● 
● 
● 
● 
● 
● 
3-Day dietary intake assessmenth (3.3) 
 
● 
● 
● 
● 
● 
● 
Administer PPI once per day (4.3.4.1) 
●i 
● 
● 
● 
● 
● 
● 
IMP immediately after mealsj (3.2) 
 
● 
● 
● 
● 
● 
● 
Laboratory tests (hematology/CBC with differential, CRP, 
serum chemistry, urinalysis) (5.5.3) 
● 
● 
 
● 
 
 
● 
Electrocardiogram (supine for 5 minutes)(5.5.2) 
● 
 
 
 
 
 
 
Immunogenicity samplek (5.2)  
 
● 
 
 
 
 
 
Optional fecal sample for microbiome analysisl (5.2) 
● 
 
 
 
 
 
 
AE = adverse event; CBC = complete blood count; CRP = C-reactive protein; DEP = dose-escalating period; DBP = diastolic blood pressure; FSH = follicle-
stimulating hormone; IMP = investigational medicinal product; iTD = individually titrated dose; Phe = phenylalanine; PPI = proton pump inhibitor; RWP = 
randomized withdrawal period; SBP = systolic blood pressure; Term = termination; Tyr = tyrosine; WOCBP = women of childbearing potential. 
a Weeks are relative to the study period (i.e., each part begins with Week 1). All assessments/visits may be conducted within a 3-day window. 
b All visits/assessments may be performed in the clinic or by a home healthcare professional at an alternate location (e.g., home, office, etc.), except for screening 
and end of DEP which must either be in person or via a virtual platform capable of completing all scheduled events including examination. 
c Participants who discontinue prematurely during the DEP will complete an early termination visit 30 days after the last dose of IMP as described in Table 6. 
Participants who discontinue prematurely during RWP will complete RWP study measures as scheduled prior to the early termination visit. Early termination 
visits are described in Section 5.6. 
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d Participants may titrate down to the previous dose level during the iTD period if there is difficulty tolerating a higher dose level or at the investigator’s 
discretion. Visits and blood draws occur after a participant has been at a dose level for 3 weeks (including the ramp of that dose level as shown in Table 4). 
Participants must remain on their iTD for 3 weeks before proceeding to Part 2. Participants cannot up-titrate again until the OLE.  
e Height and weight measured only at screening. 
f Serum pregnancy test at screening; urine pregnancy test at other visits. 
g All blood Phe should be drawn in duplicate. Baseline samples in DEP and RWP must be taken prior to the first doses in those periods. In Part 1, blood Phe will 
be measured every 3 weeks after a change in dose level during the iTD-finding period. In Part 2, blood Phe will be measured at Weeks 1, 3, and 4. See 
Section 5.4.1 of the protocol for more information. Patients enrolled in the optional DEP weekly substudy will have weekly 
 See 
Section 5.4.2 of the protocol for more information. 
h A 3-day dietary intake assessment will occur for 3 consecutive days prior to scheduled study visits and blood Phe draws. See Diet Manual for more 
information.  
i The first dose of a PPI will be taken on or before Day –7 and continue daily as described in Section 4.3.4.1.  
j Investigational medicinal product should be taken within 30 minutes of finishing a meal. See the Diet Manual for further details. 
k Samples will be used for monitoring anti-drug antibodies as needed (see Section 6.3). 
l Fecal samples can be done at any time during the screening period, no less than 5 days prior to baseline visit. 
 
 
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Table 6: 
Schedule of Events: Part 3, Open-Label Extension Period and Follow-up, 
End of Study and Early Termination Visits 
 
Quarterly Visits 
End of Study 
Early Term. 
 
Every 90 days 
 ± 5 days 
at least 30 days 
after last dose 
at least 30 days 
after last dose 
Assessmenta (Protocol Section) 
 
 
 
Vital signs (SBP/DBP, pulse, body temperature) 
(5.5.2) 
● 
● 
 
Pregnancy test (WOCBP only)b (5.5.3) 
● 
● 
 
Record concomitant medications (4.3.4) 
● 
● 
● 
Adverse event reporting (5.5.1) 
● 
● 
● 
Blood Phec and Tyr (5.4.1) 
● 
 
 
3-Day dietary intake assessmentd (3.3) 
● 
 
 
Administer PPI once per day (4.3.4.1) 
● 
 
 
IMP immediately after meals (TID) (Section 3.2.3) 
● 
 
 
Laboratory tests (hematology/CBC w/differential, 
CRP, serum chemistry) (5.5.3) 
● 
● 
 
Optional fecal sample for microbiome analysis (5.2) 
 
● 
 
AE = adverse event; CBC = complete blood count; CRP = C-reactive protein; DEP = dose-escalating period; DBP = 
diastolic blood pressure; IMP = investigational medicinal product; Phe = phenylalanine; PPI = proton pump 
inhibitor; RWP = randomized withdrawal period; SBP = systolic blood pressure; Term = termination; TID = 3 times 
per day; Tyr = tyrosine; WOCBP = women of childbearing potential.  
a All visits/assessments may be performed in the clinic or by a home healthcare professional at an alternate location 
(e.g., home, office). 
b Urine pregnancy test at quarterly visits.  
c All blood Phe should be drawn in duplicate.  
d A 3-day dietary intake assessment will occur immediately before all scheduled study visits. 
 
5.2. 
Screening and Baseline Periods 
Screening procedures must be performed to determine eligibility for enrollment within 45 days 
before the first dose of IMP. Participants who fail screening due to a transient cause or missing 
information are eligible for rescreening once, with a minimum of 7 days between screenings. 
During screening, a unique number will be assigned to each participant who signs an informed 
consent form (ICF).  
Participants meeting all the inclusion criteria and none of the exclusion criteria who are selected 
to participate in the study will be enrolled and begin the DEP baseline period. Once participants 
are enrolled in the study, they will only be identified by participant number.  
All assessments to be performed at screening and baseline are outlined in the Schedule of Events 
(Table 5). A physical examination will be performed at screening only and can be performed 
using a virtual platform. Medical history and medications taken over the past 30 days will be 
recorded at screening. Any ongoing condition as well as signs and symptoms observed prior to 
the participant signing the ICF should be recorded as medical history. 
An optional fecal sample will be collected at screening and end of study for microbiome 
analysis. Blood samples will also be collected to aid in the investigation of any immunological 
reaction seen during the study. 
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5.3. 
Randomization 
At the end of the DEP, after completing 3 weeks of dosing at their iTD, participants will be 
randomized 1:1 to receive either SYNB1934v1 at their iTD established in the DEP or placebo, 
stratified by the qualifying blood Phe value from screening. This screening value will be the 
value determined by the central lab, except for participants where the local screening value was 
> 360 μmol/L and the central screening value was ≤ 360 μmol/L, in which case the local 
screening value will be used for stratification. Stratification will be based upon screening blood 
Phe levels of 361 to 720 μmol/L or > 720 μmol/L. This stratification has been selected to 
dichotomize the participant population by control, based upon prior surveys of patients with 
PKU. 
5.4. 
Pharmacodynamic Assessments 
5.4.1. 
Blood Phenylalanine and Tyrosine 
Blood Phe and Tyr samples will be drawn
 will be 
drawn in duplicate, using separate tubes from the same venipuncture, and analyzed separately. 
The time of the last meal will be recorded when taking Phe samples. 
Baseline samples for DEP and RWP must be drawn prior to the first dose for those respective 
periods. During the DEP, blood Phe and Tyr will be measured at Week 3 of each completed dose 
level (Table 5). Blood Phe and Tyr will be measured at Weeks 1, 3, and 4 during the RWP 
(Table 5). During the OLE, blood Phe and Tyr will be measured quarterly (Table 6). 
5.4.2. 
DEP Weekly Substudy 
Participants will have the option to enroll in a substudy 
 
 at each dose level. Incentives will be provided to ensure that at least 
twenty patients are recruited into this substudy. Participants in this substudy 
 
 
 
 
 
 
 
 
 
 
   
5.5. 
Safety Assessments 
5.5.1. 
Adverse Events 
Adverse events will be assessed continuously by direct observation and participant interviews. 
For GI AEs, participants will also fill out a diary card during screening, DEP, and RWP to allow 
for enhanced capture of these AEs. The diary cards will be collected at each scheduled visit, 
excluding the initial screening visit and visits associated with the weekly substudy. The severity 
of AEs will generally be evaluated using the National Cancer Institute Common Terminology 
Criteria for Adverse Events (CTCAE), v5.0, with exceptions described in Section 6.1.1.1. All 
AEs occurring from the time a participant signs the ICF through the follow-up period will be 
recorded, regardless of causal assessment to IMP. 
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5.5.2. 
Vital Signs, Weight, Height, and Electrocardiograms 
Semi-supine vital signs (systolic blood pressure, diastolic blood pressure, pulse, and body 
temperature), weight, and height will be collected as specified in Table 5 and Table 6. 
Participants are required to remain in the semi-supine position for at least 5 minutes prior to 
obtaining vital signs.  
Supine single 12-lead electrocardiograms (ECGs) will be performed at screening as specified in 
Table 5.  
5.5.3. 
Clinical Laboratory Measurements 
Clinical safety laboratory tests will be performed at the time points specified in Table 5 and 
Table 6: chemistry panel, CBC with differential, and creatinine clearance. A pregnancy test for 
WOCBP and an FSH test for postmenopausal women will be performed at the time points 
specified in the Schedule of Events. See Appendix 1 for a list of clinical laboratory tests. 
Screening results will be assessed by the investigator for inclusion of participants in the study. 
Additionally, unscheduled clinical laboratory tests may be obtained at any time during the study 
at the investigator’s discretion. The diagnosis corresponding to any clinically significant 
abnormality or abnormality requiring treatment/intervention must be recorded as an AE. 
If genetic testing for PAH mutations was performed before screening, these data may be 
submitted at the time of screening. Genetic testing is not required for entry into the study, and no 
sample will be drawn for DNA analysis. 
5.6. 
Early Termination Assessments 
Participants who discontinue prematurely during the DEP or OLE will complete an early 
termination visit no sooner than 30 days after the last dose of IMP, as detailed in Table 6. 
Participants who discontinue prematurely during the RWP will complete RWP visits as 
scheduled prior to the early termination visit and will also complete an early termination 
assessment no sooner than 30 days after the last dose of IMP. Participants will be discharged 
from the study after this visit.  
5.7. 
End of Study Assessment 
Participants who complete the OLE will have an end of study visit no sooner than 30 days after 
the last dose of IMP, as detailed in Table 6. The end of study visit may include a fecal sample for 
microbiome analysis. 
6. 
ADVERSE EVENTS 
6.1. 
Adverse Event Definition 
An AE is any untoward medical occurrence, including the exacerbation of a preexisting 
condition, in a participant administered a pharmaceutical product, regardless of causality. 
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6.1.1. 
Assessment of Severity 
The severity rating of an AE refers to its intensity. The severity of each non-GI related TEAE 
will be categorized using the CTCAE. For any term that is not specifically listed in the CTCAE 
scale, intensity should be assigned a grade of 1 through 5 using the following CTCAE 
guidelines: 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated. 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting 
age-appropriate instrumental activities of daily living. 
Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting 
self-care activities of daily living. 
Grade 4: Life-threatening consequences; urgent intervention indicated. 
Grade 5: Death related to AE. 
6.1.1.1. 
Gastrointestinal-related Events 
Participants will be asked to fill out diary cards with information about GI AEs during screening, 
DEP, and RWP. The diary cards will be collected at each scheduled visit, excluding the initial 
screening visit and visits associated with the weekly substudy. In addition, exceptions to CTCAE 
will be implemented for grading certain GI-related TEAEs, as detailed in Table 7. 
Table 7: 
Severity Assessment of Diarrhea and Nausea 
Grade 
Diarrheaa 
Nausea 
1 
Increase of diarrhea, < 3 liquid stools a day 
over baseline; mild increase in ostomy 
output compared to baseline 
Loss of appetite without alteration in eating 
habits and/or with use of planned anti-nausea 
medication 
2 
Diarrhea ≥ 3 liquid stools daily and limiting 
instrumental ADLs 
Use of unplanned concomitant medications to 
increase oral intake 
3 
Diarrhea requiring hospitalization or 
limiting self-care ADLs 
Inadequate oral caloric or fluid intake; tube 
feeding, TPN or hospitalization indicated 
(unchanged from CTCAE) 
4 
Life-threatening consequences; urgent 
intervention indicated (unchanged from 
CTCAE) 
Not applicable 
5 
Death (unchanged from CTCAE)  
Not applicable 
ADLs = activities of daily living; CTCAE = Common Terminology Criteria for Adverse Events. 
a Diarrhea is defined as liquid stools without consistency in shape. 
6.1.2. 
Assessment of Causality 
Medical judgment should be used to determine the cause of the AE, considering all relevant 
factors such as (but not limited to) the underlying study indication, coexisting disease, 
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concomitant medication, relevant history, pattern of the AE, temporal relationship to IMP or PPI, 
and dechallenge or rechallenge. 
Related: there is a reasonable possibility that IMP or PPI caused the event; 1 or more of the 
following criteria apply: 
• The event follows a reasonable temporal sequence from administration of IMP or 
PPI. 
• The event could not be reasonably attributed to the known characteristics of the 
participant’s clinical state, environmental or toxic factors, or other modes of therapy 
administered to the participant. 
• The event follows a known pattern of response to SYNB1934v1 or PPI. 
• The event disappears or decreases on cessation or reduction in dose. (It should be 
noted that in some situations an AE will not disappear or decrease in intensity upon 
discontinuation of study dosing despite other clear indications of relatedness.) 
Unrelated: there is no reasonable possibility that the IMP or PPI caused the event; 1 or more of 
the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of 
IMP or PPI. 
• The event could be reasonably attributed to the known characteristics of the 
participant’s clinical state, concurrent illness, environmental or toxic factors, or other 
modes of therapy administered to the participant. 
• The event does not follow a known pattern of response to SYNB1934v1 or PPI. 
• The event does not disappear or decrease on cessation or reduction in dose, and it 
does not reappear or worsen when dosing is resumed. 
6.2. 
Serious Adverse Events 
An SAE is any untoward medical occurrence that meets any of the following criteria: 
• Results in death. 
• Is immediately life-threatening (refers to an event in which the participant is at risk of 
death at the time of the event; it does not refer to an event, which hypothetically 
might have caused death if it were more severe). 
• Requires inpatient hospitalization or prolongation of existing hospitalization. 
• Results in a persistent or significant incapacity or substantial disruption of the ability 
to conduct normal life functions. 
• Is a congenital anomaly/birth defect. 
• Based on appropriate medical judgment, represents an important medical event that 
may jeopardize the participant or may require intervention to prevent 1 of the other 
outcomes described above. 
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6.2.1. 
Clarification of Serious Adverse Event Definition 
• Death is an outcome of an SAE and not an SAE in itself. When death is an outcome, 
the event(s) resulting in death should be reported (e.g., “pulmonary embolism” with a 
fatal outcome). The appropriate diagnosis or term should be recorded and assigned 
severity Grade 5. 
• In instances of death due ultimately to the underlying disease, the cause of death 
should be indicated as the specific event or condition resulting in death to the extent 
possible. If no appropriate term with a Grade 5 severity in the CTCAE can be 
identified, then a term should be selected from the CTCAE category “death.” 
• “Life-threatening” means that the participant was at immediate risk of death from the 
event as it occurred. This does not include an event that might have led to death if it 
had occurred with greater severity. Grade 4 events (e.g., thrombocytopenia) are not 
always serious unless they have life-threatening consequences or result in 
hospitalization. 
• Preplanned or elective hospitalizations, including social and/or convenience situations 
(e.g., respite care), are excluded from SAE reporting. In addition, “admissions” under 
23-hour Observation or Emergency Room visits are excluded from SAE reporting; 
however, such events should still be reported on the appropriate eCRF page. 
• Overdose of either SYNB1934v1 (defined as a total dose ≥ 2 × 1013 live cells in a 
24-hour period) or concomitant medication without any overdose signs or symptoms 
unless the event meets SAE criteria (e.g., hospitalization) are excluded from SAE 
reporting; however, such events should still be reported on the appropriate eCRF 
page. 
• SAEs that occur within the safety follow-up period but are related to subsequent 
therapies are excluded from SAE reporting: 
o Participants who have completed study dosing or who terminate from study and 
then undergo subsequent therapies during the safety follow-up period and 
experience an SAE specifically related to the administration of the subsequent 
therapy will not have those events reported as SAEs. This exclusion will include 
“elective” hospitalizations necessary for the administration of such therapies. 
6.2.2. 
Serious, Unexpected, Suspected Adverse Reactions 
In accordance with regulatory requirements, the sponsor or designee will immediately notify 
regulatory authorities and the investigators, who will in turn notify their IRB/REB as necessary, 
of any AE associated with IMP administration or study procedures that is a serious, unexpected, 
suspected adverse reaction or any finding from tests in laboratory animals that suggests a 
significant risk for human participants, including reports of mutagenicity, teratogenicity, or 
carcinogenicity. An AE or suspected adverse reaction is considered “unexpected” if it is not 
listed in the Investigator’s Brochure or is not listed at the specificity or severity that has been 
previously observed. 
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6.3. 
Adverse Events of Special Interest 
An adverse event of special interest (AESI), whether serious or nonserious, is one of scientific 
and medical concern specific to SYNB1934v1 for which ongoing monitoring and rapid 
communication by the investigator may be appropriate. Based on current preclinical and clinical 
data, the following AEs will be considered AESIs and should be reported to the Sponsor within 
24 hours of awareness. 
Adverse events of special interest for SYNB1934v1 are as follows: 
• Hypersensitivity reactions due to SYNB1934v1. 
• Infections due to SYNB1934v1.  
• Grade ≥ 3 gastrointestinal AEs per protocol specified grading criteria, including nausea, 
vomiting, diarrhea, and abdominal pain. 
6.4. 
Reporting of Adverse Events 
All AEs, serious and nonserious, will be fully documented on the appropriate eCRF. For each 
AE, the investigator must provide its duration (start and end dates or ongoing), intensity, 
assessment of causality, and whether specific action or therapy was required. 
All AEs occurring from the time a participant signs the ICF through the safety follow-up period 
and at least one month following the last dose of SYNB1934v1 must be recorded on the eCRF.  
All AESIs and SAEs, regardless of relationship to SYNB1934v1 or
, must be 
reported to the Sponsor/designee on the applicable eCRF form within 24 hours of awareness. In 
addition, AESI or SAE forms should be uploaded and submitted to Synlogic.safety@arriello.com 
within 24 hours of awareness. All AESIs and SAEs, regardless of relationship to study drug, will 
be followed by the Investigator until satisfactory resolution or the Investigator deems the SAE to 
be chronic or stable. After the final follow-up visit, only IMP-related AEs/SAEs need to be 
collected and reported. 
6.5. 
Pregnancy 
Women of childbearing potential must have a negative serum pregnancy test at screening. A 
urine pregnancy test will be taken at timepoints outlined in the Schedule of Events (Table 5 and 
Table 6) and must be negative. Women of childbearing potential must agree to use acceptable 
methods of contraception, after informed consent, throughout the study, and for a minimum of 
2 weeks after dosing has been completed. Breastfeeding women or those planning to become 
pregnant are excluded from the study. 
If a participant becomes pregnant during the study, treatment must be discontinued. The 
pregnancy must be reported to the Sponsor/designee and the IRB/IEC/REB. The applicable 
eCRF and pregnancy report form should be completed within 24 hours of awareness by clinical 
site staff. Pregnancy report forms should be sent to Synlogic.safety@arriello.com. Follow-up 
evaluation of the pregnancy, fetus, and child should be performed. If the participant chooses to 
continue the pregnancy, then her pregnancy should be followed to term and 3 months after 
delivery, so that any important safety information can be obtained. 
Males participating in the study do not need to take specific precautions to prevent pregnancy. 
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6.6. 
Clinical Laboratory Abnormalities 
It is the responsibility of the investigator to assess the clinical significance of all abnormal 
laboratory values as defined by the appropriate reference ranges. All abnormal values assessed to 
be of clinical concern and at least possibly related to IMP or of uncertain causality should be 
repeated. Persistent abnormal values and changes of possible clinical concern that remain within 
the normal range should be followed at the discretion of the investigator. 
An abnormal laboratory value that is not already associated with an AE is to be recorded as an 
AE if an action on IMP dosing is made as a result of the abnormality, if intervention for 
management of the abnormality is required, or at the discretion of the investigator. 
6.7. 
Review of Safety Data 
The DMC, investigator, and sponsor medical monitor will be responsible for the ongoing review 
and evaluation of safety data, including AEs, clinical laboratory data, and any other safety 
evaluations, for the duration of the study. Details will be provided in the DMC charter. 
The first DMC meeting will occur when 
 have completed the DEP or when the 
 beyond the DEP baseline 
visit, whichever occurs first.  
The DMC will also meet after the
 participants have completed the DEP and when 
the last participant has completed the RWP.  
All meetings will be used to review safety, to ensure that there is an adequate responder rate for 
the RWP primary analysis population, and to evaluate the DEP baseline Phe values of the 
population prior to treatment. The sponsor will remain blinded to the subject-level blood-Phe 
levels; however, the DEP overall responder rates and DEP baseline Phe values as well as the 
interim analysis results will be provided to the sponsor by the DMC. 
7. 
ESTIMANDS 
The following are the descriptions of the estimands for the primary and key secondary endpoints 
in the study, based on the methods described in ICH-E9 (R1). 
7.1. 
Part 1: Primary and Key Secondary Efficacy Objective Estimands 
The estimands corresponding to the Part 1 DEP primary and key secondary objectives and 
endpoints are described in Table 8 and Table 9, respectively. 
Table 8: 
Estimand for the Part 1 Primary Objective 
Objective 
To assess the percentage change in blood Phe level 
A. Population 
The population is the DEP full analysis set (defined in Section 8.2) characterized by 
the inclusion/exclusion criteria provided in Section 4.2. 
B. Variable 
Continuous variable: Percent change from DEP baseline in blood Phe level at the last 
measurement of the iTD of SYNB1934v1 
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C. Intercurrent events 
• 
Participant does not reach an iTD or iTD dose is dropped: As described in 
Section 3.1, the participant’s iTD will be the highest dose tolerated. Additionally, 
as described in Section 8.12, doses may be dropped at the DEP interim analysis. A 
principal stratum approach will be taken in that the target population is the set of 
participants who reach an iTD and the corresponding dose is not dropped at the 
interim analysis.  
• 
Treatment discontinuation: If a participant permanently discontinues study drug in 
the DEP, the participant will be asked to have an early termination visit 30 days 
after last dose of study drug (see Section 3.5). Treatment discontinuation will 
follow the principal stratum approach in that if the participant has reached an iTD 
(defined as having reached an iTD if they tolerate 3 weeks at a dose, regardless of 
whether other doses are tolerated) the participant will be included in the analysis.  
• 
Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not 
be considered (i.e., all observed data will be used regardless of antibiotic use). 
• 
Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered (i.e., all observed data will be used 
regardless of prohibited medication use). 
D. Population-level 
summary 
The population-level summary is the least-squares mean for the percent change from 
baseline in blood Phe level at the last measurement of the iTD of SYNB1934v1 
derived from an MMRM using an observed case approach. The MMRM model is 
described in Section 8.4.1. 
DEP = dose-escalating, open-label period; iTD = individually titrated dose; MMRM = mixed model with repeated 
measures; Phe = phenylalanine. 
 
Table 9: 
Estimands for the Part 1 Key Secondary Objectives 
Objective 
To assess the absolute change in Phe level 
A. Population 
The population is the DEP full analysis set (defined in Section 8.2) characterized by 
the inclusion/exclusion criteria provided in Section 4.2.  
B. Variable 
Continuous variable: Change from DEP baseline in blood Phe level at the last 
measurement of the iTD of SYNB1934v1 
C. Intercurrent events 
• 
Participant does not reach an iTD or iTD dose is dropped: As described in 
Section 3.1, the participant’s iTD will be the highest dose tolerated. Additionally, 
as described in Section 8.12, doses may be dropped at the DEP interim analysis. A 
principal stratum approach will be taken in that the target population is the set of 
participants who reach an iTD and the corresponding dose is not dropped at the 
interim analysis.  
• 
Treatment discontinuation: If a participant permanently discontinues study drug in 
the DEP, the participant will be asked to have an early termination visit 30 days 
after last dose of study drug (see Section 3.5). Treatment discontinuation will 
follow the principal stratum approach in that if the participant has reached an iTD 
(defined as having reached an iTD if they tolerate 3 weeks at a dose, regardless of 
whether other doses are tolerated) the participant will be included in the analysis 
• 
Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not 
be considered (i.e., all observed data will be used regardless of antibiotic use). 
• 
Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered (i.e., all observed data will be used 
regardless of prohibited medication use). 
D. Population-level 
summary 
The population-level summary is the least-squares mean for the change from baseline 
in blood Phe level at the last measurement of the iTD of SYNB1934v1 derived from 
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an MMRM using an observed case approach. The MMRM model is described in 
Section 8.5.1. 
Objective 
To determine the responder population 
A. Population 
The population is the DEP full analysis set (defined in Section 8.2) characterized by 
the inclusion/exclusion criteria provided in Section 4.2. 
B. Variable 
Dichotomous variable: A ≥ 20% reduction from baseline in blood Phe level at any time 
in the DEP 
C. Intercurrent events 
• 
Participant does not reach an iTD or iTD dose is dropped: As described in 
Section 3.1, the participant’s iTD will be the highest dose tolerated. Additionally, 
as described in Section 8.12, doses may be dropped at the DEP interim analysis. A 
principal stratum approach will be taken in that the target population is the set of 
participants who reach an iTD and the corresponding dose is not dropped at the 
interim analysis.  
• 
Treatment discontinuation: If a participant permanently discontinues study drug in 
the DEP, the participant will be asked to have an early termination visit 30 days 
after last dose of study drug (see Section 3.5). Treatment discontinuation will 
follow the principal stratum approach in that if the participant has reached an iTD 
(defined as having reached an iTD if they tolerate 3 weeks at a dose, regardless of 
whether other doses are tolerated) the participant will be included in the analysis 
• 
Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not 
be considered (i.e., all observed data will be used regardless of antibiotic use). 
• 
Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered (i.e., all observed data will be used 
regardless of prohibited medication use). 
D. Population-level 
summary 
The proportion of participants meeting the ≥ 20% reduction from baseline in blood Phe 
level at any time in the DEP will be summarized along with 95% confidence intervals. 
DEP = dose-escalating, open-label period; iTD = individually titrated dose; MMRM = mixed model with repeated 
measures; Phe = phenylalanine. 
7.2. 
Part 2: Primary and Key Secondary Efficacy Objectives Estimands 
The estimands corresponding to the Part 2 RWP primary and key secondary objectives and 
endpoints are described in Table 10 and Table 11, respectively. 
Table 10: 
Estimand for the Part 2 Primary Objective 
Objective 
To evaluate efficacy of SYNB1934v1 versus placebo in the responder population 
A. Population 
The population is the RWP responder full analysis set (defined in Section 8.2) 
characterized by the inclusion/exclusion criteria provided in Section 4.2.  
B. Variable 
Continuous variable: Change from RWP baseline to Week 4 in blood Phe level 
C. Intercurrent events 
• 
Treatment discontinuation: consistent with a treatment-policy strategy, treatment 
discontinuation will not be considered. 
• 
Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not 
be considered. 
• 
Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered. 
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Objective 
To evaluate efficacy of SYNB1934v1 versus placebo in the responder population 
D. Population-level 
summary 
The population-level summary is the least-squares means and difference in the change 
from baseline to Week 4 in blood Phe levels between the SYNB1934v1 and placebo 
treatment groups. An MMRM using an observed case approach is being applied. The 
post-baseline blood Phe levels at Weeks 1, 3, and 4 will be used. The MMRM model is 
described in Section 8.4.2. 
MMRM = mixed model with repeated measures; Phe = phenylalanine; RWP = randomized withdrawal period. 
 
Table 11: 
Estimands for the Part 2 Key Secondary Objectives 
Objective 
To evaluate the efficacy of SYNB1934v1 versus placebo in the responder population 
with regard to the proportion of participants with a blood Phe level ≤ 360 µmol/L 
A. Population 
The population is the RWP responder full analysis set (defined in Section 8.2) 
characterized by the inclusion/exclusion criteria provided in Section 4.2. 
B. Variable 
Dichotomous variable: blood Phe level ≤ 360 μmol/mL at Week 4 
C. Intercurrent events 
• Treatment discontinuation: consistent with a treatment-policy strategy, treatment 
discontinuation will not be considered. 
• Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not be 
considered. 
• Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered. 
D. Population-level 
summary 
Difference in proportions between the SYNB1934v1 and placebo treatment groups. 
The analysis method is described in Section 8.5. 
Objective 
To evaluate the efficacy of SYNB1934v1 versus placebo in the responder population 
with regard to the change from DEP baseline in blood Phe level 
A. Population 
The population is the RWP responder full analysis set (defined in Section 8.2) 
characterized by the inclusion/exclusion criteria provided in Section 4.2. 
B. Variable 
Continuous variable: Change from DEP baseline to Week 4 in blood Phe level. 
C. Intercurrent events 
• Treatment discontinuation: consistent with a treatment-policy strategy, treatment 
discontinuation will not be considered. 
• Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not be 
considered. 
• Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered. 
D. Population-level 
summary 
The population-level summary is the least-squares means and difference in the change 
from DEP baseline to Week 4 in blood Phe levels between the SYNB1934v1 and 
placebo treatment groups. An MMRM using an observed case approach is being 
applied. The post-baseline blood Phe levels at Weeks 1, 3, and 4 will be used. The 
MMRM model is described in Section 8.5. 
Objective 
To evaluate the efficacy of SYNB1934v1 versus placebo in the responder population 
with regard to the percent change from DEP baseline in blood Phe level 
A. Population 
The population is the RWP responder full analysis set (defined in Section 8.2) 
characterized by the inclusion/exclusion criteria provided in Section 4.2. 
B. Variable 
Continuous variable: Percent change from DEP baseline to Week 4 in blood Phe level. 
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C. Intercurrent events 
• Treatment discontinuation: consistent with a treatment-policy strategy, treatment 
discontinuation will not be considered. 
• Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not be 
considered. 
• Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered. 
D. Population-level 
summary 
The population-level summary is the least-squares means and difference in percent 
change from DEP baseline to Week 4 in blood Phe levels between the SYNB1934v1 
and placebo treatment groups. An MMRM using an observed case approach is being 
applied. The post-baseline blood Phe levels at Weeks 1, 3, and 4 will be used. The 
MMRM model is described in Section 8.5. 
DEP = dose-escalating, open-label period; MMRM = mixed model with repeated measures; Phe = phenylalanine; 
RWP = randomized withdrawal period. 
8. 
STATISTICAL METHODS AND DETERMINATION OF 
SAMPLE SIZE 
8.1. 
General Statistical Methods 
All evaluations and tabulations will be carried out as described in detail in a statistical analysis 
plan (SAP), which will be finalized and approved prior to the DEP interim analysis and database 
lock and unblinding.  
A disposition of participants will be provided for each of the 3 parts of the study and will include 
a breakdown of participants who were randomized, treated, discontinued treatment, lost to 
follow-up, or withdrew consent (as appropriate). 
Continuous variables will be summarized using the number of observations, mean, standard 
deviation (SD), median, minimum, and maximum. Categorical variables will be summarized 
using frequency counts and percentages. 
Baseline for blood Phe level in each of the 3 parts of the study will be defined as the mean of the 
duplicate blood Phe level measurements obtained immediately prior to administration of the first 
dose of each part. If only one blood Phe level measurement is available, then that measure will 
be used as baseline. Similarly, for the determination of responders and participants who achieve 
a specific reduction in Phe (e.g., Phe level ≤ 360 µmol/L at any time in the DEP, or a 
≥ 20% reduction from baseline) the mean of the duplicate blood Phe level measurements at the 
applicable post-baseline timepoint (e.g., Week 3) will be used. If only one of the duplicate blood 
Phe levels is available at a particular post-baseline timepoint, then that measurement will be 
used. In describing the endpoints, the baseline and week numbering are specific to the 
corresponding study part, unless explicitly noted otherwise.  
8.2. 
Populations for Analysis 
Analysis populations will be defined for each of the 3 parts of the study (DEP, RWP, and OLE).  
Full analysis set (FAS): The full analysis sets are based on the intention-to-treat principle. The 
DEP FAS includes all participants enrolled in the DEP.  
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The RWP FAS is defined as all participants randomized into the RWP. Additionally: 
• The responder RWP FAS is defined as participants in the RWP FAS who are 
responders 
• The nonresponder RWP FAS is defined as participants in the RWP FAS who are 
nonresponders 
A responder is defined as a participant who achieves a ≥ 20% reduction in blood Phe level 
compared to DEP baseline on SYNB1934v1 (i.e., this is an exploratory endpoint for the DEP, 
see Table 1). 
The responder RWP FAS will be the primary efficacy analysis population for the RWP. 
Analyses on the RWP FAS will be performed according to the randomized treatment group. 
In the event that, based on the interim analysis results utilizing the criteria provided in Table 12, 
the DMC recommends dropping either the 1 × 1012 or the 1 × 1012 and 6 × 1011 doses, the DEP 
FAS will exclude all participants who were enrolled and treated at a dropped dose in the DEP. 
Additionally, the RWP FAS will exclude all participants who have been randomized in the RWP 
up to this time. The participants in the DEP who have an iTD corresponding to a dropped dose 
will go directly into the OLE part of the study (i.e., as opposed to being randomized into the 
RWP).  
These participants who are excluded from the DEP FAS and RWP FAS will be summarized 
separately.  
Safety analysis set (SAS): For each of the 3 study parts, the SAS will include all participants 
who received any amount of IMP in the corresponding study part with treatment assignment 
based on the treatment received. 
Per-protocol analysis set (PPS): The DEP PPS is defined as all DEP FAS participants, who had 
no major protocol deviations that would affect efficacy, completed their final 3-week iTD, whose 
dietary Phe remained within 20% of their DEP baseline value, and who did not require systemic 
antibiotic treatment during DEP. The DEP PPS will be used as a sensitivity analysis for the DEP 
primary efficacy endpoint.  
The responder RWP PPS is defined as all responder RWP FAS participants, who had no major 
protocol deviations that would affect efficacy, had at least 75% treatment compliance, whose 
dietary Phe remained within 20% of their DEP baseline value, and who did not require systemic 
antibiotic treatment during DEP or RWP (see Section 4.3.4.2). The responder RWP PPS will be 
used as a sensitivity analysis for the RWP primary efficacy endpoint. Major protocol deviations 
will be determined prior to the RWP database lock. Analyses on the responder RWP PPS will be 
performed according to the randomized treatment group. 
8.3. 
Determination of Sample Size 
 
. This assumes a dropout rate of
 and 
approximately 50% of participants are responders. If, during the course of the study, it becomes 
apparent that the assumptions were not accurate or a dose level is dropped after the interim 
analysis, the number of participants enrolled into the DEP may be adjusted. The study has over 
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. The mean percent reduction and standard deviation in blood Phe values used in the 
power calculations are based on results from a similar study in participants with PKU.26 
A sample size of 
 
 
 
 
, a 
 The power was calculated 
via a t-test. The mean treatment group difference and standard deviation in blood Phe values 
used in the power calculations are based on results from a similar study in participants with 
PKU.26 
8.4. 
Primary Efficacy Endpoint Analyses 
The primary efficacy analyses for DEP and RWP are presented in the sections below. Sensitivity 
and supplemental analyses to support these analyses will be presented in the SAP. 
8.4.1. 
Part 1: DEP 
The DEP primary efficacy endpoint is the percent change from DEP baseline in blood Phe level 
at the last measurement of the iTD of SYNB1934v1. The last measurement is the participant’s 
last Week 3 blood Phe level at the iTD of SYNB1934v1 (ie, as discussed in Section 3.2.1).  
The mean percent change from baseline at the participant’s iTD will be tested by an MMRM 
analysis. The MMRM model will have DEP baseline blood Phe level and iTD dose level as fixed 
effects. The dependent variable is the percent change from baseline in blood Phe level at the last 
measurement of the iTD of SYNB1934v1.  
Least-squares means for the percent change from baseline overall and at each iTD dose level, 
along with the corresponding 
 and p-values testing a percent change 
from baseline equal to 0, will be calculated. The primary efficacy analysis will test the 
least-squares means percent change from baseline equal to 0 for the blood Phe level at the 
participants’ last week (Week 3) at his or her established iTD. 
At each of the scheduled assessments blood Phe will be drawn in duplicate. The MMRM will 
incorporate these participant repeated measures. For the MMRM analysis, the results of the 
post-baseline duplicate samples from the Week 3 visit will be included in the statistical model 
(i.e., without averaging the results within a given visit before inclusion into the MMRM). 
The DEP FAS will be the primary efficacy analysis population. 
8.4.2. 
Part 2: RWP 
The RWP primary efficacy endpoint is the change from RWP baseline to Week 4 in blood Phe 
level. An MMRM analysis will be used to compare the mean change in blood Phe level between 
the placebo and SYNB1934v1 dose groups. The MMRM model will have treatment group, RWP 
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baseline blood Phe level, iTD dose level, visit, and visit × treatment group (interaction effect), 
visit × iTD dose level (interaction effect), and visit × RWP baseline blood Phe level (interaction 
effect) as fixed effects. The MMRM will also incorporate the participant’s repeated measures at 
visits Weeks 1, 3, and 4, with visit being treated as a categorical variable. Least-squares means 
for each treatment group and the SYNB1934v1 treatment group difference from placebo, along 
with the corresponding
 will be calculated for Weeks 1, 3, 
and 4. The primary efficacy analysis will compare the least-squares means treatment difference 
between SYNB1934v1 and placebo in the change from baseline in the blood Phe level at 
Week 4. 
At each of the scheduled assessments, blood Phe will be drawn in duplicate. The MMRM will 
incorporate these participant repeated measures. For the MMRM analysis, the results of each of 
the post-baseline duplicate samples from a visit will be included in the statistical model 
(i.e., without averaging the results within a given visit before inclusion into the MMRM). 
The responder RWP FAS will be the primary efficacy analysis population. 
8.5. 
Key Secondary Efficacy Endpoint Analyses 
8.5.1. 
Part 1: DEP 
The key secondary efficacy endpoints for the DEP are: 
• Change from baseline in blood Phe level at last measurement of the iTD of 
SYNB1934v1 
• A ≥ 20% reduction from baseline in blood Phe level at any time of the iTD of 
SYNB1934v1 
• A ≥ 20% reduction from baseline in blood Phe level at any time in the DEP.  
The key secondary endpoint, change from baseline in blood Phe level at last measurement of the 
iTD of SYNB1934v1, will be analyzed using an MMRM analysis similar to the model described 
for the DEP primary efficacy analysis (Section 8.4), with the dependent variable being the 
change in blood Phe level from DEP baseline. 
For the 2 key secondary endpoints, a ≥ 20% reduction from baseline in blood Phe level at any 
time of the iTD of SYNB1934v1, and a ≥ 20% reduction from baseline in blood Phe level at any 
time in the DEP. The proportion of participants meeting these criteria will be summarized along 
with 95% confidence intervals.  
These analyses will be performed on the DEP FAS. 
8.5.2. 
Part 2: RWP 
The key secondary efficacy endpoints for the RWP are: 
• Percent change from DEP baseline in blood Phe level at Week 4. 
• Change from DEP baseline to Week 4 in blood Phe level 
• Blood Phe level ≤ 360 µmol/L at Week 4. 
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The proportion of participants meeting the key secondary endpoint, a blood Phe level 
≤ 360  µmol/L at Week 4, will be tested between the placebo and SYNB1934v1 dose groups 
using a Fisher’s Exact test. The key secondary endpoint, change from DEP baseline to Week 4 in 
blood Phe level, will be analyzed using an MMRM analysis similar to the model described for 
the RWP primary efficacy analysis (Section 8.4), with the dependent variable being the change 
in blood Phe level from DEP baseline, and the DEP baseline blood Phe level as the baseline 
blood Phe level covariate. The key secondary endpoint, percent change from DEP baseline in 
blood Phe level at Week 4, will be analyzed using an MMRM analysis similar to the model 
described for the RWP primary efficacy analysis (Section 8.4), with the dependent variable being 
the percent change in blood Phe level from DEP baseline, and the DEP baseline blood Phe level 
as the baseline blood Phe level covariate.  
These analyses will be performed on the responder RWP FAS. 
8.6. 
Other Secondary Efficacy Endpoint Analyses 
8.6.1. 
Part 1: DEP 
The secondary efficacy endpoints for the DEP are: 
• Blood Phe level ≤ 360 μmol/L at any time of the iTD of SYNB1934v1 
• Blood Phe level ≤ 360 µmol/L at any time in the DEP. 
For each of these 2 endpoints, the proportion of participants meeting the endpoint will be 
summarized along with 
. These analyses will be performed on the DEP 
FAS. 
8.6.2. 
Part 2: RWP 
The additional secondary efficacy endpoints for the RWP are: 
• A ≥ 20% reduction from DEP baseline in blood Phe level at Week 4. 
The proportion of participants achieving a ≥ 20% reduction in blood Phe level from baseline to 
Week 4 will be tested between the placebo and SYNB1934v1 dose groups using a Fisher’s Exact 
test. 
These analyses will be performed on the responder RWP FAS.  
8.7. 
Exploratory Endpoint Analyses 
The analysis methods for the exploratory endpoints will be presented in the SAP. 
8.8. 
Missing Data 
The primary efficacy endpoint analyses for the DEP and RWP will be an observed-case analysis. 
Missing data will not be imputed for the primary efficacy analyses.  
For the following key secondary efficacy analyses, an observed-case analysis will also be 
applied:  
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• DEP Part 1: Change from baseline in blood Phe level at last measurement of the iTD 
of SYNB1934v1 
• RWP Part 2: Change from DEP baseline to Week 4 in blood Phe level 
• RWP Part 2: Percent change from DEP baseline in blood Phe level at Week 4 
For the following dichotomous key secondary efficacy analyses, an observed-case approach is 
being applied such that if a participant is missing the data needed to determine the endpoint, they 
will be considered as not having met the endpoint. 
• DEP Part 1: A ≥ 20% reduction from baseline in blood Phe level at any time of the 
iTD of SYNB1934v1 
• DEP Part 1: A ≥ 20% reduction from baseline in blood Phe level at any time in the 
DEP 
• RWP Part 2: Blood Phe level ≤ 360 µmol/L at Week 4. 
Sensitivity analyses considering different methods of handling missing data for the primary and 
key secondary efficacy endpoints will be specified in the SAP. Additional details on the handling 
of missing data will be provided in the SAP. 
8.9. 
Multiplicity 
The overall Type I error rate for the DEP will be controlled at the 2-sided 0.05 level for the 
primary endpoint. If the DEP primary endpoint analysis is significant at the 2-sided 0.05 level 
the key secondary endpoint, change from baseline in blood Phe level at last measurement of the 
iTD of SYNB1934v1, will be tested at the 2-sided 0.05 level. Note that the 2 dichotomous DEP 
key secondary endpoints; a ≥ 20% reduction from baseline in blood Phe level at any time of the 
iTD of SYNB1934v1, and a ≥ 20% reduction from baseline in blood Phe level at any time in the 
DEP - do not have an associated inferential test.  
The overall Type I error rate for the RWP will also be controlled at the 2-sided 0.05 level for the 
primary endpoint (i.e., DEP, and RWP, will each have a 2-sided Type I error rate of 0.05). The 
control of multiplicity at 0.05 within each study part is consistent with this study having 3 parts, 
with each study part having a distinct set of objectives, endpoints, and analysis populations. 
For the RWP, the 3 key secondary endpoints will be tested at the 2-sided 0.05 level using a fixed 
sequential testing methodology in this order:  
1) Percent change from DEP baseline to Week 4 in blood Phe level 
2) Change from DEP baseline to Week 4 in blood Phe level  
3) Blood Phe level ≤ 360 μmol/mL at Week 4  
These key secondary endpoints will be tested following this prespecified order only if the RWP 
primary efficacy endpoint null hypothesis is rejected. 
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8.10. 
Safety Analysis 
Safety will be evaluated by scheduled monitoring of AEs, vital signs, and clinical laboratory 
measurements. Safety parameters will be summarized descriptively by the treatment regimen 
(dose and frequency) that the participant was on at the time of the safety measurement. 
By-participant listings of all measurements and parameters will be presented in tabular format, 
including absolute values and changes from baseline (if applicable), by dose cohort and study 
day. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities, and 
severity of AEs and laboratory abnormalities will be graded using the National Cancer Institute 
CTCAE, v5.0, with certain exceptions as listed in Table 7. Adverse events will be tabulated by 
study period, treatment group, system organ class, and preferred term. Incidence tables of 
participants with AEs will be presented for all AEs by maximum severity, AESIs, SAEs, AEs 
assessed as related to IMP, and AEs resulting in discontinuation of study dosing. 
For the DEP, safety analyses will be performed on the DEP SAS. For the RWP and OLE study 
parts, safety analyses will be performed on the corresponding SAS, with results being presented 
for the responder and nonresponder populations as well as overall population. 
8.11. 
Randomization and Blinding 
In the RWP, participants will be randomized in a 1:1 ratio to receive either SYNB1934v1 or 
matching placebo, stratified on screening Phe level (361 to 720 μmol/L, or > 720 μmol/L). 
Participants, investigators, and the sponsor will be blinded to randomized IMP assignment during 
the RWP. In the case of an emergency where information regarding treatment assignment would 
impact the care provided to a participant during the RWP of the study, the investigator will have 
immediate access to unblind the treatment code in the interactive response system (IXRS). The 
instructions for unblinding a subject in the IXRS can be found in the IXRS User Guide. In the 
event unblinding is necessary, the investigator is strongly encouraged to contact the medical 
monitor to discuss the situation and the subjects medical status prior to unblinding. It is 
mandatory that any personnel involved in the unblinding or who have access to the unblinded 
treatment assignment maintain the confidentiality of the information. 
During the DEP and RWP, blood Phe levels will be blinded to the participant, investigator and 
sponsor. During the DEP and RWP, the investigator will receive an email alert from the central 
laboratory if a blood Phe is > 1.5 × the DEP baseline, if Phe is < 30 μmol/L, or if Tyr is < 20 
μmol/L. After the receipt of one of these trigger values, the investigator should arrange for a 
retest of Phe and Tyr. If the trigger value is confirmed to be out of range, the individual 
participant will be discontinued from the study.  
8.12. 
Interim Analysis 
Once the first
 have attained an iTD and completed the DEP or the 
 
participant has a baseline DEP and has been in the study 
, whichever occurs earlier, 
the DEP data will be evaluated by an independent DMC. The DMC will use the below 
pre-specified set of criteria based on both safety and efficacy to determine whether the dose 
regimen is appropriate or whether any doses should be discontinued as well as whether the study 
should be stopped for futility. Enrollment will not be paused during the interim analysis.  
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If a dose level is dropped, participants who are in the DEP and have an iTD corresponding to a 
dropped dose will not be randomized into the RWP but will instead enter the OLE directly. In 
addition, participants who are in the OLE at a dose level that has been dropped will be 
transitioned to the highest dose level retained. 
The size of the DEP interim analysis cohort and corresponding criteria for dropping a dose 
(Table 12) provide reasonable operating characteristics for proper determination if one or more 
doses should be dropped. 
Table 12: 
DMC Criteria for Dose Changes and Futility 
Evaluation by DMC 
Criteria to Drop the Dose 
1 × 1012 dose 
< 20% of the participants who attain an iTD are at the 1 × 1012 dose 
OR 
> 75% of participants who have 1 × 1012 as their iTD had a greater 
Phe reduction at their 6 × 1011 dose versus their 1 × 1012 dose. 
6 × 1011 dose 
 
Note: considered only 
if the Drop 1 × 1012 
Criteria has been met 
< 30% of the participants who attain an iTD have 6 × 1011 or 1 × 1012 
as their iTD 
OR 
> 75% of participants who have 6 × 1011 or 1 × 1012 as their iTD had a 
greater Phe reduction at their 3 × 1011 dose versus their 6 × 1011 dose 
Stop for futility 
<30% of participants enrolled into the DEP attain an iTD and 
complete the DEP 
OR 
< 25% of participants who attain an iTD and complete the DEP are 
responders 
DEP = dose-escalating, open-label period; DMC = data monitoring committee; iTD = individually titrated dose; 
Phe = phenylalanine 
 
In the event that, based on the interim analysis results utilizing the criteria provided in Table 12, 
the DMC recommends dropping either the 1 × 1012 or the 1 × 1012 and 6 × 1011 doses, the RWP 
FAS will be modified as described in Section 8.2. 
In addition, the DMC will review interim data at defined intervals in the study. The Sponsor will 
remain blinded to the subject-level blood Phe levels during the DEP and RWP but will have 
access to the DEP interim analysis results. 
The data from the OLE (Part 3) will be reviewed for safety and tolerability on an ongoing basis. 
9. 
CHANGES IN THE CONDUCT OF THE STUDY OR PLANNED 
ANALYSIS 
Only the sponsor may modify the protocol. Protocol deviations can be made when a participant’s 
safety is compromised. In these circumstances, the investigator should inform the sponsor and 
the full IRB/IEC/REB within 1 working day after the emergency occurred. All amendments that 
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have an impact on participant risk or the study objectives or require revision of the ICF must 
receive approval from the IRB/IEC/REB prior to implementation. 
10. 
DATA RECORDING, RETENTION, AND MONITORING 
10.1. 
Case Report Forms 
Data will be collected using source documents through an electronic data capture system at the 
clinical site. The investigator or designee will record data specified in the protocol using eCRFs. 
Changes or corrections to eCRFs will be made by the investigator or an authorized member of 
the study staff according to the eCRF completion guidelines. It is expected that the site will enter 
all data within 5 business days of a participant visit. 
It is the investigator’s responsibility to ensure eCRFs are complete and accurate, regardless of 
whether this responsibility has been delegated in whole or in part. Following review and 
approval, the investigator or designee will electronically sign and date the pages, which certifies 
that the investigator has thoroughly reviewed and confirmed all data on the eCRF. 
10.2. 
Data Retention 
Data retention practices will follow International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use (ICH) guidelines, which note that essential 
documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing 
applications in an ICH region or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the IMP. However, these documents should be retained for a longer 
period if required by the applicable legal requirements. 
10.3. 
Data Monitoring 
This study will be closely monitored by representatives of the sponsor or designee throughout its 
duration. Monitoring will include personal visits with the investigator and study staff as well as 
appropriate communications by telephone, teleconference, fax, mail, email, use of the electronic 
data capture system, or any other method as applicable. It is the monitor’s responsibility to 
inspect eCRFs at regular intervals throughout the study to verify the completeness, accuracy, and 
consistency of the data and to confirm adherence to the study protocol and Good Clinical 
Practice (GCP) guidelines. The investigator agrees to cooperate with the monitor to ensure that 
any problems detected during the course of this study are resolved promptly. The investigator 
and site will permit study-related monitoring, audits, IRB/IEC/REB review, and regulatory 
inspection, including direct access to source documents. 
It is understood that study monitors and any other personnel authorized by the sponsor may 
contact and visit the investigator and will be permitted to inspect all study records (including 
eCRFs and other pertinent data) on request, provided that participant confidentiality is 
maintained, and that the inspection is conducted in accordance with local regulations. 
Every effort will be made to maintain the anonymity and confidentiality of participants during 
this study. However, because of the experimental nature of the IMP, the investigator agrees to 
allow representatives of the sponsor and authorized representatives of regulatory authorities to 
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inspect the facilities used in the conduct of this study and to inspect, for purposes of verification, 
the hospital or clinic records of all participants enrolled in the study. 
10.4. 
Quality Control and Quality Assurance 
Quality control procedures will be conducted according to the sponsor and CRO’s internal 
procedures. The study site may be audited by a quality assurance representative of the sponsor. 
All necessary data and documents will be made available for inspection. 
11. 
REGULATORY, ETHICAL, AND LEGAL OBLIGATIONS 
11.1. 
Good Clinical Practice 
The study will be performed in accordance with the protocol, guidelines for GCP established by 
the ICH and applicable local regulatory requirements and laws with all clinical study files 
maintained in accordance with ICH/GCP guidelines. 
11.2. 
Institutional Review Board/Independent Ethics Committee/Research 
Ethics Board Approval 
The investigator must inform and obtain approval from the IRB/IEC/REB for the conduct of the 
study at named sites, for the protocol, ICF, Investigator Brochure, and any other written 
information that will be provided to the participants and for any advertisements that will be used. 
Written approval must be obtained prior to recruitment of participants into the study. 
Proposed amendments to the protocol (see Section 9) and aforementioned documents must be 
submitted to the sponsor for review and approval, then to the IRB/IEC/REB. Amendments may 
be implemented only after a copy of the approval letter from the IRB/IEC/REB has been 
transmitted to the sponsor. 
In accordance with GCP guidelines, the investigator will be responsible for ensuring that annual 
updates are provided to the IRB/IEC/REB (or more frequently in accordance with the 
requirements, policies, and procedures as established by the IRB/IEC/REB) until the study is 
completed (i.e., finalization of the clinical study report) to facilitate continuing review of the 
study and that the IRB/IEC/REB is informed about the end of the study. Copies of the update, 
subsequent approvals, and final letter must be sent to the sponsor. 
11.3. 
Regulatory Authority Approval 
The study will be performed in accordance with the requirements of the local health authority 
(e.g., US FDA, Health Canada) and will also meet all requirements of ICH GCP guidance. 
Amendments to the protocol will be submitted to the health authority prior to implementation, in 
accordance with applicable regulations. 
11.4. 
Other Required Approvals 
In addition to IRB/IEC/REB and regulatory authority approval, the investigator will be 
responsible for ensuring that all other required approvals (e.g., approval from the local research 
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and development board or scientific committee) will be obtained prior to recruitment of 
participants into the study. 
11.5. 
Informed Consent 
Informed consent is a process that is initiated prior to the participants agreeing to participate in 
the study and continues throughout the participants’ study participation. It is the investigator’s 
responsibility (or designee) to obtain written informed consent (the “informed consent form” or 
ICF) from each participant after adequate explanation of the aims, methods, anticipated benefits, 
and potential hazards of the study and before any study procedures are initiated. The investigator 
or his/her designee will explain the nature of the study to the participant and answer all questions 
regarding the study. The investigator is responsible for ensuring that the participant fully 
understands the nature and purpose of the study. Information should be given in both oral and 
written form whenever possible. No participant should be obliged to participate in the study. The 
participant must be informed that participation is voluntary, that they may withdraw at any time, 
and that choosing not to participate will not affect the care the participant will receive.  
Each participant should be given a copy of the ICF and associated materials. The original copy 
of the signed and dated ICF must be retained at the site and is subject to inspection by 
representatives of the sponsor, regulatory authorities, or IRB/IEC/REB. If any amendments occur 
throughout the course of the study that affect the ICF (i.e., when new study procedures or 
assessments have been added), all active participants should be reconsented using the same 
process for the initial consent. 
11.6. 
Participant Confidentiality 
The investigator must ensure that the participant’s privacy is maintained and that all necessary 
and appropriate measures consistent with applicable law will be employed to safeguard their 
personal, health, and medical information. On eCRFs and other documents submitted to the 
sponsor, participants will be identified by their assigned participant number. Documents that are 
not submitted to the sponsor (e.g., signed ICFs) should be kept in a confidential file by the lead 
investigator. 
All personal data gathered in this trial will be treated in the strictest confidence by investigators, 
monitors, and all associated personnel. No data will be disclosed to any third party without the 
express permission of the participant concerned, except that the investigator shall permit 
authorized representatives of the sponsor, regulatory authorities, and the IRB/IEC/REB to review 
the portion of the participant’s medical record that is directly related to the study. As part of the 
required content of the ICF, the participant must be informed that his/her records will be 
reviewed in this manner. 
For trials being conducted under Regulation EU No 536/2014, as per Annex I, D (17am), the 
contract between the Sponsor and vendors or trial sites will specify the responsibilities of the 
parties related to data protection, including the handling of data security breaches and respective 
communication and cooperation of the parties. 
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11.7. 
Study Confidentiality and Disclosure of Information 
Information concerning the study, the progress of the study, protocol, processes, assessments, 
IMP, scientific data, or other non-public information related to the study or the sponsor and its 
representatives is confidential and remains the property of the sponsor. The lead investigator and 
his or her designees may use this information for the purposes of the study only.  
It is understood by the lead investigator that the sponsor will use information obtained in this 
clinical study in connection with the clinical development program, and therefore may disclose it 
as it sees fit, including to other clinical investigators and to regulatory authorities. In order to 
allow the use of the information derived from this clinical study, the lead investigator 
understands that he/she has an obligation to provide complete test results and all data obtained 
during this study to the sponsor. 
Verbal or written discussion of the progress of the study, or the study results, prior to study 
completion and full reporting, should only be undertaken with written consent from the sponsor. 
11.8. 
Publication of Study Data 
The sponsor encourages the scientific publication of data from clinical research studies in a 
relevant peer-reviewed journal. However, investigators may not present or publish partial or 
complete study results individually without participation of the sponsor. The lead investigator 
and sponsor may propose appropriate scientific manuscripts or abstracts from the study data. All 
proposed publications must be reviewed and commented on by the sponsor before submission 
for publication. The detailed procedures for the review of publications are set out in the clinical 
trial agreement entered into with the sponsor in connection with this study. These procedures are 
in place to ensure (a) coordination of study data publication, (b) adequate review of data for 
publication against the validated study database for accuracy, and (c) that confidentiality of 
sponsor business information or participant personal information is maintained. 
Qualification of authorship will follow the requirements of the International Committee of 
Medical Editors (www.icmje.org). The names of investigators and sponsor representatives 
responsible for designing the study and analyzing the results will be included in the 
publication(s). This custom can be adjusted upon mutual agreement of the authors and Synlogic. 
In addition, this clinical trial must be registered with ClinicalTrials.gov, which will be done by 
the sponsor or its representative and will be registered in any applicable international database or 
website as required by law or regulation. The results of and data from this study belong to 
Synlogic. 
11.9. 
Ethical Standards 
We are committed to designing, implementing, conducting, analyzing, and reporting these trials 
in compliance with the highest ethical and scientific standards. Protection of participant safety is 
the overriding concern in the design of clinical trials. In all cases, Synlogic clinical trials will be 
conducted in compliance with local and/or national regulations and in accordance with the 
consensus ethical principles that have their origin in the Declaration of Helsinki. 
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12. 
REFERENCES 
1. 
NNSGRC. Annual Report. National Newborn Screening and Genetics Resource Center. 
May 2009. 
2. 
Lindegren ML, Krishnaswami S, Reimschisel T, Fonnesbeck C, Sathe NA, McPheeters 
ML. A systematic review of BH4 (sapropterin) for the adjuvant treatment of 
phenylketonuria. JIMD Rep. 2013;8:109-19. 
3. 
Anikster Y, Haack TB, Vilboux B, et al. Biallelic mutations in DNAJC12 cause 
hyperphenylalaninemia, dystonia, and intellectual disability. Am J Hum Genet. 2017;100: 
257-66. 
4. 
Blau N, Martinez A, Hoffmann GF, Thöny B. DNAJC12 deficiency: A new strategy in the 
diagnosis of hyperphenylalaninemias. Mol Genet Metab. 2018;123: 1-5. 
5. 
Blau N, Longo N. Alternative therapies to address the unmet medical needs of patients 
with phenylketonuria. Expert Opin Pharmacother. 2015;16(6):791-800. 
6. 
Vernon HJ, Koerner CB, Johnson MR, Bergner A, Hamosh A. Introduction of sapropterin 
dihydrochloride as standard of care in patients with phenylketonuria. Mol Genet Metab. 
2010;100(3):229-33. 
7. 
Vockley J, Andersson HC, Antshel KM, et al. Phenylalanine hydroxylase deficiency: 
diagnosis and management guideline. Genet Med. 2014;16(2):188-200. 
8. 
Ashe K, Kelso W, Farrand S, er al. Psychiatric and cognitive aspects of phenylketonuria: 
the limitations of diet and promise of new treatments. Front Psych. 2019;10:561. 
9. 
van Spronsen FJ, van Wegberg AM, Ahring K, et al. Key European guidelines for the 
diagnosis and management of patients with phenylketonuria. Lancet Diabetes Endocrinol. 
2017;5:743-56. 
10. 
Santos LL, Fonsecca CG, Starling ALP, et al. Variations in genotype-phenotype 
correlations in phenylketonuria patients. Genet. Mol. Res. 2010;9(1):1-8. 
11. 
Bilder DA, Noel K, Baker ER, et al. Systematic review and meta-analysis of 
neuropsychiatric symptoms and executive functioning in adults with phenylketonuria. Dev 
Neurol. 2016;41:245-60. 
12. 
Gentile JK, ten Hoedt AE, Bosch AM. Pscyhosocial aspects of PKU: hidden disabilities—
a review. Mol Genet Metab. 2010;99(suppl):S64-67. 
13. 
Griffiths PV, Demellweek C, Fay N, Robinson PH, Davidson DC. Weschler subscale IQ 
and subtest in early treated phenylketonuria. Arch Dis Child. 2000;82(3):209-15. 
14. 
Waisbren SE, Noel K, Fahrbach K, et al. Phenylalanine blood levels and clinical outcomes 
in phenylketonuria: a systematic literature review and meta-analysis. Mol Genet Metab. 
2007;92:63-70. 
15. 
Huijbregts SCJ, de Sonneville LMJ, Licht R, van Spronsen FJ, Verkerk PH, Sergeant JA. 
Sustained attention and inhibition of cognitive interference in treated phenylketonuria: 
associations with concurrent and lifetime phenylalalnine concentrations. 
Neuropscyhologia. 2002;40(1):7-15. 
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16. 
Singh RH, Rohr F, Frazier D, et al. Recommendations for the nutrition management of 
phenylalanine hydroxylase deficiency. Genet Med. 2014; 16(2):121-31. 
17. 
van Wegberg AMJ, MacDonald A, Ahring K, et al. The complete European guidelines on 
phenylketonuria: diagnosis and treatment. Orphanet J Rare Dis. 2017;12:162 
18. 
Grech A, Colline CE, Holmes A, et al. Maternal exposures and the infant gut microbiome: 
a systematic review and meta-analysis. Gut Microbes. 2021;13: e1897210. 
https://doi.org/10.1080/19490976.2021.1897210. 
19. 
Kuvan prescribing information (United States). BioMarin Pharmaceutical Inc, July 2015. 
20. 
Burton BK, Grange DK, Milanowski A, et al. The response of patients with 
phenylketonuria and elevated serum phenylalanine to treatment with oral sapropterin 
dihydrochloride (6R-tetrahydrobiopterin): a phase II, multicentre, open-label, screening 
study. J Inherit Metab Dis. 2007;30(5):700-7. 
21. 
Palynziq prescribing information (United States). BioMarin Pharmaceutical Inc, May 
2018. 
22. 
Eijgelshoven I, Demridas S, Smith TA,van Loon JMT, Latour S, Bosch AM. The time 
consuming nature of phenylketonuria: a cross-sectional study investigating time burden 
and costs of phenylketonuria in the Netherlands. Mol Genet Metab. 2013;109(3):237-42. 
23. 
Thomas J, Levy H, Amato S, et al. Pegvaliase for the treatment of phenylketonuria: results 
of a long-term phase 3 clinical trial program (PRISM). Mol Genet Metab. 2018;124:27-38. 
24. 
FDA. Enrichment strategies for clinical trials to support approval of human drugs and 
biological products. Guidance for industry. March 2019. Available: https://www.fda.gov/ 
regulatory-information/search-fda-guidance-documents/enrichment-strategies-clinical-
trials-support-approval-human-drugs-and-biological-products. 
25. 
Adams TB, Cohen SM, Doull J, et al. The FEMA GRAS assessment of cinnamyl 
derivatives used as flavor ingredients. Food Chem Toxicol. 2004;42(2):157-85. 
26. 
FDA. Drug approval package: Kuvan (sapropterin dihydrochloride) tablets. 13 December 
2007. Available: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2007/ 
022181TOC.cfm. 
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APPENDIX 1. CLINICAL LABORATORY TESTS 
Hematology 
(CBC with 
differential) 
Basophils% 
Basophils 
Eosinophils% 
Eosinophils 
Hematocrit 
Hemoglobin 
Lymphocytes% 
Lymphocytes 
Mean corpuscular hemoglobin 
Mean corpuscular volume 
Monocytes% 
Monocytes 
Neutrophils% 
Neutrophils 
Platelet count 
Red blood cells 
White blood cells 
Serum 
chemistry 
Glucose 
BUN 
Creatinine with eGFR 
Sodium 
Potassium 
Chloride 
Calcium 
Total protein 
Albumin 
Fractionated bilirubin (total direct 
and indirect) 
Follicle-stimulating hormone (for 
postmenopausal women only)a 
Alkaline phosphatase 
Aspartate aminotransferase 
Alanine aminotransferase 
Pregnancy (for WOBCP only) 
Glucose 
C-reactive protein 
Urinalysis 
Specific gravity 
pH 
Glucose 
Bilirubin 
Ketones 
Occult blood 
Protein 
Nitrite 
Leukocyte esterase 
Pregnancy (for WOBCP only) 
Abbreviations: BUN = blood urea nitrogen; eGFR = estimated glomerular filtration rate; WOBCP = women of 
childbearing potential. 
a Performed at screening only. 
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SIGNATURE PAGE 
 
 
 
 
 
 
 
Date 
 
Prepared by: 
 
Senior Statistical Consultant for Synlogic 
 
 
 
 
 
 
 
 
Date 
 
 
Medical Director 
Synlogic Operating Company, Inc. 
 
 
 
 
 
 
 
Date 
 
  
Senior Director Biostatistics 
Synlogic Operating Company, Inc. 
 
 
 




 
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Table of Contents 
1 
INTRODUCTION .............................................................................................. 11 
2 
STUDY SUMMARY.......................................................................................... 11 
2.1 
STUDY OBJECTIVES AND ENDPOINTS ............................................... 11 
2.2 
STUDY DESIGN ......................................................................................... 14 
2.2.1 
Number of Participants ......................................................................... 16 
2.2.2 
Randomization and Blinding Procedures.............................................. 16 
2.2.2.1 De-Escalation in the DEP After Randomization .................................. 16 
2.2.3 
Unblinding after the RWP .................................................................... 16 
2.3 
Efficacy Assessments ................................................................................... 17 
2.3.1 
Blood Phenylalanine and Tyrosine ....................................................... 17 
2.3.2 
DEP Weekly Substudy .......................................................................... 17 
2.4 
Safety Assessments ...................................................................................... 17 
2.4.1 
Adverse Events ..................................................................................... 17 
2.4.2 
Vital Signs, Weight, Height, and Electrocardiograms .......................... 18 
2.4.3 
Clinical Laboratory Measurements ....................................................... 18 
2.5 
Study Estimands ........................................................................................... 18 
2.5.1 
Part 1: Primary and Key Secondary Efficacy Objective Estimands ..... 18 
2.5.2 
Part 2: Primary and Key Secondary Efficacy Objectives Estimands ... 21 
3 
STATISTICAL METHODS ............................................................................... 24 
3.1 
General Methods .......................................................................................... 24 
3.1.1 
Computing Environment ....................................................................... 24 
3.1.2 
Reporting of Numerical Values ............................................................ 24 


 
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3.2 
Baseline Value and Change from Baseline .................................................. 25 
3.3 
Blood Phe Values at Baseline and Post-Baseline Visits .............................. 25 
3.4 
Handling of Missing/Incomplete Values...................................................... 25 
3.4.1 
Missing Dates........................................................................................ 26 
3.4.2 
Missing Data for Efficacy Analyses ..................................................... 26 
3.4.3 
Calculations for the DEP Responder .................................................... 26 
3.5 
Analysis Populations .................................................................................... 26 
3.5.1 
Subgroups ............................................................................................. 28 
3.6 
Participants Disposition and Evaluability .................................................... 28 
3.6.1 
Participant Disposition .......................................................................... 28 
3.6.2 
Protocol Deviations ............................................................................... 30 
3.7 
Demographics............................................................................................... 30 
3.8 
Baseline Characteristics ............................................................................... 30 
3.9 
Medical History ............................................................................................ 31 
3.10 Prior and Concomitant Medications/Procedures .......................................... 31 
3.10.1 PPI and H2 Blocker Use ....................................................................... 31 
3.10.2 
 ................................................................................... 31 
3.11 Dietary Phe ................................................................................................... 32 
3.12 Systemic Antibiotic Treatment..................................................................... 32 
3.13 Treatment Compliance and Exposure .......................................................... 32 
3.13.1 Compliance to Study Treatment ........................................................... 32 
3.13.2 Treatment Duration ............................................................................... 34 


 
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3.13.3 Distribution of SYNB1934v1 iTD in the RWP .................................... 34 
3.14 Efficacy Analysis ......................................................................................... 34 
3.14.1 Part 1: DEP ........................................................................................... 34 
3.14.1.1 Weekly DEP Substudy and Missing Data ............................................ 34 
3.14.1.2 Primary Efficacy Endpoint Analysis .................................................... 34 
3.14.1.3 Primary Efficacy Sensitivity and Supplementary Analysis .................. 35 
3.14.1.4 Key Secondary Efficacy Endpoints Analysis ....................................... 37 
3.14.1.5 Key Secondary Sensitivity and Supplementary Analysis ..................... 37 
3.14.1.6 Control of Multiplicity .......................................................................... 39 
3.14.1.7 Secondary Efficacy Endpoints Analysis ............................................... 39 
3.14.1.8 Exploratory Endpoints Analysis ........................................................... 39 
3.14.1.9 Subgroup Analyses ............................................................................... 40 
3.14.2 Part 2: RWP .......................................................................................... 41 
3.14.2.1 Primary Efficacy Endpoint Analysis .................................................... 41 
3.14.2.2 Primary Efficacy Analysis Model Does Not Converge ........................ 42 
3.14.2.3 Primary Efficacy Sensitivity and Supplementary Analysis .................. 42 
3.14.2.4 Key Secondary Efficacy Endpoint Analyses ........................................ 43 
3.14.2.5 Key Secondary Sensitivity and Supplementary Analysis ..................... 43 
3.14.2.6 Control of Multiplicity .......................................................................... 45 
3.14.2.7 Secondary Efficacy Endpoints Analysis ............................................... 45 
3.14.2.8 Exploratory Endpoints Analysis ........................................................... 46 
3.14.2.9 Subgroup Analyses ............................................................................... 47 


 
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3.14.3 Part 3: OLE ........................................................................................... 47 
3.15 Safety Analysis ............................................................................................. 49 
3.15.1 Adverse Events ..................................................................................... 49 
3.15.1.1 Adverse Events Severity ....................................................................... 50 
3.15.1.2 Adverse Events Causality ..................................................................... 51 
3.15.1.3 Adverse Events of Special Interest ....................................................... 51 
3.15.1.4 Adverse Event Missing Date Imputation .............................................. 51 
3.15.1.5 Adverse Event Summaries .................................................................... 52 
3.15.1.6 Adverse Events by Dose Ramp Day ..................................................... 54 
3.15.1.7 Adverse Event Summaries Across Study Periods ................................ 55 
3.15.1.8 GI Daily Diary Card.............................................................................. 55 
3.15.2 Clinical Laboratory Evaluation ............................................................. 56 
3.15.3 Vital Signs, Weight and Height ............................................................ 56 
3.15.4 ECG....................................................................................................... 56 
3.15.5 Physical Examination............................................................................ 56 
3.16 Sample Size Justification ............................................................................. 57 
3.17 Interim Analysis ........................................................................................... 57 
3.18 Multiple Imputation Methodology ............................................................... 58 
3.18.1 Multiple Imputation of Blood Phe for the DEP .................................... 59 
3.18.2 Multiple Imputation of Blood Phe for the RWP ................................... 59 
3.18.2.1 Missing at Random Multiple Imputation .............................................. 60 
3.18.2.2 Missing Not at Random Multiple Imputation ....................................... 61 


 
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3.18.3 Multiple Imputation Random Number Seeds ....................................... 61 
3.19 Tables Listings and Figures ......................................................................... 61 
3.20 Changes from the Protocol Planned Analyses ............................................. 62 
4 
REFERENCES ................................................................................................... 63 
5 
APPENDIX 1: SCHEDULE OF EVENTS ........................................................ 64 
6 
APPENDIX 2: CLINICAL LABORATORY TESTS ........................................ 67 
7 
APPENDIX 3: DMC Deliverables ..................................................................... 68 
 


 
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LIST OF ABBREVIATIONS 
 
Abbreviation 
Full Term  
AE 
adverse event 
AraC 
arabinose-responsive transcriptional activator 
ADLs 
activities of daily living 
ANCOVA 
analysis of covariance 
BID 
twice per day 
CBC 
complete blood count 
CI 
confidence interval 
CSR 
clinical study report 
CTCAE 
 Common Terminology Criteria for Adverse Events  
DAP 
diaminopimelate 
DEP 
dose-escalating, open-label period 
DMC 
data monitoring committee 
ECG 
electrocardiogram 
EcN 
escherichia coli Nissle 1917 
eCRF 
electronic case report form 
FAS 
full analysis set 
FDA 
Food and Drug Administration 
GI 
gastrointestinal 
GPV 
genotypic phenotype values 
ICH 
International Conference on Harmonisation 
IMP 
investigational medicinal product 
IPTG 
isopropyl ß-D-1-thiogalactopyranoside 
iTD 
individually titrated dose  
IXRS 
interactive response system  
LAAD 
L-amino acid deaminase  
MAR 
Missing at Random 
MCMC 
Markov chain Monte Carlo  
MI 
multiple imputation 
MMRM 
mixed-model with repeated measures 
MNAR 
missing not at Random 
mPAL 
modified phenylalanine ammonia lyase  
NCI 
National Cancer Institute 
OLE 
open-label extension  
PAL 
phenylalanine ammonia lyase  


 
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Phe 
phenylalanine 
pks 
polyketide synthase 
PP  
phenylpyruvate 
PPI 
proton pump inhibitor 
PPS 
per protocol analysis set 
PT 
preferred term 
QD 
once per day 
RWP 
randomized withdrawal period 
SAE 
serious adverse event 
SAS 
safety analysis set 
SD 
standard deviation  
SOC 
system organ class 
TCA 
trans-cinnamic acid  
TEAE 
treatment emergent adverse events  
TID 
three times per day 
Tyr 
tyrosine 
US 
United States 
 
 
 


 
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1 INTRODUCTION 
SYNB1934 was derived from Escherichia coli Nissle 1917 (EcN) in a series of genetic manipulations 
designed to allow enhanced degradation of Phe within the human gut. The degradation of Phe is carried 
out by the activity of engineered genes encoding phenylalanine ammonia lyase (PAL), which catalyzes 
the conversion of Phe to trans-cinnamic acid (TCA), and L-amino acid deaminase (LAAD), which 
catalyzes the conversion of Phe to phenylpyruvate (PP). The deletion of the pks island resulted in a 
modified strain of SYNB1934v1, which is referred to as SYNB1934v1. 
Study SYNB1934-CP-003 is a 3-part, Phase 2b/3 study consisting of a dose-escalating, open-label 
period (DEP; Part 1) of up to 15 weeks, followed by a 4-week, double-blind, placebo-controlled, 
randomized withdrawal period (RWP; Part 2), and an open-label extension (OLE; Part 3) of up to 36 
months. 
This Statistical Analysis Plan (SAP) describes data-handling and statistical procedures to be used for the 
analysis and reporting of efficacy and safety data collected in this Phase 3 study, SYNB1934-CP-003 
(protocol version 4.0 01JUL2023), and to be presented in the clinical study report (CSR). The methods 
are based on those presented in Section 8 of the study protocol. Note that there are multiple country-
specific version 4.0 protocols. A notable difference between the United States (US) protocol v4.0 and 
the non-US v4.0 protocols is that, in the non-US protocols, participants 12 to 17 years of age may be 
enrolled after the first 20 participants have completed the DEP, with approval of the DMC. Any post-
hoc or exploratory analyses not specified in this SAP will be identified as such when they are presented 
in the CSR.  
This SAP was written in accordance with the recommendations outlined in the International Conference 
on Harmonisation (ICH) E9 Guideline entitled “Guidance for Industry: Statistical Principles for Clinical 
Trials” and the ICH-E3 Guideline, entitled “Guidance for Industry: Structure and Content of Clinical 
Study Reports.”  
2 STUDY SUMMARY 
2.1 STUDY OBJECTIVES AND ENDPOINTS 
This study has 3 parts, and each part has a distinct set of objectives, endpoints, and analysis populations. 
The determination of the primary evidence of efficacy will be the comparison of SYNB1934v1 versus 
placebo in the randomized withdrawal period (Part 2).  
Objectives and endpoints are presented in Table 1 to Table 3 for the respective parts of the study.  


 
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Table 1:  Objectives and Endpoints: Part 1, Dose Escalating, Open-Label Period (DEP; Part 1) 
Objective 
Endpoint 
Primary 
 
• 
To assess the percentage change in blood 
phenylalanine (Phe) level 
• 
Percent change from baseline in blood Phe level at 
last measurement of the individually titrated dose 
(iTD) of SYNB1934v1  
Key Secondary: 
 
• To assess the absolute change in Phe level 
• Change from baseline in blood Phe level at last 
measurement of the iTD of SYNB1934v1 
• To determine the responder population 
• A ≥ 20% reduction from baseline in blood Phe level 
at any time in the DEP  
Other Secondary: 
 
• To assess proportion of participants achieving 
blood Phe level ≤ 360 µmol/L 
• Blood Phe level ≤ 360 µmol/L at any time of the iTD 
of SYNB1934v1 
• Blood Phe level ≤ 360 µmol/L at any time in the 
DEP 
• To assess the safety and tolerability of 
SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from baseline in clinical laboratory 
parameters 
• Changes from baseline in vital signs measurements 
Exploratory: 
 
• 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Note: baseline and week numbering are specific to study Part 1, unless explicitly noted otherwise in the endpoint. 
 
Table 2:  Objectives and Endpoints: Part 2, Randomized Withdrawal (RWP; Part 2) 
Objective 
Endpoint 


 
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Primary: To evaluate efficacy of SYNB1934v1 versus 
placebo in the responder population 
• Change from baseline to Week 4 in blood Phe 
level 
Key Secondary: 
 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard to 
percent change from DEP baseline in blood Phe level 
• Percent change from DEP baseline in blood 
Phe level at Week 4 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard to 
change from DEP baseline in blood Phe level 
• Change from DEP baseline to Week 4 in blood 
Phe level 
• To evaluate the efficacy of SYNB1934v1 versus 
placebo in the responder population with regard to the 
proportion of participants with a blood Phe level ≤ 
360 µmol/L 
• Blood Phe level ≤ 360 µmol/L at Week 4 
Other Secondary Objectives: 
 
• To assess the safety and tolerability of SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from baseline in clinical laboratory 
parameters 
• Change from baseline in vital signs 
measurements 
• To evaluate efficacy of SYNB1934v1 versus placebo 
in the responder population with regard to the 
proportion of participants with a ≥ 20% reduction 
from DEP baseline in blood Phe level at Week 4 
• A ≥ 20% reduction from DEP baseline in 
blood Phe level at Week 4 
Exploratory: 
 
• 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
  
 
 
  
 
 
  
• 
 
 
 
 
 
 
 
 
 
 


 
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• 
 
 
 
 
 
 
Note: baseline and week numbering are specific to study Part 2, unless explicitly noted otherwise in the endpoint. 
 
Table 3:  Objectives and Endpoints: Part 3, Open-label Extension (OLE; Part 3) 
Objective 
Endpoint 
• To assess the safety and tolerability of SYNB1934v1 
• Incidence and severity of adverse events 
• Changes from DEP baseline in clinical 
laboratory parameters 
• Changes from DEP baseline in vital signs 
measurements 
• 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Note: baseline and week numbering are specific to study Part 3, unless explicitly noted otherwise in the endpoint. 
 
2.2 STUDY DESIGN 
This is a 3-part, Phase 3 study consisting of a dose-escalating, open-label period (DEP; Part 1) of up to 
15 weeks, followed by a 4-week, double-blind, placebo-controlled, randomized withdrawal period 
(RWP; Part 2), and an open-label extension (OLE; Part 3) of up to 36 months. Figure 1 presents a study 
schematic. 
In the DEP, all enrolled participants will maintain a stable diet reflecting their baseline Phe intake and 
receive escalating doses of SYNB1934v1 from approximately 3 to 15 weeks to determine an iTD, which 
is defined as the highest dose the participant is able to tolerate. A participant will be defined as having 
reached an iTD if they tolerate 3 weeks at a dose, regardless of whether other doses are tolerated. 
Blood Phe level will be measured at each dose level after 3 weeks at that level. A responder will be 
defined as a participant who achieves a ≥ 20% reduction in blood Phe level compared to DEP baseline 
on their iTD. For inclusion in the RWP primary efficacy analysis population, response will be assessed 
at the third week of a participant’s established iTD for SYNB1934v1. 


 
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If at any time during a 3-week dosing interval in the DEP the participant or investigator considers a dose 
level to be intolerable, the dose may be de-escalated after discussion with the medical monitor. In this 
case, the participant will restart 3 weeks at the lower level iTD. After de-escalation, participants cannot 
escalate to a higher dose again during. 
The DEP will contain an optional substudy (DEP weekly substudy) to evaluate the range and variability 
of 
 at each dose level. The 
participants in this substudy will have 
weekly Phe and Tyr value assessments during the DEP (i.e., with Tyr assessments being primarily 
assessed for safety). 
Once the first 
 participants have attained an iTD and completed the DEP or the 
 has a 
baseline DEP and has been in the study for 
, whichever occurs earlier, the DEP data will be 
evaluated by an independent Data Monitoring Committee (DMC). The DMC will use a pre-specified set 
of criteria based on both safety and efficacy to determine whether the dose regimen is appropriate or 
whether any doses should be discontinued as well as whether the study should be stopped for futility. 
Enrollment will not be paused during the interim analysis. 
Participants who complete at least 3 weeks at their iTD during the DEP will enter a 4-week RWP. 
Participants will be randomized 1:1 to receive SYNB1934v1 at their iTD determined in the DEP or 
placebo TID. Randomization will be stratified on screening Phe level. Blood Phe level will be measured 
at Weeks 1, 3, and 4 of the RWP. Participants will remain on the same diet they consumed during the 
DEP. 
Participants who complete the 4-week RWP may enter the OLE and receive SYNB1934v1 for up to 36 
months at their iTD. (At the sponsor’s discretion, enrollment of new participants directly into OLE may 
be opened.) The iTD in Parts 1 and 3 can differ. Participants will be allowed to modify their standard 
diet if their blood Phe level is < 240 µmol/L, as determined by either local or central laboratory testing, 
with guidance from the investigator as outlined in the study-specific Diet Manual. The 3-day dietary 
intake assessments will be conducted per the Schedule of Events 
 
Figure 1:  Study SYNB1934-CP-003 Schematic 
 
 


 
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2.2.1 Number of Participants 
This study plans to enroll approximately 
 participants. More participants may be 
enrolled to achieve 
 randomized in Part 2 (RWP). 
2.2.2 Randomization and Blinding Procedures 
In the RWP, participants will be randomized in a 1:1 ratio to receive either SYNB1934v1 or matching 
placebo, stratified on screening Phe level (
 Participants, 
investigators, and the sponsor will be blinded to randomized IMP assignment during the RWP. In the 
case of an emergency where information regarding treatment assignment would impact the care 
provided to a participant during the RWP of the study, the investigator will have immediate access to 
unblind the treatment code in the interactive response system (IXRS). The instructions for unblinding a 
participant in the IXRS can be found in the IXRS User Guide. In the event unblinding is necessary, the 
investigator is strongly encouraged to contact the medical monitor to discuss the situation and the 
participants medical status prior to unblinding. It is mandatory that any personnel involved in the 
unblinding or who have access to the unblinded treatment assignment maintain the confidentiality of the 
information. 
During the DEP and RWP a participant’s blood Phe levels will be blinded to the participant, investigator 
and sponsor. During the DEP and RWP, the investigator will receive an email alert from the central 
laboratory if a blood Phe is > 1.5 × the DEP baseline, if Phe is < 30 μmol/L, or if Tyr is < 20 μmol/L. 
After the receipt of one of these trigger values, the investigator should arrange for a retest of Phe and 
Tyr. If the trigger value is confirmed to be out of range, the individual participant will be discontinued 
from the study.  
The DEP overall responder rates and DEP baseline Phe values as well as the group-level DEP interim 
analysis results will be provided to the sponsor by the DMC. 
Both the CRO’s blinded and unblinded teams will have access to the participant-level blood-Phe levels. 
2.2.2.1 De-Escalation in the DEP After Randomization 
When a participant in the DEP study completes the 2nd week of their 3-week iTD dosing period, the 
participant’s randomization into the RWP will occur (i.e., the RWP randomization occurs prior to RWP 
baseline to ensure the participant is provided with the appropriate study medication). If, subsequent to 
RWP randomization, the participant needs to de-escalate to a lower dose, the participant will not enter 
the RWP, but instead either discontinue the study or enter the OLE portion of the study. The participant, 
site, and sponsor will remain blinded to the participant’s randomized RWP study medication. 
2.2.3 Unblinding after the RWP 
Once all participants have completed the RWP, the DEP and RWP parts of the study will be locked, 
unblinded, and analyzed.  


 
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2.3 Efficacy Assessments 
The following are the list of efficacy/pharmacodynamic assessments: 
2.3.1 Blood Phenylalanine and Tyrosine 
Blood Phe and Tyr samples will be drawn 2.5 to 5 hours after the last meal. Phe and Tyr samples will be 
drawn in duplicate, using separate tubes from the same venipuncture, and analyzed separately. The time 
of the last meal will be recorded when taking Phe samples. 
Baseline samples for DEP and RWP must be drawn prior to the first dose for those respective periods. 
During the DEP, blood Phe and Tyr will be measured at Week 3 of each completed dose level. During 
the OLE, blood Phe and Tyr will be measured quarterly. Blood Phe and Tyr will also be measured at 
early termination. 
For all Tyr summaries the average value will be used, with the individual duplicate values being 
presented in the listings.  
The individual duplicate blood Phe values will be used in some analyses, and the average of the 
duplicate values used for other analyses and summaries (see Section 3.3).  
2.3.2 DEP Weekly Substudy 
Participants will have the option to enroll in a substudy to evaluate 
 
. Incentives will be provided to ensure that at least twenty participants are 
recruited into this substudy. Participants in this substudy will have 
 
assessments during the DEP. These weekly values may be performed during a 3-day window around the 
 
 
 
 These samples will be drawn 2.5 to 5 hours after the last meal. P
 
 
 
Note: for 3 
consecutive days prior to scheduled study visits and blood Phe draws, all participants will have a 3-day 
dietary intake assessment (see Section 3.10.2).  
2.4 Safety Assessments 
2.4.1 Adverse Events 
Adverse events will be assessed continuously by direct observation and participant interviews. For GI 
AEs, participants will also fill out a diary card during screening, DEP, and RWP to allow for enhanced 
capture of these AEs. The severity of AEs will generally be evaluated using the National Cancer 
Institute Common Terminology Criteria for Adverse Events (CTCAE), v5.0, with exceptions described 
in Table 9. All AEs occurring from the time a participant signs the ICF through the follow-up period 
will be recorded, regardless of causal assessment to IMP. 










 
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C. Intercurrent events 
• Treatment discontinuation: consistent with a treatment-policy strategy, treatment 
discontinuation will not be considered (i.e., all observed data will be used 
regardless of treatment discontinuation). 
• Antibiotic use: consistent with a treatment-policy strategy, antibiotic use will not 
be considered (i.e., all observed data will be used regardless of antibiotic use). 
• Prohibited medication use: consistent with a treatment-policy strategy, prohibited 
medication use will not be considered (i.e., all observed data will be used 
regardless of prohibited medication use). 
D. Population-level 
summary 
The population-level summary is the least-squares means and difference in the 
change from baseline to Week 4 in blood Phe levels between the SYNB1934v1 and 
placebo treatment groups. An MMRM using an observed case approach is being 
applied. The post-baseline blood Phe levels at Weeks 1, 3, and 4 will be used. The 
MMRM model is described in Section 3.14.2.1. 
MMRM = mixed model with repeated measures; Phe = phenylalanine; RWP = randomized withdrawal period. 
 
 






 
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Proportions will be presented as percentages. 
3.2 Baseline Value and Change from Baseline 
Unless otherwise explicitly defined otherwise, the baseline value is defined as the most recent non-
missing value obtained immediately prior to administration of first dose.  
• Change from baseline will be calculated by subtracting the baseline value from the post-dose 
assessment for each participant (i.e., post-dose – baseline).  
• Percent change from baseline will be calculated by multiplying the change from baseline by 100 
and dividing by the baseline value (i.e., =100  change from baseline /baseline)  
3.3 Blood Phe Values at Baseline and Post-Baseline Visits 
Baseline for blood Phe level in each of the 3 parts of the study will be defined as the mean of the 
duplicate blood Phe level measurements at the first visit of that part. If only one blood Phe level 
measurement is available, then that measure will be used as baseline. If the measurement at the first visit 
is missing, the most recent non-missing blood Phe levels from the central lab to the visit will be used 
(i.e., blood Phe levels from the local labs will not be used for analyses but can be used as for the 
inclusion criterion).  
As defined in the schedule of events (Table 11and Table 12), the Part 2 RWP baseline is the End of the 
DEP visit, and the OLE baseline is the Part 2 RWP Week 4 visit.  
Similarly, for the determination of responders and participants who achieve a specific reduction in Phe 
(e.g., Phe level ≤ 360 µmol/L at any time in the DEP, or a ≥ 20% reduction from baseline), and in 
situations where a single-Phe value is needed for a for a particular visit (e.g., summary statistics over 
time), the mean of the duplicate blood Phe level measurements at the applicable post-baseline timepoint 
(e.g., Week 3) will be used. If only one of the duplicate blood Phe levels is available at a particular post-
baseline timepoint, then that measurement will be used. Unless otherwise specified in the SAP, only the 
MMRM analyses of blood Phe will use the duplicate blood Phe assessments, all other summaries will 
use the average of the duplicate blood Phe measurements.  
3.4 Handling of Missing/Incomplete Values 
Unless otherwise specified, missing data will not be imputed; the analyses will be performed on the non-
missing values (i.e., this will be referred to as observed-case).  
If a non-numeric clinical laboratory value is reported for an endpoint of the form “< X.XX”, “≤ X.XX”, 
“>X.XX”, or “≥X.XX” and the endpoint is being summarized as a numeric quantitatively (i.e., assuming 
the endpoint response is numeric) the numeric value portion of the result being reported will be imputed. 
For example, an endpoint non-numeric values of “<2.00” and “>100.00” may be reported; in these 
instances, the values of 2.00 and 100.00, respectively, will be imputed. 


 
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3.4.1 Missing Dates  
The imputation of missing date components (i.e., missing day, month, or year values) for start/stop date 
of adverse events and prior/concomitant medications are described in the corresponding section where 
the date is discussed. 
3.4.2 Missing Data for Efficacy Analyses 
The methods for the handling of missing data for the efficacy analyses are described in the 
corresponding section where the analysis is discussed. 
3.4.3 Calculations for the DEP Responder 
As discussed in Section 2.2, a responder is a defined as a participant who achieves a ≥ 20% reduction in 
blood Phe level compared to DEP baseline. The following are additional details regarding the definition 
of a responder: 
• A participant will be considered a responder if they met the ≥ 20% reduction in blood Phe level 
at any time in the DEP. 
• This will include the scheduled Week 3 blood Phe assessments at the end of each 3-week dosing 
period.  
• This will not include assessments that were made specific to the participants included in the 
DEP Weekly substudy (Section 2.3.2).  
3.5 Analysis Populations  
Analysis populations will be defined for each of the 3 parts of the study (DEP, RWP, and OLE).  
Full analysis set (FAS): The full analysis sets are based on the intention-to-treat principle. The DEP 
FAS includes all participants enrolled in the DEP who take at least one dose of DEP study medication.  
The RWP FAS is defined as all participants randomized into the RWP who take at least one dose of 
RWP study medication. Additionally: 
• The responder RWP FAS is defined as participants in the RWP FAS who are responders 
• The nonresponder RWP FAS is defined as participants in the RWP FAS who are 
nonresponders. 
A responder is defined as a participant who achieves a ≥ 20% reduction in blood Phe level compared to 
DEP baseline on SYNB1934v1.  
The responder RWP FAS will be the primary efficacy analysis population for the RWP. Analyses on the 
RWP FAS will be performed according to the randomized treatment group. 
In the event that, based on the interim analysis results utilizing the criteria provided in Table 10, the 
DMC recommends dropping either the 1 × 1012 or the 1 × 1012 and 6 × 1011 doses the following will 
occur: 


 
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• The participants in the DEP who have an iTD corresponding to a dropped dose will go directly 
into the OLE part of the study (i.e., as opposed to being randomized into the RWP).  
• The RWP FAS, responder RWP FAS, and nonresponder RWP FAS, will exclude all participants 
whose iTD dose level was dropped. 
These participants who are excluded from the RWP FAS will be summarized separately.  
Safety analysis set (SAS): For each of the 3 study parts, the SAS will include all participants who 
received any amount of IMP in the corresponding study part with treatment assignment based on the 
treatment received. 
For the RWP the safety analyses will be presented for the RWP SAS overall as well as by 
responder/nonresponder subgroups. Similarly, the OLE safety analyses will be presented for the OLE 
SAS overall as well as by responder/nonresponder subgroups.  
A participant will be considered receiving treatment if the eCRF question “did the patient receive any 
study drug doses” is answered “Yes” and there is a corresponding non-missing date.  
Per-protocol analysis set (PPS): The DEP PPS is defined as all DEP FAS participants, who had no 
major protocol deviations that would affect efficacy, completed their final 3-week iTD, whose dietary 
Phe remained within 20% of their DEP baseline value (see Section 3.10.2), and who did not require 
systemic antibiotic treatment during DEP (see Section 3.12). The DEP PPS will be used as a sensitivity 
analysis for the DEP primary efficacy endpoint.  
The responder RWP PPS is defined as all responder RWP FAS participants, who had no major protocol 
deviations that would affect efficacy, had at least 75% treatment compliance (see Section 3.13.1), whose 
dietary Phe remained within 20% of their DEP baseline value (see Section 3.10.2), and who did not 
require systemic antibiotic treatment during DEP or RWP (see Section 3.12). 
The responder RWP PPS will be used as a sensitivity analysis for the RWP primary efficacy endpoint. 
RWP major protocol deviations will be determined in a blinded manner prior to the RWP database lock. 
Analyses on the responder RWP PPS will be performed according to the randomized treatment group. 
Following the protocol deviations handling plan, major protocol deviations that would affect efficacy 
will be determined by a review of the functional team members (i.e., the set of functional team members 
will be defined in the protocol deviations handling plan), which will be blinded for the RWP.  
RWP Randomized analysis set: The RWP Randomized analysis set is defined as all participants 
randomized into the RWP. The responder Randomized analysis set is defined as participants in the 
Randomized analysis set who are responders. The nonresponder Randomized analysis set is defined as 
participants in the Randomized analysis set who are nonresponders.  
 


 
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3.5.1 Subgroups 
For selected efficacy and safety analyses (see Section 3.14 and Section 3.15, respectively) the 
summaries will be performed overall and by responder/nonresponder subgroups (i.e., as defined in 
Section 3.5). 
The DEP will contain an optional substudy (DEP weekly substudy) to evaluate the range and variability 
of Phe and Tyr response at each dose level (see Section 2.3.2). The twenty participants in this substudy 
will have weekly Phe and Tyr value assessments during the DEP. The weekly Phe and Tyr assessments 
will be summarized for this subgroup.  
The following additional subgroups will also be considered: 
• Adolescents: Note that, for non-US sites, participants 12 to 17 years of age may be enrolled after 
the first 20 participants have completed the DEP, with approval of the DMC.  
• Participants on sapropterin dihydrochloride (Kuvan) at the DEP baseline visit (i.e., DEP study 
day 1). 
• Genotypic phenotype values (GPV) subgroups: For the subset of participants who have non-
missing results for allele #1 and allele #2 (i.e., from the Genotyping eCRF page), their GPV will 
be calculated. The GPV will be calculated by entering the allele information into the following 
website http://www.biopku.org/biopku/search-allele-start.asp. The GPV is a number from 0 to 
10; the following 3 subgroups will be considered based on the GPV score: 0-2.7, 2.8-6.6, 6.7-
10.0.  
• Participants in the DEP who had an iTD of a dose that was discontinued (i.e., this subgroup will 
only be utilized if a dose is discontinued as per the DMCs recommendation as defined in Section 
3.17). 
• Participants in the RWP who were excluded from the RWP FAS (i.e., this subgroup will only be 
utilized if a dose is discontinued as per the DMCs recommendation as defined in Section 3.17). 
The analyses to be performed on these subgroups will be discussed in the corresponding efficacy and 
safety sections.  
3.6 Participants Disposition and Evaluability 
3.6.1 Participant Disposition 
The following participant disposition is summarized using the analysis populations defined in Section 
3.5. 
• 
The number of participants who entered the Screening Period (i.e., as defined by signing informed 
consent) 
• 
The number and percent of participants rescreened (using the number screened as the denominator). 
 


 
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The following percentages will use the DEP FAS as the denominator: 
• 
The number of participants enrolled in DEP (i.e., defined as being assigned study drug) 
• 
The number and percent of participants completing/not completing the DEP (i.e., completed the 
DEP is defined as having completed the End of DEP/RWP Baseline Visit as indicated in the eCRF) 
• 
The number and percent of participants who achieve an iTD  
• 
The number and percent of responders. 
 
The following percentages will use the RWP FAS as the denominator: 
• 
The number of participants randomized into the RWP overall and by responder/nonresponder status 
• 
The number and percent of participants completing/not completing the RWP (i.e., completed the 
RWP is defined as having a non-missing RWP Week 4 visit) overall and by 
responder/nonresponder. 
 
The following percentages will use the OLE SAS as the denominator: 
• 
The number of participants enrolled in OLE (i.e., defined as being assigned OLE study drug) 
• 
The number and percent of participants completing the OLE/not completing the OLE (i.e., 
completing the study as determined by the eCRF question “Did the subject complete the study per 
protocol?”). 
 
Note: The number and percent of participants who did not complete each of the 3 study parts will be 
summarized along with the reason: 
o LOST TO FOLLOW-UP 
o PROTOCOL DEVIATION 
o ADVERSE EVENT 
o LOW LEVELS OF PHE OR TYR OR PROFOUND ELEVATION IN PHE  
o PREGNANCY  
o STUDY TERMINATED BY SPONSOR 
o PHYSICIAN DECISION 
o WITHDRAWAL BY SUBJECT 
o NON-COMPLIANCE WITH STUDY DRUG 
o DEATH 
o OTHER.  
The summaries will be overall for Part 1; overall, responder status, and treatment group (i.e., active or 
placebo) for Part 2; and overall and responder status for Part 3.  


 
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3.6.2 Protocol Deviations 
The protocol deviations will be shown in a participant listing, by study part and treatment group (for the 
RWP).  
For the RWP, the determination of major protocol deviations and major protocol deviations that would 
impact efficacy (i.e., a criterion for the RWP PPS as defined in Section 3.5) will be performed in a 
blinded manner.  
3.7 Demographics  
Demographics will be summarized for the following analysis Populations: 
• DEP: FAS and SAS 
• RWP: FAS, SAS, PPS 
• OLE: SAS. 
For each analysis population the summary will be overall and by treatment group for the RWP (i.e., 
SYNB1934v1 and Placebo). For the RWP, results will also be presented by responder/nonresponder.  
Descriptive statistics for continuous variables will be provided for age (at time of informed consent), 
height, weight, and BMI. Frequency counts and percentages will be tabulated for sex, fertility status (for 
females), race, and ethnicity.  
3.8 Baseline Characteristics  
Baseline characteristics will be summarized for the DEP FAS, DEP SAS, RWP FAS, RWP SAS, and 
responder RWP FAS analysis populations.  
The following information will be summarized for the DEP populations: 
• Screening Phe 
• DEP Baseline Phe  
• DEP Baseline Phe intake 
• DEP Baseline tyrosine (Tyr) 
• Sapropterin dihydrochloride (Kuvan) at DEP Baseline (Yes/No) 
• Current Proton pump inhibitor (PPI) Use at DEP Screening (Yes/No). 
The following information will be summarized for the RWP populations: 
• Screening Phe 
• DEP Baseline Phe  
• DEP Baseline Phe intake 
• RWP Baseline Phe  
• DEP Baseline tyrosine (Tyr) 
• RWP Baseline tyrosine (Tyr) 
• Sapropterin dihydrochloride (Kuvan) at DEP Baseline (Yes/No) 


 
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• Current Proton pump inhibitor (PPI) Use at DEP Screening (Yes/No). 
3.9 Medical History 
Medical history will be summarized for the DEP SAS. Medical history will be coded using MedDRA 
version 25.1 (or higher). The coded Preferred Terms (PT) will be summarized overall for the DEP SAS 
and, for the RWP SAS, by responder/nonresponder within each treatment group, and overall. A 
participant will be counted only once for each preferred term. The summary will present the results 
alphabetically by System Organ Class (SOC) and, within SOC, by decreasing frequency for the PT.  
3.10 Prior and Concomitant Medications/Procedures 
Prior and concomitant medications will be coded using WHO Drug (Version: B3 Global September 
2022) 
The incidence of prior medication will be presented by therapeutic class, and preferred (generic) drug 
name for the DEP SAS. 
For the DEP and OLE study periods, the incidence of concomitant medication will be presented by 
therapeutic class, and preferred (generic) drug name for the corresponding SAS. A participant is counted 
only once for each therapeutic class and for each preferred drug name.  
A participant is counted only once for each therapeutic class and for each preferred drug name.  
Prior medications are defined as those taken and stopped prior to the date of first study treatment in the 
DEP. Any medication given at least once on or after the DEP first study treatment date will be defined 
as a concomitant medication including those which were started before first study treatment date and 
continued after first study treatment date.  
Where a medication start date is partially or fully missing, and it is unclear as to whether the medication 
is prior or concomitant, it will be assumed that it is a concomitant medication.  
Note that PPI and H2 blocker use will be summarized separately (see Section 3.10.1) 
3.10.1 PPI and H2 Blocker Use 
PPI and H2 blocker use is captured on a specific eCRF page. PPI and H2 use will be summarized 
(separately) using the same methodology described in Section 3.10 (separate listings as well).  
3.10.2 
 Use 
use is captured on a specific eCRF page.
 use will be summarized (separately) 
using the same methodology described in Section 3.10 (separate listings as well).  


 
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3.11 Dietary Phe 
For 3 consecutive days prior to all scheduled study visits and blood Phe draws (see Schedule of Events 
Table 11 and Table 12), all participants will have a 3-day dietary intake assessment. Participants will 
follow their usual diet (including Phe, protein, and medical food intake) from baseline in the DEP to the 
end of the RWP.  
The following information will be summarized for the 3-day DEP Baseline Period: 
• Calories (KCAL) 
• Total Protein (grams) 
• Natural Protein (grams) 
• Medical Food Protein (grams) 
• Phe (grams). 
The following information will be summarized for the DEP, RWP, and OLE Periods using the 
corresponding SAS: 
• At each scheduled visit, the percent change from baseline in 3-day dietary Phe levels will be 
summarized 
• For the DEP and RWP, the number and of times a participant’s 3-day Phe exceeded the within  
20% (inclusive) DEP baseline Phe criterion.  
Note: the 3-day dietary endpoints to be summarized are the 3-day averages which are provided on the 
corresponding eCRF page. 
For the DEP and OLE, the results will be presented overall. For the RWP, the results will be 
summarized by treatment group and responder/nonresponder within each treatment group.  
3.12 Systemic Antibiotic Treatment  
Systemic antibiotic treatment is defined as any concomitant medication antibiotic in which the route is 
oral, intravenous, or intramuscular. Any topical use is specifically not systemic. This ‘topical use’ 
includes antibiotics applied to any reachable area (most often the eye and ear canal). 
The number and percent of participants using systemic antibiotic treatment will be summarized for the 
DEP, RWP, and OLE Periods using the corresponding SAS. For the DEP and OLE, and the results will 
be presented overall. For the RWP, the results will be summarized by treatment group and 
responder/nonresponder within each treatment group.  
3.13 Treatment Compliance and Exposure 
3.13.1 Compliance to Study Treatment 
Compliance rates will be calculated for each of the 3 study periods (DEP, RWP, and OLE) using the 
following methodology: 


 
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For a participant, the compliance at each of the 3 individual dose levels received (3x1011, 6x1011, 
1x1012), will be calculated by the following: 
% Compliance =100  
# of sachets given − (# of sachets returned + # of sachets lost or destroyed) 
Expected # of sachets to be taken 
Here: 
# of sachets given: is determined by the number of kits given for the corresponding dose level 
For each dose level kit there are 30 additional sachets provided in case of delayed visits, destroyed 
sachets, etc. In addition, for dose levels 6x1011 and 1x1012 there are 30 additional sachets in each kit 
for the lower dose levels of 3x1011 and 6x1011, respectively, in case a dose reduction is needed. If a 
participant requires a dose reduction, they will use the additional sachets from the existing kit until a 
new kit is received. Study drug administration and study drug accountability will be completed at the 
dose level in EDC.  
# of sachets returned: is determined by the “Number of [individual dose level] sachets returned” field on 
the drug accountability CRF page for the corresponding dose level 
# of sachets lost or destroyed: is determined by the “Number of sachets lost or destroyed” field on the 
drug accountability CRF page for the corresponding dose level 
Expected # of sachets to be taken: is determined by taking the difference between the “Start date” and 
“Stop date” on the study drug administration CRF for the corresponding dose level and accounting 
for either the dose ramp schedule for the DEP and OLE or three sachets a day for the RWP. 
For the DEP and OLE and the expected number of sachets follows the following dose ramp schedule. 
Dose frequency is linked to an individual sachet (e.g., TID is 3 sachets). De-escalation of dose is 
possible at any point during the dose ramp. When a participant has de-escalated the expected # of 
sachets to be taken will be calculated by the actual number of days spent at the dose level.  
Table 8: DEP and OLE Dose Ramp Schedule 
 
Dose Level 1 
Dose Level 2 
Dose Level 3 
Timing 
D1-9 
D10-14 
D15-21 Wk 4 
Wk 5 
Wk 6 
Wk 7 
Wk 8 
Wk 9 
Dose (live 
cells) 
  
3 x 1011*  
3 × 1011 
3 × 1011 6 × 1011 
6 × 1011 6 × 1011 1 × 1012 
1 × 1012 1 × 1012 
Frequency QD, D1-3 QD, D10-11 TID 
QD × 3 days TID 
TID 
QD × 3 days 
TID 
TID 
 
BID, D4-6  BID, D12-14  
BID × 4 days  
 
BID × 4 days  
 
 
TID, D7-9  
 
 
 
 
 
 
 
BID = twice per day; D = day; QD = once per day; TID = 3 times per day. 
* See Section 4.3.1 of the study protocol for details.  
For the RWP Period, the compliance rate will be the compliance rate for the corresponding dose level 
the participant was on during the RWP (i.e., dose modification is not allowed during the RWP).  


 
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For each of the 3 study periods (DEP, RWP, and OLE), compliance will be summarized using the SAS 
(defined in Section 3.5), with results also being presented by: 
• For the DEP: By dose level 
• For the RWP: By treatment group, responder/nonresponder subgroups, and treatment group 
within responder/nonresponder subgroups 
• For the OLE: By dose level, responder/nonresponder subgroups 
In addition to summary statistics for the compliance rate, the number and percentage of participants 
whose compliance rate is ≥ 75% will be reported.  
3.13.2 Treatment Duration 
For each of the 3 study periods treatment duration will be Last dose date – First dose date + 1. 
For each of the 3 study periods (DEP, RWP, and OLE), treatment duration will be summarized using the 
SAS populations (defined in Section 3.5), with results also being presented by responder/nonresponder 
and treatment group within responder/nonresponder subgroups for the RWP and OLE.  
Additionally for the DEP, the duration of time (i.e., days) that a participant was on a dose level that the 
participant discontinued from will be summarized by dose level and overall.  
3.13.3 Distribution of SYNB1934v1 iTD in the RWP 
The number and percentage of participants at each of the 3 individual dose levels (3x1011, 6x1011, 
1x1012) will be summarized for the RWP using the FAS population (defined in Section 3.5), with results 
also being presented by responder/nonresponder and treatment group within responder/nonresponder 
subgroups. 
3.14 Efficacy Analysis 
3.14.1 Part 1: DEP 
3.14.1.1 Weekly DEP Substudy and Missing Data 
Unless otherwise explicitly specified, the additional weekly blood Phe data collected in the subgroup of 
participants in the DEP Weekly Substudy (see Section 2.3.2) will not be considered when imputing 
missing blood Phe data in the DEP efficacy analyses.  
3.14.1.2 Primary Efficacy Endpoint Analysis 
The DEP primary efficacy endpoint is the percent change from DEP baseline in blood Phe level at the 
last measurement of the iTD of SYNB1934v1. The last measurement is the participant’s last Week 3 
blood Phe level at the iTD of SYNB1934v1.  
The mean percent change from baseline at the participant’s iTD will be tested by an MMRM analysis. 
The MMRM model will have DEP baseline blood Phe level as a fixed effect. The dependent variable is 


 
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the percent change from baseline in blood Phe level at the last measurement of the iTD of SYNB1934v1 
(i.e., with duplicate blood Phe assessments being made at the last measurement).  
Least-squares means for the percent change from baseline, along with the corresponding 
 
confidence intervals and p-values testing a percent change from baseline equal to 0, will be calculated. 
The primary efficacy analysis will test the least-squares means percent change from baseline equal to 0 
for the blood Phe level at the participants’ last week (Week 3) at his or her established iTD. 
At each of the scheduled assessments blood Phe will be drawn in duplicate. The MMRM will 
incorporate the participant repeated/duplicate measures. For the MMRM analysis, the results of the post-
baseline duplicate samples from the Week 3 visit will be included in the statistical model (i.e., without 
averaging the results within a given visit before inclusion into the MMRM).  
The MMRM analysis will be based on the restricted maximum likelihood method assuming an 
unstructured covariance structure to model the repeated-measures. A Kenward-Roger approximation 
will be used for the denominator degrees of freedom. If there is a convergence problem due to the 
unstructured covariance matrix, a variance components covariance structure will be used.  
The DEP FAS will be the primary efficacy analysis population (defined in Section 3.5). As the primary 
efficacy analysis is considering only participants who reach an iTD (see Section 2.5.1) missing data for 
the DEP Week 3 blood Phe will generally be limited to those scenarios where the blood Phe sample is 
not analyzed. In the scenario that a participant is missing both blood Phe values at the DEP Week 3 for 
their iTD, the following algorithm will be applied: 
• If the participant has a corresponding previous non-missing Week 3 blood Phe value at the iTD 
dose (e.g., the participant escalates to a higher dose then subsequently de-escalates back to a 
lower dose, which is their iTD) this value will be used.  
• Otherwise, the value will remain missing, and an observed-case approach will be used.  
3.14.1.3 Primary Efficacy Sensitivity and Supplementary Analysis  
The following sensitivity and supplementary analyses will be performed for the primary efficacy 
endpoints.  
• Analysis by iTD: The primary efficacy endpoint analysis will be performed on each iTD subgroup.  
 
• Imputation of Missing Data: Multiple imputation will be used to impute missing blood Phe data 
during the DEP. The imputation of missing data analysis will assume the data is missing at random 
(MAR). The methodology for the multiple imputation is provided in Section 3.18.1. As discussed in 
Section 3.14.1.1, the imputation will not utilize the data from the DEP Weekly substudy. The 
analysis model will be the same model as the DEP primary efficacy endpoint analysis. The DEP 
FAS will be the primary efficacy analysis population for this analysis. 
 
• Analysis of Covariance: The primary efficacy endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model. The percent change from DEP baseline in blood Phe level at the last 


 
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measurement of the iTD of SYNB1934v1 will be the dependent variable is a factor and DEP 
baseline Phe is a covariate. For this analysis the 2 Phe assessments occurring at the last measurement 
of the iTD of SYNB1934v1 will be averaged (i.e., if just 1 Phe assessment at this timepoint, that 
value will be used).  
 
Least-squares means for the percent change from baseline overall dose level, along with the 
corresponding
confidence intervals and p-values testing a percent change from baseline equal 
to 0, will be calculated. 
 
This will be an observed-case analysis using the DEP FAS population, missing data will not be 
imputed. 
 
• Excluding Participants on a Dropped Dose: The primary efficacy endpoint analysis will be 
performed on the DEP FAS using an observed case analysis which excludes participants who were.  
 
• Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The primary efficacy endpoint analysis will be performed on the DEP FAS using an 
observed case analysis which excludes observations after the following intercurrent events occur in 
the DEP: Treatment discontinuation, antibiotic use, prohibited medication use.  
 
• RWP PPS Analysis: The primary efficacy endpoint analysis will be performed using the responder 
DEP PPS analysis set (defined in Section 3.5).  
 
• MMRM Analysis by Dose Level: The percent change in blood Phe level will be analyzed using an 
MMRM model. All available Week 3 blood Phe data will be used. The model will have percent 
change from baseline as the dependent variable. Dose level (as a categorical variable) and baseline 
blood Phe will be fixed effects. The MMRM analysis will be based on the restricted maximum 
likelihood method. A Kenward-Roger approximation will be used for the denominator degrees of 
freedom. The covariance structure for the repeated-measures will be a direct product of unstructured 
for the multiple Week 3 visits (i.e., a total of 5 possible Week 3 visits, 3 for each dose level and 2 for 
possible repeats of the 3×1011 and 6×1011 doses) and compound symmetry for the repeated 
assessments at a particular visit. As such the covariance structure will use the direct product structure 
“UN@CS” as defined in SAS PROC Mixed.  
This will be an observed-case analysis, this analysis will be performed using the DEP FAS and also 
using the responder RWP FAS population, missing data will not be imputed.  
If the MMRM analysis does not converge, the repeated measures at each blood Phe assessment will 
be averaged together and the average of the repeated measures will be used to calculate the percent 
change in blood Phe level, the dependent variable. The model will have percent change from 
baseline as the dependent variable (i.e., using the averaged value at Week 3 visit). The MMRM 
model will be the same (i.e., same set of fixed effects) with the exception that the covariance 
structure for the repeated-measures will be unstructured for the multiple Week 3 visits (i.e., a total of 


 
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5 possible Week 3 visits, 3 for each dose level). If this model does not converge a compound 
symmetry covariance structure will be used.  
  
Additionally, histograms illustrating the DEP Week 3 change and percent change from baseline in blood 
Phe by treatment group will be produced, overall and by iTD.  
3.14.1.4 Key Secondary Efficacy Endpoints Analysis  
The key secondary efficacy endpoint analyses for the DEP are provided below. These analyses will be 
performed on the DEP FAS. 
• Change from baseline in blood Phe level at last measurement of the iTD of SYNB1934v1 
The key secondary endpoint, change from baseline in blood Phe level at last measurement of the iTD 
of SYNB1934v1, will be analyzed using an MMRM analysis similar to the analysis methodology 
model described for the DEP primary efficacy analysis (Section 8.4), with the dependent variable 
being the change in blood Phe level from DEP baseline.  
• A ≥ 20% reduction from baseline in blood Phe level at any time in the DEP 
The number and proportion of participants meeting this criterion will be summarized along with 
). As discussed in Section 3.14.1.1, this analysis will not consider the 
additional blood Phe data from DEP Weekly substudy. Note this will consider all Week 3 post-
baseline blood Phe values occurring in the DEP. This will be an observed-case analysis approach 
using the DEP FAS such that if a participant is missing the data needed to determine the endpoint, they 
will be considered as not having met the endpoint. 
3.14.1.5 Key Secondary Sensitivity and Supplementary Analysis  
• Change from baseline in blood Phe level at last measurement of the iTD of SYNB1934v1 
o Analysis by iTD: The analysis will be performed on each iTD subgroup. 
o Imputation of Missing Data: Multiple imputation will be used to impute missing blood Phe data 
during the DEP. The imputation of missing data will assume the data is missing at random 
(MAR). The methodology for the multiple imputation and analysis is provided in Section 3.18.1. 
The analysis model will be the same model as the DEP key secondary endpoint analysis. This 
analysis will be performed on the DEP FAS population.  
o Analysis of Covariance: The change from baseline in blood Phe level at last measurement of the 
iTD of SYNB1934v1 endpoint will be analyzed using an ANCOVA model. The change from 
DEP baseline in blood Phe level at the last measurement of the iTD of SYNB1934v1 will be the 
dependent variable, using the average of the duplicate blood Phe levels (i.e., if one of the values 
is missing, the non-missing value will be used). The ANCOVA has Treatment as a fixed effect 
and DEP baseline Phe is a covariate. This will be an observed-case analysis using the DEP FAS 
population, missing data will not be imputed.  


 
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o Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The key secondary efficacy endpoint analysis will be performed on the DEP FAS 
using an observed case analysis which excludes observations after the following intercurrent 
events occur in the DEP: Treatment discontinuation, antibiotic use, prohibited medication use.  
o RWP PPS Analysis: The analysis will be performed using the responder RWP PPS analysis set 
(defined in Section 3.5).  
o MMRM Analysis by Dose Level: The change in blood Phe level will be analyzed using an 
MMRM model. All available Week 3 blood Phe data will be used. The model will have change 
from baseline as the dependent variable. Dose level (as a categorical variable) and baseline blood 
Phe will be fixed effects. The MMRM analysis will be based on the restricted maximum 
likelihood method. A Kenward-Roger approximation will be used for the denominator degrees of 
freedom. The covariance structure for the repeated-measures will be a direct product of 
unstructured for the multiple Week 3 visits (i.e., a total of 5 possible Week 3 visits, 3 for each 
dose level and 2 for possible repeats of the 3×1011 and 6×1011 doses) and compound symmetry 
for the repeated assessments at a particular visit. As such the covariance structure will use the 
direct product structure “UN@CS” as defined in SAS PROC Mixed.  
This will be an observed-case analysis, this analysis will be performed using the DEP FAS and 
also using the responder RWP FAS population, missing data will not be imputed.  
If the MMRM analysis does not converge, the repeated measures at each blood Phe assessment 
will be averaged together and the average of the repeated measures will be used to calculate the 
percent change in blood Phe level, the dependent variable. The model will have change from 
baseline as the dependent variable (i.e., using the averaged value at Week 3 visit). The MMRM 
model will be the same (i.e., same set of fixed effects) with the exception that the covariance 
structure for the repeated-measures will be unstructured for the multiple Week 3 visits (i.e., a 
total of 5 possible Week 3 visits, 3 for each dose level). If this model does not converge a 
compound symmetry covariance structure will be used.  
• A ≥ 20% reduction from baseline in blood Phe level at any time in the DEP 
o Analysis by iTD: The analysis will be performed on each iTD subgroup. 
o Imputation of Missing Data: Multiple imputation will be used to impute missing blood Phe data 
during the DEP. The imputation of missing data analysis will assume the data is missing at 
random (MAR). The methodology for the multiple imputation is provided in Section 3.18.1. The 
number and proportion of participants meeting this criterion will be summarized along with 
 
 based on the imputed data. This analysis will be performed on the DEP FAS population. 
o Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The key secondary efficacy endpoint analysis will be performed on the DEP FAS 
using an observed case analysis which excludes observations after the following intercurrent 
events occur in the DEP: Treatment discontinuation, antibiotic use, prohibited medication use.  


 
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o RWP PPS Analysis: The analysis will be performed using the responder RWP PPS analysis set 
(defined in Section 3.5).  
3.14.1.6 Control of Multiplicity 
The overall Type I error rate for the DEP will be controlled at the 2-sided
 for the primary 
endpoint. If the DEP primary endpoint analysis is significant at the 2-sided 
 the key secondary 
endpoint, change from baseline in blood Phe level at last measurement of the iTD of SYNB1934v1, will 
be tested at the 2-sided 
level.  
Note that the dichotomous DEP key secondary endpoint, a ≥ 20% reduction from baseline in blood Phe 
level at any time in the DEP, does not have an associated inferential test.  
3.14.1.7 Secondary Efficacy Endpoints Analysis 
The following are the analysis methods for the DEP secondary efficacy endpoints.  
• Blood Phe level ≤ 360 µmol/L at any time of the iTD of SYNB1934v1 
• Blood Phe level ≤ 360 µmol/L at any time in the DEP 
For each of these 2 endpoints, the proportion of participants meeting the endpoint will be summarized 
along with 
confidence intervals. This will be an observed-case analysis approach using the DEP 
FAS such that if a participant is missing the data needed to determine the endpoint, they will be considered 
as not having met the endpoint. 
3.14.1.8 Exploratory Endpoints Analysis 
The following are the analysis methods for the set of DEP exploratory endpoints. These analyses will be 
performed on the DEP FAS. For the dichotomous endpoints, this will be an observed-case analysis 
approach using the DEP FAS such that if a participant is missing the data needed to determine the 
endpoint, they will be considered as not having met the endpoint. For the continuous endpoint, this will be an 
observed-case analysis. As discussed in Section 3.14.1.1, these analyses will not consider the additional 
blood Phe data from DEP Weekly substudy. 
• A ≥ 20% reduction from baseline in blood Phe level at the third week of a participant’s last 3 
weeks at the iTD of SYNB1934v1 
Note that if a participant has 2 Week 3 blood Phe visits at their iTD (i.e., the participant escalated in 
dose then de-escalated back to their iTD) both Week 3 values will be considered such that the 
participant can reach the ≥ 20% reduction at either of these assessments. 
• Blood Phe level ≤ 600 µmol/L at any time of the iTD of SYNB1934v1 
• Blood Phe level ≤ 600 µmol/L at any time in the DEP 
For each of these endpoints, the number and proportion of participants meeting the corresponding 
criteria will be summarized along with 
 For these endpoints using a ≤ 600 
µmol/L criterion, the analysis will be performed on those participants with a baseline DEP blood Phe 
value > 600 µmol/L.  


 
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• Dose level at first blood Phe level ≤ 360 µmol/L  
The distribution of the dose levels (i.e., 3x1011, 6x1011, 1x1012) when a participant first had a blood 
Phe level ≤ 360 µmol/L will be summarized. The percentage will be two presented ways: 
1) Based on the DEP FAS with a category for participants who did not reach a blood Phe level 
≤ 360 µmol/L 
2) Based on the sub-set of participants who reached a blood Phe level ≤ 360 µmol/L. 
• Dose level at first ≥ 20% reduction in blood Phe level 
The distribution of the dose levels (i.e., 3x1011, 6x1011, 1x1012) when a participant first had a blood 
Phe reduction of level ≥ 20% will be summarized. The percentage will be presented ways: 
1) Based on the DEP FAS with a category for participants who did not reach a blood Phe level 
≥ 20% 
2) Based on the sub-set of participants who reached a blood Phe level ≥ 20%. 
• Change in weekly blood Phe levels during each step of the dose ramp 
For the participants that participate in the DEP Weekly blood Phe substudy (see Section 2.3.2). 
Summary statistics of the Weekly blood Phe, change from baseline in blood Phe, and percent change 
from baseline in blood Phe will be provided for each week the blood Phe is assessed.  
3.14.1.9 Subgroup Analyses 
The DEP primary and key secondary efficacy analyses will be performed for the following subgroups 
(as defined in Section 3.5.1):  
• Participants on sapropterin dihydrochloride (Kuvan) at the DEP baseline visit 
• Genotypic phenotype values (GPV) subgroups. 
Additionally, if a dose was discontinued (see Section 3.17) the DEP primary and key secondary efficacy 
endpoint analyses will be performed on the subgroup of participants who had an iTD of a discontinued 
dose. For the DEP primary endpoint analysis, the MMRM analysis would be performed including the 
participants who had an iTD corresponding to a discontinued dose(s), in addition to the participants with 
an iTD corresponding to the retained doses. 
As a supplemental analysis, summary statistics for the following endpoints will be performed on the 
DEP FAS adolescent subgroup: 
• Change and percent change from baseline in blood Phe level at last measurement of the iTD of 
SYNB1934v1.  
• A ≥ 20% reduction from baseline in blood Phe level at any time in the DEP 


 
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3.14.2 Part 2: RWP 
The efficacy analyses for the RWP will be based on the responder RWP FAS (defined in Section 3.5) 
unless explicitly stated otherwise. 
3.14.2.1 Primary Efficacy Endpoint Analysis  
The RWP primary efficacy endpoint is the change from RWP baseline to Week 4 in blood Phe level. An 
MMRM analysis will be used to compare the mean change in blood Phe level between the placebo and 
SYNB1934v1 dose groups. The MMRM model will have treatment group, Screening Blood Phe (as a 
dichotomous variable: ≤ / > 720 μmol/L), RWP baseline blood Phe level, iTD dose level, visit, and visit 
× treatment group (interaction effect), visit × iTD dose level (interaction effect), and visit × RWP 
baseline blood Phe level (interaction effect) as fixed effects. The MMRM will also incorporate the 
participant’s repeated measures at visits Weeks 1, 3, and 4, with visit being treated as a categorical 
variable. Least-squares means for both treatment groups and the SYNB1934v1 treatment group 
difference from placebo, along with the corresponding 
confidence intervals and p-values will be 
calculated for Weeks 1, 3, and 4. The primary efficacy analysis will compare the least-squares means 
treatment difference between SYNB1934v1 and placebo in the change from baseline in the blood Phe 
level at Week 4. 
At each of the scheduled assessments, blood Phe will be drawn in duplicate. The MMRM will 
incorporate these participant repeated measures. For the MMRM analysis, the results of each of the post-
baseline duplicate samples from a visit will be included in the statistical model (i.e., without averaging 
the results within a given visit before inclusion into the MMRM). 
The MMRM analysis will be based on the restricted maximum likelihood method. A Kenward-Roger 
approximation will be used for the denominator degrees of freedom. The covariance structure for the 
repeated-measures will be a direct product of unstructured (i.e., for the visits) and compound symmetry 
for the repeated assessments at a particular visit (i.e., the subject-level repeated measures has 2 
dimensions, post-baseline Weeks 1, 3, and 4, as well as the 2 assessments at each visit. As such the 
covariance structure will use the direct product structure “UN@CS” as defined in SAS PROC Mixed).  
For the dependent variable, this will be an observed-case analysis using the responder RWP FAS 
population, missing data will not be imputed.  
In the event that a participant is missing their RWP baseline blood Phe (i.e., which is their DEP iTD 
Week 3 blood Phe value), the participant’s last non-missing DEP Week 3 blood Phe value (i.e., the most 
recent non-missing Week 3 blood Phe value from a previous dose level) will be used as the RWP 
baseline blood Phe covariate value (i.e., as specified in Section 3.3, the baseline RWP covariate value 
will be the mean of the corresponding duplicate blood Phe assessments). In the unlikely event that the 
participant does not have any non-missing DEP Week 3 blood Phe values, the blood Phe covariate value 
will remain missing, and the participant will be excluded from the RWP primary efficacy analysis, 
which is an observed case analysis. Note that the multiple imputation sensitivity analysis Section 
3.14.2.3 will impute missing RWP baseline blood Phe.  


 
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3.14.2.2 Primary Efficacy Analysis Model Does Not Converge  
If the RWP primary efficacy MMRM analysis does not converge, the repeated measures at each blood 
Phe assessment will be averaged together and the average of the repeated measures will be used to 
calculate the change from baseline in blood Phe level, the dependent variable. The model will have 
change from baseline as the dependent variable. The model will have percent change from baseline as 
the dependent variable (i.e., using the averaged value at each of the Week 1, 3, and 4 visits). The 
MMRM model will be the same (i.e., same set of fixed effects as described in Section 3.14.2.1) with the 
exception that the covariance structure for the repeated-measures will be unstructured to account for the 
within-subject Week 1, 3, and 4 assessments. If this model does not converge, a compound symmetry 
covariance structure will be used.  
3.14.2.3 Primary Efficacy Sensitivity and Supplementary Analysis  
The following sensitivity and supplementary analyses will be performed for the RWP primary efficacy 
endpoint.  
• Imputation of Missing Data: Multiple imputation methods will be used to impute missing blood Phe 
data during the RWP. The imputation of missing data analysis will be performed twice, once 
assuming the data is missing at random (MAR), and also assuming missing not at random (MNAR). 
The methodology for the multiple imputation is provided in Section 3.18.2. The analysis model will 
be the same model as the RWP primary efficacy endpoint analysis. 
 
• Analysis of Covariance: The primary efficacy endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model. The change from RWP baseline to Week 4 in blood Phe level is the 
dependent variable, using the average of the duplicate Week 4 blood Phe levels (i.e., if one of the 
values is missing, the non-missing value will be used). The ANCOVA has treatment as a fixed effect 
and RWP baseline Phe is a covariate. This will be an observed-case analysis using the responder 
RWP FAS population, missing data will not be imputed. 
 
• Responder RWP PPS Analysis: The primary efficacy endpoint analysis will be performed using the 
responder RWP PPS analysis set (defined in Section 3.5).  
 
• Responder Randomized set Analysis: The primary efficacy endpoint analysis will be performed 
using the responder Randomized analysis set (defined in Section 3.5). Missing data will be imputed 
assuming the data is missing at random (MAR). The methodology for the multiple imputation is 
provided in Section 3.18.2.  
 
• Responder RWP FAS Analysis Including Participants in a Dropped Dose: The primary efficacy 
endpoint analysis will be performed using the responder RWP FAS analysis set (defined in Section 
3.5) with the modification that participants whose dose was dropped at the DEP interim analysis are 
included in the analysis (i.e., this analysis will only be performed if one or more doses are dropped at 
the DEP interim analysis  (see Section 3.17).  
 


 
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• Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The primary efficacy endpoint analysis will be performed on the responder RWP FAS 
using an observed case analysis which excludes observations after the following intercurrent events 
occur in the RWP: Treatment discontinuation, antibiotic use, prohibited medication use.  
 
Additionally, histograms illustrating the Week 4 change and percent change from baseline in blood Phe 
by treatment group will be produced.  
3.14.2.4 Key Secondary Efficacy Endpoint Analyses 
The key secondary efficacy endpoint analyses for the RWP are provided below. These analyses will be 
performed on the responder RWP FAS. 
• Percent change from DEP baseline in blood Phe level at Week 4 
The percent change from DEP baseline to Week 4 in blood Phe level, will be analyzed using an 
MMRM analysis similar to the model described for the RWP primary efficacy analysis (Section 
3.14.2.1), with the dependent variable being the percent change in blood Phe level from DEP 
baseline, and the DEP baseline blood Phe level as the baseline blood Phe level covariate. This will 
be an observed-case analysis using the DEP FAS population, missing data will not be imputed. Note 
that if the MMRM model does not converge, the same methodology as described for the RWP 
primary efficacy analysis (Section 3.14.2.1) will be applied. 
• Change from DEP baseline to Week 4 in blood Phe level 
The change from DEP baseline to Week 4 in blood Phe level, will be analyzed using an MMRM 
analysis similar to the model described for the RWP primary efficacy analysis (Section 3.14.2.1), 
with the dependent variable being the change in blood Phe level from DEP baseline, and the DEP 
baseline blood Phe level as the baseline blood Phe level covariate. This will be an observed-case 
analysis using the responder RWP FAS population, missing data will not be imputed. Note that if the 
MMRM model does not converge, the same methodology as described for the RWP primary 
efficacy analysis (Section 3.14.2.1) will be applied. 
• Blood Phe level ≤ 360 µmol/L at Week 4 
The proportion of participants meeting the key secondary endpoint, Blood Phe level ≤ 360 µmol/L at 
Week 4, will be tested between the placebo and SYNB1934v1 treatment groups using a Fisher’s 
Exact test. This will be an observed-case analysis approach using the responder RWP FAS such that 
if a participant is missing the data needed to determine the endpoint, they will be considered as not 
having met the endpoint. 
3.14.2.5 Key Secondary Sensitivity and Supplementary Analysis  
o The following are the set of sensitivity and supplementary analysis for the RWP Key secondary 
endpoints:  
• Percent change from DEP baseline in blood Phe level at Week 4 


 
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o Imputation of Missing Data: Multiple imputation methods will be used to impute missing blood 
Phe data during the RWP. The imputation of missing data analysis will be performed twice, once 
assuming the data is missing at random (MAR), and also assuming missing not at random 
(MNAR). The methodology for the multiple imputation and analysis is provided in Section 
3.18.2. The analysis model will be the same model as the corresponding key secondary endpoint 
analysis. 
o Analysis of Covariance: The percent change from DEP baseline to Week 4 in blood Phe level 
endpoint will be analyzed using an ANCOVA model. The percent change from DEP baseline to 
Week 4 in blood Phe level will be the dependent variable, using the average of the duplicate 
Week 4 blood Phe levels (i.e., if one of the values is missing, the non-missing value will be 
used). The ANCOVA has treatment as a fixed effect, and DEP baseline Phe is a covariate.  
o RWP PPS Analysis: The key secondary efficacy MMRM analysis will be performed using the 
responder RWP PPS analysis set (defined in Section 3.5).  
Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The key secondary efficacy endpoint analysis will be performed on the responder 
RWP FAS using an observed case analysis which excludes observations after the following 
intercurrent events occur in the RWP: Treatment discontinuation, antibiotic use, prohibited 
medication use.  
• Change from DEP baseline to Week 4 in blood Phe level 
o Imputation of Missing Data: Multiple imputation methods will be used to impute missing blood 
Phe data during the RWP. The imputation of missing data analysis will be performed twice, once 
assuming the data is missing at random (MAR), and also assuming missing not at random 
(MNAR). The methodology for the multiple imputation and analysis is provided in Section 
3.18.2. The analysis model will be the same model as the corresponding key secondary endpoint 
analysis.  
o Analysis of Covariance: The change from DEP baseline to Week 4 in blood Phe level endpoint 
will be analyzed using an ANCOVA model. The change from DEP baseline to Week 4 in blood 
Phe level is the dependent variable, using the average of the duplicate Week 4 blood Phe levels 
(i.e., if one of the values is missing, the non-missing value will be used). The ANCOVA has 
treatment as a fixed effect, and DEP baseline Phe is a covariate. This will be an observed-case 
analysis using the responder RWP FAS population, missing data will not be imputed. 
o RWP PPS Analysis: The key secondary efficacy MMRM analysis will be performed using the 
responder RWP PPS analysis set (defined in Section 3.18.2).  
o Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The key secondary efficacy endpoint analysis will be performed on the responder 
RWP FAS using an observed case analysis which excludes observations after the following 
intercurrent events occur in the RWP: Treatment discontinuation, antibiotic use, prohibited 
medication use.  


 
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• Blood Phe level ≤ 360 µmol/L at Week 4 
o Exact Logistic Regression: The Blood Phe level ≤ 360 µmol/L at Week 4 endpoint will be 
tested between the placebo and SYNB1934v1 treatment groups using an exact logistic regression 
with treatment group as a factor and RWP Baseline Blood Phe (≤360/>360 µmol/L) as a 
dichotomous covariate. This will be an observed-case analysis approach using the responder 
RWP FAS such that if a participant is missing the data needed to determine the endpoint, they 
will be considered as not having met the endpoint. 
o RWP PPS Analysis: The Fisher’s exact test will be performed using the responder RWP PPS 
analysis set.  
o Excluding Observations after Treatment Discontinuation, Antibiotic use, or Prohibited 
Medication: The Blood Phe level ≤ 360 µmol/L at Week 4 endpoint will be tested via Fisher’s 
Exact Test using an observed case analysis which excludes observations after the following 
intercurrent events occur in the RWP: Treatment discontinuation, antibiotic use, prohibited 
medication use. This will be an observed-case analysis approach using the responder RWP FAS 
such that if a participant is missing the data needed to determine the endpoint, they will be 
considered as not having met the endpoint. 
3.14.2.6 Control of Multiplicity 
The overall Type I error rate for the RWP will also be controlled at the 
 for the 
primary endpoint (i.e., DEP, and RWP, will each have a 2-sided Type I error rate of
). The control 
of multiplicity at 
 within each study part is consistent with this study having 3 parts, with each study 
part having a distinct set of objectives, endpoints, and analysis populations. 
For the RWP, the 3 key secondary endpoints will be tested at the 
level using a fixed 
sequential testing methodology in this order:  
1) Percent change from DEP baseline to Week 4 in blood Phe level.  
2) Change from DEP baseline to Week 4 in blood Phe level  
3) Blood Phe level ≤ 360 μmol/mL at Week 4 
These key secondary endpoints will be tested following this prespecified order only if the RWP primary 
efficacy endpoint null hypothesis is rejected. An endpoint analysis will be considered statistically 
significant if the corresponding test for that analysis and all previous endpoint analyses are statistically 
significant using a 
Type I error rate 
  
3.14.2.7 Secondary Efficacy Endpoints Analysis 
The additional secondary efficacy endpoint for the RWP is: 
• A ≥ 20% reduction from DEP baseline in blood Phe level at Week 4. 
The proportion of participants achieving a ≥ 20% reduction in blood Phe level from baseline to Week 4 
will be tested between the placebo and SYNB1934v1 treatment groups using a Fisher’s Exact test. 


 
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This will be an observed-case analysis approach using the responder RWP FAS such that if a participant 
is missing the data needed to determine the endpoint, they will be considered as not having met the 
endpoint. 
3.14.2.8 Exploratory Endpoints Analysis 
The following are the analysis methods for the set of Exploratory endpoints.  
For the dichotomous endpoints, this will be an observed-case analysis approach such that if a participant 
is missing the data needed to determine the endpoint, they will be considered as not having met the endpoint. 
For the continuous endpoint, this will be an observed-case analysis.  
The following analyses will be performed on the responder RWP FAS 
• Change from baseline to Week 1 in blood Phe level  
The analysis of the change from RWP baseline to Week 1 in blood Phe level will be a part of the 
RWP primary efficacy analysis, described in Section 3.14.2.1 for responder RWP FAS.  
• Percent change from baseline to Week 1, and Week 4 in blood Phe level 
The percent change from RWP baseline to Week 1, Week 3, and Week 4 in blood Phe level, will be 
analyzed using an MMRM analysis similar to the model described for the RWP primary efficacy 
analysis (Section 3.14.2.1), with the dependent variable being the percent change in blood Phe level 
from RWP baseline, and the RWP baseline blood Phe level as the baseline blood Phe level covariate. 
• Blood Phe level ≤ 600 µmol/L at Week 4 
The proportion of participants meeting the Blood Phe level ≤ 600 µmol/L at Week 4 criteria, will be 
tested between the placebo and SYNB1934v1 treatment groups using a Fisher’s Exact test.  
The following analyses will be performed on the nonresponder RWP FAS. 
• Change from baseline to Week 1 and Week 4 in blood Phe level  
The analysis of the change from RWP baseline to Weeks 1 and 4 in blood Phe level will be analyzed 
as described in Section 3.14.2.1 using the nonresponder RWP FAS.  
• Percent change from baseline to Week 1 and Week 4 in blood Phe level  
The percent change in blood Phe level from RWP baseline to Week 1 and from RWP baseline to 
Week 4, will be analyzed using an MMRM analysis similar to the model described for the RWP 
primary efficacy analysis (Section 3.14.2.1), with the dependent variable being the percent change in 
blood Phe level from RWP baseline, and the RWP baseline blood Phe level as the baseline blood 
Phe level covariate. 
• Blood Phe level ≤ 360 μmol/L at Week 4 
The proportion of participants meeting the Blood Phe level ≤ 360 µmol/L at Week 4 criteria, will be 
tested between the placebo and SYNB1934v1 treatment groups using a Fisher’s Exact test. The 
analysis will be repeated using a criterion of ≤ 360 µmol/L.  


 
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• A ≥ 20% reduction from DEP baseline in blood Phe level at Week 4 
The proportion of participants achieving a ≥ 20% reduction in blood Phe level from baseline to 
Week 4 will be tested between the placebo and SYNB1934v1 treatment groups using a Fisher’s 
Exact test. 
• Change from DEP baseline to Week 4 in blood Phe level 
The change from DEP baseline to Week 4 in blood Phe level, will be analyzed using an MMRM 
analysis similar to the model described for the RWP primary efficacy analysis (Section 3.14.2.1), 
with the dependent variable being the change in blood Phe level from DEP baseline, and the DEP 
baseline blood Phe level as the baseline blood Phe level covariate.  
• Percent change from DEP baseline in blood Phe level at Week 4 
The percent change from DEP baseline to Week 4 in blood Phe level, will be analyzed using an 
MMRM analysis similar to the model described for the RWP primary efficacy analysis (Section 
3.14.2.1), with the dependent variable being the percent change in blood Phe level from DEP 
baseline, and the DEP baseline blood Phe level as the baseline blood Phe level covariate.  
3.14.2.9 Subgroup Analyses 
The RWP primary and key secondary efficacy analyses will be performed for the following subgroups 
(as defined in Section 3.5.1):  
• Participants on sapropterin dihydrochloride (Kuvan) at the DEP baseline visit 
• Genotypic phenotype values (GPV) subgroups. 
Additionally, if a dose was discontinued (see Section 3.17) the RWP primary and key secondary 
efficacy endpoint analyses will be performed on the subgroup of participants who had been excluded 
from the RWP FAS. 
As a supplemental analysis, summary statistics for the following endpoints will be performed on the 
RWP FAS adolescent subgroup: 
• Change and percent change from RWP baseline to Week 4.  
• Change and percent change from DEP baseline to Week 4.  
• Blood Phe Level ≤ 360 µmol/L at Week 4. 
 
3.14.3 Part 3: OLE 
The following set of OLE endpoints will be performed separately for the responder and nonresponder 
OLE FAS. For the dichotomous endpoints, this will be an observed-case analysis approach such that if a 
participant is missing the data needed to determine the endpoint, they will be considered as not having met 
the endpoint. For the continuous endpoint, this will be an observed-case analysis.  
• Change from DEP baseline in blood Phe levels 


 
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At each scheduled OLE assessment timepoint, summary statistics for change from DEP will be 
provided.  
 
Additionally, the change from DEP baseline in blood Phe levels will be analyzed using MMRM. The 
dependent variable is the change from DEP baseline in blood Phe level at each of the scheduled 
assessment timepoints in the OLE. Least-squares means for the change from baseline, along with the 
corresponding
 intervals and p-values testing a change from baseline equal to 0, will 
be calculated.  
 
Note that the least-squares means will use the observed margins approach (i.e., as opposed to equal 
weighting across the 3 iTD dose levels, the weighting will be proportional to the number non-
missing percent change from baseline observations in each of the 3 iTD dose levels). 
 
• Blood Phe level ≤ 360 μmol/L  
At each scheduled OLE assessment timepoint, the number and proportion of participants meeting the 
corresponding criteria will be summarized along with 
 (i.e., 
  
 
In addition to the analysis being performed on the responder/nonresponder OLE FAS populations, 
this analysis will be performed stratified on if a participant had a had an increase in 3-day dietary 
Phe (i.e. as discussed in Section 3.10.2) relative to DEP Baseline at each particular visit (i.e., at each 
timepoint summaries will be performed for the following groups: 
o Responder OLE 
o Nonresponder OLE 
o Responder OLE participants who had an increase in 3-day dietary Phe*  
o Responder OLE participants who did not have an increase in 3-day dietary Phe*  
o Nonresponder OLE participants who had an increase in 3-day dietary Phe* 
o Nonresponder OLE participants who did not have an increase in 3-day dietary Phe*  
* Note: if a participant has a missing value for the 3-day dietary Phe at the OLE timepoint or at 
DEP baseline, the participant will be excluded from the analysis. 
• Change from DEP baseline in blood Phe levels over time 
At each scheduled OLE assessment timepoint, summary statistics for change from DEP will be 
provided overall and stratified on the participants increase in dietary Phe (see Section 3.10.2) using 
the following categories: 
o 0 mg/kg/day (i.e., no increase) 
o 1-20 mg/kg/day 
o 21-40 mg/kg/day 
o >40 mg/kg/day. 


 
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• Change from DEP baseline in blood Phe level over time until first protocol-specified increase in 
dietary protein intake. 
At each scheduled OLE assessment timepoint, summary statistics for change from DEP in blood Phe 
level will be provided. This analysis will exclude observations which occur after the participant had 
their first protocol-specified increase in dietary protein.  
• Change from DEP baseline in dietary Phe intake 
As discussed in Section 3.10.2, at each scheduled OLE assessment timepoint, participants will have 
a 3-day dietary intake assessment. At each scheduled visit, the percent change from baseline in 3-day 
dietary Phe levels will be summarized. 
3.15 Safety Analysis 
Safety will be evaluated by scheduled monitoring of AEs, vital signs, clinical laboratory measurements, 
ECGs, and PEs. By-participant listings of all measurements and parameters will be presented in tabular 
format, including absolute values and changes from baseline (if applicable), by study period, dose cohort 
(as applicable), and study day. 
Safety results will be presented separately for each of the 3 study Periods (DEP, RWP, and OLE). For 
the DEP, safety analyses will be performed on the DEP SAS. For the RWP and OLE study parts, safety 
analyses will be performed on the corresponding SAS, with results being presented for the responder 
and nonresponder populations as well as the overall population. 
Additionally, adverse events will also be presented across the 3 Study Periods (i.e., DEP, RWP, and 
OLE) as discussed in Section 3.15.1.7.  
3.15.1 Adverse Events 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
Version 25.1 (or higher) and will be classified using SOC and PT (with the exception of diarrhea and 
nausea AEs as discussed in Section 3.15.1.1). Severity of AEs and laboratory abnormalities will be 
graded using the National Cancer Institute Common Terminology Criteria for Adverse Events. Adverse 
events causality will be dichotomous: Related or Unrelated (i.e., as defined in Section 6.1.2 of the study 
protocol).  
Treatment emergent adverse events (TEAEs) are any adverse events occurring or worsening in severity 
after the first administration of SYNB1934v1 study medication.  
TEAEs will be tabulated by study Part, treatment group (for Part 2), system organ class, and preferred 
term. Incidence tables of participants with TEAEs will be presented for all TEAEs by maximum 
severity, serious AEs (SAEs), TEAEs assessed as related to IMP, and TEAEs resulting in 
discontinuation of study dosing. 
The number and percentage of participants who experienced at least one TEAE as well as the number 
and percentage of participants who experienced each specific SOC and PT will be presented. For the 


 
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presentation of TEAE incidences, the SOCs will be sorted alphabetically, and within SOC, the preferred 
term (PT) will be used and presented by decreasing total frequency. 
3.15.1.1 Adverse Events Severity 
The severity rating of an AE refers to its intensity. The severity of each AE will be categorized using the 
NCI CTCAE. For any term that is not specifically listed in the CTCAE scale, intensity should be 
assigned a grade of 1 through 5 using the following CTCAE guidelines: 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated. 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate 
instrumental activities of daily living. 
Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self-care activities of daily 
living. 
Grade 4: Life-threatening consequences; urgent intervention indicated. 
Grade 5: Death related to AE. 
 
Exceptions to CTCAE criteria will be implemented for grading certain gastrointestinal-related TEAEs, 
as detailed in Table 9.  
Table 9: Severity Assessment of Diarrhea and Nausea 
Grade 
Diarrheaa 
Nausea 
1 
Increase of diarrhea, < 3 liquid stools a day 
over baseline; mild increase in ostomy 
output compared to baseline 
Loss of appetite without alteration in eating 
habits and/or with use of planned anti-nausea 
medication 
2 
Diarrhea ≥ 3 liquid stools daily and limiting 
instrumental ADLs 
Use of unplanned concomitant medications to 
increase oral intake 
3 
Diarrhea requiring hospitalization or 
limiting self-care ADLs 
Inadequate oral caloric or fluid intake; tube 
feeding, TPN or hospitalization indicated 
(unchanged from CTCAE) 
4 
Life-threatening consequences; urgent 
intervention indicated (unchanged from 
CTCAE) 
Not applicable 
5 
Death (unchanged from CTCAE)  
Not applicable 
ADLs = activities of daily living; CTCAE = Common Terminology Criteria for Adverse Events, TPN= Total parenteral 
nutrition. 
a Diarrhea is defined as liquid stools without consistency in shape. 


 
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3.15.1.2 Adverse Events Causality  
An adverse event will be assessed a causality of Related/Not Related to each the following: 
• SYNB1934v1 
• PPI. 
3.15.1.3 Adverse Events of Special Interest 
As discussed in Section 6.3 of the study protocol, adverse events of special interest (AESIs) for 
SYNB1934v1 are as follows:  
• Hypersensitivity reactions due to SYNB1934v1 
• Infections due to SYNB1934v1 
• Grade ≥ 3 gastrointestinal AEs per protocol specified grading criteria, including nausea, 
vomiting, diarrhea, and abdominal pain.  
3.15.1.4 Adverse Event Missing Date Imputation 
The following rules apply when determining if an AE is treatment-emergent in the scenario where the 
start date is missing or partially missing. These rules provide an algorithm to “impute” a complete AE 
start date which will then be used to determine if the AE is treatment emergent.  
AE Start Date Imputation  
AE start date missing day and month: 
• If the year is the same as the year of the treatment start date, the day and month of the date of 
treatment start date will be assigned to the missing fields 
• If the year is prior to the year of the treatment start date, December 31 will be assigned to the 
missing fields 
• If the year is after the date of the treatment start date, January 1 will be assigned to the missing 
fields.  
AE start date missing month only: 
• The day will be treated as missing, and both month and day will be replaced according to the 
above procedure.  
AE start date missing day only: 
• If the month and year are the same as the month and year of the treatment start date, the day of 
the treatment start date will be assigned to the missing day  
• If either the year is before the year of the date of the treatment start date or if both years are the 
same, but the month is before the month of the treatment start date, the last day of the month will 
be assigned to the missing day 


 
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• If either the year is after the year of the treatment start date or if both years are the same, but the 
month is after the month of the treatment start date, the first day of the month will be assigned to 
the missing day.  
AE start date completely missing: 
• If the AE end date is complete and after the treatment start date, the treatment start date will be 
assigned to the missing start date 
• If the end date is complete and before the treatment start date, the end date will be assigned to the 
missing start date  
• Otherwise, the AE start date will be assigned the treatment start date.  
If the end date is complete and the imputed start date as above is after the end date, the start date will be 
imputed by the end date.  
 
AE End Date Imputation  
The following is the algorithm to impute a missing AE end date.  
AE end date year is missing or AE is “ongoing”: 
• If the AE end date year is missing or is indicated as “ongoing” the AE end date will not be 
imputed.  
AE end date missing day and month: 
• If the AE end date day and month is missing the month and day should be set to the earliest of 
the following: 31DECYYYY, date of death, participant’s last visit date.  
AE end date missing month only: 
• The day will be treated as missing, and both month and day will be replaced according to the 
above procedure.  
AE end date missing day only: 
• If the AE end date day is missing the day should be set to the earliest of the following: last day of 
the month, date of death, participant’s last visit date.  
3.15.1.5 Adverse Event Summaries  
The following are the set of adverse event summaries.  
A. Overall summary of TEAEs 
B. Incidence of TEAEs by SOC and PT  
C. Incidence of TEAEs by SOC, PT, and Severity  
D. Incidence of TEAEs by SOC, PT, and Related to SYNB1934v1 


 
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E. Incidence of TEAEs by SOC, PT, and Related to PPI 
F. Incidence of TEAEs Leading to Drug Interruption by SOC and PT  
G. Incidence of TEAEs Leading to Dose Reduction by SOC and PT  
H. Incidence of TEAEs Leading to Drug Discontinuation by SOC and PT  
I. Incidence of AESIs by SOC, PT, and Severity  
J. Incidence of Serious Adverse Events by SOC and PT  
K. Incidence of SYNB1934v1 Related Serious Adverse Events by SOC and PT  
L. Incidence of PPI Related Serious Adverse Events by SOC and PT.  
 
For the DEP only:  
M. Incidence of Adverse Events prior to first dose of SYNB1934v1 by SOC and PT by Dose Level 
N. Incidence of Adverse Events prior to first dose of SYNB1934v1 which are PPI related by SOC 
and PT by Dose Level 
O. Incidence of TEAEs by SOC and PT by Dose Level 
P. Incidence of TEAEs by SOC, PT, and Severity by Dose Ramp Period 
Q. Incidence of TEAEs by SOC, PT, and Related to SYN1934 by Dose Ramp Period. 
This set of analyses will be performed for each of the 3 study periods (DEP, RWP, OLE) separately 
using the corresponding SAS. The following conventions will be applied: 
• For the DEP, the AEs summaries will be presented by SYNB1934v1 dose group and overall. The 
SYNB1934v1 dose group will be determined by the dose the participant was on at the TEAE 
start date 
• For the RWP, the AEs summaries will be presented by SYNB1934v1 (i.e., each individual iTD 
dose and overall) and placebo and within these 2 treatment groups by: responder, nonresponder, 
and overall 
• For the OLE, the AE summaries will be presented by responder, nonresponder, and overall. The 
SYNB1934v1 dose group will be determined by the dose the participant was on at the TEAE 
start date. 
For the presentation of TEAE incidences, the SOCs will be sorted alphabetically, and within SOC, the 
PT will be used and presented by decreasing total frequency based on the total population. 
For the overall summary of TEAEs (i.e., item A above), the following information will be provided:  
• Number and percent of participants with a TEAE 
• Number and percent of participants with a TEAE of Grade 1 


 
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• Number and percent of participants with a TEAE of Grade 2 
• Number and percent of participants with a TEAE of Grade 3  
• Number and percent of participants with a TEAE of Grade 4 
• Number and percent of participants with a SYNB1934v1 related TEAE 
• Number and percent of participants with a PPI related TEAE 
• Number and percent of participants with an AE leading to treatment interruption 
• Number and percent of participants with an AE leading to treatment discontinuation  
• Number and percent of participants with an AE leading to dose reduction 
• Number and percent of participants with an SAE 
• Number and percent of participants with a SYNB1934v1-related SAE 
• Number and percent of participants who died. 
 
Additionally, for the adolescent subgroup, TEAEs will be summarized by PT and SOC for each of the 3 
study periods (DEP, RWP, and OLE).  
• For the DEP, the TEAEs summaries will be presented by SYNB1934v1 dose group and overall. 
The SYNB1934v1 dose group will be determined by the dose the participant was on at the TEAE 
start date.  
• For the RWP, the TEAEs summaries will be presented by SYNB1934v1 (i.e., each individual 
iTD dose and overall) and placebo and within these 2 treatment groups.  
• For the OLE the TEAEs summaries will be presented by SYNB1934v1 dose group and overall. 
The SYNB1934v1 dose group will be determined by the dose the participant was on at the TEAE 
start date.  
3.15.1.6 Adverse Events by Dose Ramp Day 
For the DEP, adverse events will be analyzed relative to the Dose Ramp Schedule (see Table 8).  
Specifically, TEAEs will be summarized by dose level (3 × 1011, 6 × 1011, 1 × 1012) and within dose 
level by the dose frequency schedule.  
The Dose Ramp Days will be partitioned as follows for each of the 3 doses (i.e., as described in Table 4 
of the study protocol): 
• 3× 1011: Days 1-3, 4-6, 7-9, 10-11, 12-14, and 15-21 (as well as “Overall”) 
• 6 × 1011: Days 1-3, 4-7, and 8-21 (as well as “Overall”) 
• 1 × 1012: Days 1-3, 4-7, and 8-21 (as well as “Overall”). 


 
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The SYNB1934v1 dose level and Day period will be determined by the dose and dose ramp day the 
participant was on at the TEAE start date. The denominator will include all participants who received at 
least one dose within the corresponding dose ramp day period (i.e., this calculation will be based on a 
participant’s first and last dose day within a dose level). This will also include TEAEs which occur 
while a participant on at particular dose level (e.g., if a participant is on 3 × 1011 at two different times 
during the DEP, both of these periods will be included when assessing participants TEAEs at the 3 × 
1011 dose).  
These summaries will be: 
• Incidence of TEAEs by PT and SOC  
• Incidence of TEAEs Leading to Dose Reduction by SOC and PT  
• Incidence of TEAEs Leading to Drug Discontinuation by SOC and PT.  
Additionally, within each dose ramp, a “per-day” rate of adverse events will be calculated with the 
numerator being the number of participants with the corresponding TEAE and the denominator being 
the sum of the number of days the participants were in the dose ramp period. The summaries will be:   
• Per-Day rate of TEAEs by PT and SOC  
• Per-Day rate of TEAEs Leading to Dose Reduction by SOC and PT  
• Per-Day rate of TEAEs Leading to Drug Discontinuation by SOC and PT.  
3.15.1.7 Adverse Event Summaries Across Study Periods 
The analyses specified in Section 3.15.1.5 will be repeated based on the data across all periods (DEP, 
RWP, and OLE). TEAEs will be summarized across study periods by dose group: placebo, 3x1011, 
6x1011, 1x1012 and SYNB1934v1 overall. The dose group will be determined by the dose the participant 
was on at the TEAE start date.  
3.15.1.8 GI Daily Diary Card 
As discussed in Section 5.5.1 of the study protocol, participants will fill out a diary card during 
screening, DEP, and RWP to allow for enhanced capture of the gastrointestinal (GI) AEs. This diary will 
contain the GI event description start/stop date and the question “how much did this event bother you?” 
with the response being a 0–10-point numeric rating scale with 0 bothered” and 10 bothered”. When the 
investigator records this event as an AE, the following summaries of the tolerability will be performed: 
• For GI TEAEs with a start date in the Screening Period, the tolerability will be summarized by 
PT and overall. 
• For GI TEAEs with a start date in the DEP, the tolerability will be summarized by PT and 
overall, for each iTD and overall. 
• For GI TEAEs with a start date in the RWP, the tolerability will be summarized by PT and 
overall, for treatment group, and with the active arm, for each iTD and overall. 


 
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Note: the other information provided in the GI Daily Diary Card will be incorporated in the adverse 
event database. For AEs occurring over multiple days, the highest bothersomeness score will be 
recorded. Should the investigator determine that the event recorded on the daily diary card is not an 
adverse event, the corresponding GI Daily Diary card entry, including the bothersomeness data, will not 
be recorded in the adverse event database. 
3.15.2 Clinical Laboratory Evaluation 
Continuous laboratory data will be summarized using descriptive statistics of actual values and changes 
from baseline over time at each scheduled assessment timepoint (i.e., as defined in the Schedule of 
Events; Table 11and Table 12). These data will also be categorized as low, normal, or high based on the 
reference ranges of the central laboratory. 
The set of clinical laboratory tests are provided in Appendix 2. Note, the lab parameter Tyr, will also be 
summarized as a clinical laboratory test, but in a separate table. 
For each of the 3 study periods (i.e., DEP, RWP, and OLE), shift tables for hematology and serum 
chemistry parameters summarizing the number and percentages of participants who had both DEP 
baseline and at least one post baseline measurements will be displayed (i.e., using the DEP baseline as 
the reference for all 3 study parts). Both the lowest and highest value of post-baseline assessments will 
be used. For this analysis, any lab collection performed on unscheduled analysis visits will be included. 
The set of hematology and serum chemistry parameters which have corresponding Low/Normal/High 
assessments will be included.  
For the RWP the results will be presented by treatment group; and presented overall for the DEP and 
OLE.  
3.15.3 Vital Signs, Weight and Height 
Semi-supine vital signs (systolic blood pressure, diastolic blood pressure, pulse, and body temperature), 
weight, and height will be collected as specified in the Schedule of Events (Table 11 and Table 12). 
Participants are required to remain in the semi-supine position for at least 5 minutes prior to obtaining 
vital signs. Body mass index will be calculated based on weight and height as specified in the Schedule 
of Events. 
This data will be summarized using descriptive statistics of actual values and changes from baseline 
over time at each scheduled assessment timepoint.  
3.15.4 ECG 
Supine single 12-lead electrocardiograms (ECGs) will be performed at baseline as specified in the 
Schedule of Events (Table 11 and Table 12). A listing of the ECG data will be presented.  
3.15.5 Physical Examination 
A physical examination will be performed at screening only, these results will be presented in a listing.  


 
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3.16 Sample Size Justification 
Approximately 
participants will be enrolled in the DEP to ensure that approximately 
 
are randomized into the RWP. This assumes a dropout rate of 
 and approximately 
 
of participants are responders. If, during the course of the study, it becomes apparent that the 
assumptions were not accurate, or a dose level is dropped after the interim analysis, the number of 
participants enrolled into the DEP may be adjusted. The study has over 
 in testing that the 
percent mean change from baseline at the last measurement of the iTD in blood Phe level is different 
from 0. This assumes a mean percent reduction in blood Phe levels of 
 a standard deviation of 
 drop-out rate, and a 2-sided Type I error rate of
 mean percent reduction and 
standard deviation in blood Phe values used in the power calculations are based on results from a similar 
study in participants with PKU FDA (2007).  
A sample size of 
 in the responder RWP FAS yields approximately 
 power to detect a 
difference in the mean change in blood Phe level from baseline to Week 4 between the SYNB1934v1 
and placebo treatment groups. This assumes a mean treatment group difference in blood Phe of -
 between the SYNB1934v1 and placebo groups, a change from baseline standard deviation 
of 
 1:1 randomization of SYNB1934v1 and placebo, a
, and a 
2-sided Type I error rate of
. The power was calculated via a t-test. The mean treatment group 
difference and standard deviation in blood Phe values used in the power calculations are based on results 
from a similar study in participants with PKU FDA (2007).  
3.17 Interim Analysis 
Once the first 
 participants have attained an iTD and completed the DEP or the 
 has a 
baseline DEP and has been in the study for 
, whichever occurs earlier, the DEP data will be 
evaluated by an independent DMC. The DMC will use the below pre-specified set of criteria based on 
both safety and efficacy to determine whether the dose regimen is appropriate or whether any doses 
should be discontinued as well as whether the study should be stopped for futility. Enrollment will not 
be paused during the interim analysis.  
If a dose level is dropped/discontinued, participants who are in the DEP and have an iTD corresponding 
to a dropped dose will not be randomized into the RWP but will instead enter the OLE directly. In 
addition, participants who are in the OLE at a dose level that has been dropped will be transitioned to the 
highest dose level retained. 
The size of the DEP interim analysis cohort and corresponding criteria for dropping a dose (Table 10) 
provide reasonable operating characteristics for proper determination if one or more doses should be 
dropped. 
Table 10: 
DMC Criteria for Dose Changes and Futility 
Evaluation by DMC 
Criteria to Drop the Dose 


 
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1 × 1012 dose 
< 20% of the participants who attain an iTD are at the 1 × 1012 
dose 
OR 
> 75% of participants who have 1 × 1012 as their iTD had a greater 
Phe reduction at their 6 × 1011 dose versus their 1 × 1012 dose. 
6 × 1011 dose 
 
Note: considered only 
if the Drop 1 × 1012 
Criteria has been met 
< 30% of the participants who attain an iTD have 6 × 1011 or 1 × 
1012 as their iTD 
OR 
> 75% of participants who have 6 × 1011 or 1 × 1012 as their iTD 
had a greater Phe reduction at their 3 × 1011 dose versus their 6 × 
1011 dose 
Stop for futility 
<30% of participants enrolled into the DEP attain an iTD and 
complete the DEP 
OR 
< 25% of participants who attain an iTD and complete the DEP 
are responders 
Note: these dose-dropping criteria are only applied for the first interim analysis.  
DEP = dose-escalating, open-label period; DMC = data monitoring committee; iTD = individually 
titrated dose; Phe = phenylalanine 
In the event that, based on the interim analysis results utilizing the criteria provided in Table 10, the 
DMC recommends dropping either the 1 × 1012 or the 1 × 1012 and 6 × 1011 doses, the RWP FAS will be 
modified as described in Section 3.5. 
In addition, the DMC will review interim data at defined intervals in the study (i.e., as defined in the 
DMC charter). The Sponsor will remain blinded to the subject-level blood Phe levels during the DEP 
and RWP but will have access to the DEP interim analysis results. 
The data from the OLE (Part 3) will be reviewed for safety and tolerability on an ongoing basis. 
3.18 Multiple Imputation Methodology 
This section describes the methodology for multiple imputation for missing blood Phe data for both the 
DEP and RWP Periods. Based on previous studies in this indication, FDA (2007), it is anticipated that 
the amount of missing data will be relatively small (
).  


 
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3.18.1  Multiple Imputation of Blood Phe for the DEP 
For sensitivity and supplementary analysis of the DEP primary and key secondary efficacy endpoint 
analyses, MI will be used to handle missing. The following is a summary of the Missing at Random 
imputation method being used for the DEP.  
Missing Blood Phe values will be imputed via multiple imputation (MI) assuming MAR for each 
scheduled assessment visit (DEP Baseline and Week 3). Note that at each scheduled assessment visit 
there are duplicate blood Phe measurements and Week 3 refers to the participants last Week 3 blood Phe 
value at the iTD.  
Fifty imputed datasets will be created. 
The following are the set of variables to be used in the multiple imputation model: 
• Screening blood Phe  
• Previous DEP Baseline Blood Phe values. For the duplicate blood Phe measures occurring at each 
scheduled visit, they will be “ordered” by the corresponding vial number: 
o DEP BaselineA and DEP BaselineB 
o Week 3A and Week3B 
Here the “A” values correspond to the duplicate blood Phe value with the smallest vial number and the 
“B” values correspond to the duplicate blood Phe value with the largest vial number. If there is just a 
single corresponding vial for an assessment it will be assigned “A”. Week 3 refers to the participants last 
Week 3 blood Phe value at the iTD.  
The imputations will be performed using monotone linear regression imputation methods which will 
impute the participants’ blood Phe values at each of the scheduled assessment visits in the study 
(including the duplicate assessments at each scheduled visit).  
If the missing data does not follow a monotonic pattern, a sequential approach to imputing the data via a 
Markov chain Monte Carlo (MCMC) method to produce a monotone missing data pattern will be 
applied using the MCMC impute=monotone option in Proc MI (SAS System). Then the monotone linear 
regression imputation methods will be applied.  
Note: if the multiple imputation model does not converge or produce estimates (i.e., due to over-
specification) the set of imputation variables may be modified. 
Based on the multiple imputed complete blood Phe datasets, the change and percent change from 
baseline in blood Phe levels will be calculated and the corresponding analyses performed and combined 
(i.e., via MIANALYZE in SAS).  
3.18.2 Multiple Imputation of Blood Phe for the RWP 
For sensitivity and supplementary analysis of the RWP primary and key secondary efficacy endpoint 
analyses, MI methods will be used to handle missing. The following is a summary of the two imputation 
methods being used in the RWP, Missing at Random (MAR) and Missing Not at Random (MNAR).  


 
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The MAR and MNAR MI will each create 50 imputed datasets. The imputed datasets will be analyzed 
using the specified primary or key secondary efficacy endpoint analysis methodology. The results across 
the multiple imputed data sets will be combined using SAS Proc MIANALYZE. 
3.18.2.1 Missing at Random Multiple Imputation 
Missing Blood Phe values will be imputed via multiple imputation (MI) assuming MAR for each of the 
following assessment visits (DEP Screening, DEP Baseline, RWP Baseline, RWP Week 1, RWP Week 
3, and RWP Week 4). Note that at each scheduled assessment visit there are duplicate blood Phe 
measurements.  
Fifty imputed datasets will be created. 
The following are the set of variables to be used in the multiple imputation model: 
• DEP Screening blood Phe  
• DEP Baseline blood Phe (i.e., using the average of the duplicate values) 
• iTD Dose level 
• Randomized Treatment group (SYNB1934v1 or placebo) 
• Previous RWP Baseline blood Phe values. For the duplicate blood Phe measures occurring at each 
scheduled visit, they will be “ordered” by the corresponding vial number: 
o RWP BaselineA and RWP BaselineB 
o Week 1A and Week1B 
o Week 3A and Week3B 
o Week 4A and Week4B 
Here the “A” values correspond to the duplicate blood Phe value with the smallest vial number and the 
“B” values correspond to the duplicate blood Phe value with the largest vial number. If there is just a 
single corresponding vial for an assessment it will be assigned “A”.  
The imputations will be performed using monotone linear regression imputation methods which will 
impute the participants’ blood Phe values at each of the scheduled assessment visits in the study 
(including the duplicate assessments at each scheduled visit).  
If the missing data does not follow a monotonic pattern, a sequential approach to imputing the data via a 
Markov chain Monte Carlo (MCMC) method to produce a monotone missing data pattern will be 
applied using the MCMC impute=monotone option in Proc MI (SAS System). Following the method 
described in Smith (2017) the non-monotonic data imputation will be performed “by” the categorical 
treatment group variable. Then the monotone linear regression imputation methods will be applied.  
Note: if the multiple imputation model does not converge or produce estimates (i.e., due to over-
specification) the set of imputation variables may be modified. 


 
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Based on the complete Blood Phe datasets, the change and percent change from baseline in blood Phe 
levels will be calculated.  
3.18.2.2 Missing Not at Random Multiple Imputation 
Missing Blood Phe values will be imputed via multiple imputation (MI) assuming MNAR for each 
scheduled assessment visit (RWP Baseline, Week 1, Week 3, and Week 4). Note that at each scheduled 
assessment visit there are duplicate blood Phe measurements.  
The imputation of monotonic missing scores in the SYNB1934v1 treatment group will be based on the 
distribution in the placebo group. This method assumes that the trajectory of withdrawals from the 
SYN1934 treatment arm follows the distribution of the placebo participants. The MNAR option in Proc 
MI (SAS system) will be used to impute the placebo distribution as described (Yuan, 2014).  
Fifty imputed datasets will be created.  
The set of variables for this MNAR multiple imputation model is the same set as defined for the MAR 
multiple imputation (Section 3.18.2.1) with the exception of Treatment group, which is not included in 
the MNAR imputation (i.e., the placebo distribution is used to impute the data).  
The imputations will be performed using monotone linear regression imputation methods which will 
impute the participants’ blood Phe values at each of the scheduled assessment visits in the study 
(including the duplicate assessments at each scheduled visit).  
If the missing data does not follow a monotonic pattern, a sequential approach to imputing the data via a 
Markov chain Monte Carlo (MCMC) method to produce a monotone missing data pattern will be 
applied using the MCMC impute=monotone option in Proc MI (SAS System). Then the monotone linear 
regression imputation methods will be applied.  
Note: if the multiple imputation model does not converge or produce estimates (i.e., due to over-
specification) the set of imputation variables may be modified. 
3.18.3 Multiple Imputation Random Number Seeds 
The following random number seeds will be used for the multiple imputations; these seeds were 
randomly generated. 
• OLE MAR: 337623 
• RWP MAR: 599067  
• RWP MNAR: 658167 
3.19 Tables Listings and Figures 
The set of tables, listings, figures, and associated shells will be provided in a separate document. 


 
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3.20 Changes from the Protocol Planned Analyses 
The following are the set of changes from the protocol planned analyses as specified in the study 
protocol.  
These changes will be incorporated into the next study protocol amendment.  
 
• In Section 2.2.2.1, the handling of the scenario when a participant in the DEP de-escalates after 
RWP randomization is described.  
• In Section 2.5.1, the intercurrent events for the estimands were updated. In particular, 
participants whose dose is dropped during the interim analysis will not be excluded from the 
analyses.  
• In Section 3.5, the DEP FAS was updated to no longer exclude participants whose dose was 
dropped at the interim analysis.  
• In Section 3.5, the DEP FAS was updated to exclude those participants who did not take at least 
one dose of DEP study medication.  
• In Section 3.5, the RWP FAS, responder RWP FAS, and nonresponder RWP FAS were updated 
to exclude those participants whose dose was dropped at the DEP interim analysis.  
• In Section 3.5, the RWP FAS, responder RWP FAS, and nonresponder RWP FAS were updated 
to exclude those participants who did not take at least one dose of RWP study medication.  
• In Section 3.14.1.2, the DEP primary efficacy analysis, iTD was removed as a covariate from the 
MMRM.  
• In Section 3.14.2.1, the RWP primary efficacy analysis, Screening blood Phe was added as a 
covariate to the MMRM (i.e., as a dichotomous variable: ≤ / > 720 μmol/L). 
• In Section 3.14.2.6, the testing order of the 3 key secondary endpoints in the RWP was not 
consistent within the study protocol between the synopsis and Section 8.9 of protocol. The 
correct testing order (i.e., consistent with Section 8.9 of the study protocol) is provided in this 
SAP.  


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4 REFERENCES 
 
FDA. Drug approval package: Kuvan (sapropterin dihydrochloride) tablets. 13 December 2007. Available: 
https://www.accessdata.fda.gov/drugsatfda_docs/nda/2007/022181TOC.cfm  
Smith, C., Kosten S. (2017). "Multiple Imputation: A Statistical Programming Story." PharmaSUG: 2017 - Paper SP2001. 
 
Yuan, Y. (2014). "Sensitivity Analysis in Multiple Imputation for Missing Data." SUGI paper: SAS270-2014. 
 
 
 
 


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5 APPENDIX 1: SCHEDULE OF EVENTS 
Table 11: Schedule of Events: Dose-escalation and Randomized Withdrawal (Parts 1 and 2) 
 
 
DEP (Part 1)  
RWP (Part 2) 
Description/Weeka 
Screenb 
Study/DEP 
Baseline 
iTD-Finding 
Period 
End of DEPb 
(RWP 
Baseline)c 
Wk 1 
Wk 3 
Wk 4 (OLE 
Baseline)c 
Study Day 
-45 to -1 
1 
Up to 105 
daysd 
21 days after 
iTD 
7 
21 
28 
Assessmentb (Protocol Section) 
 
 
 
 
 
 
 
Informed consent (11.5) 
● 
 
 
 
 
 
 
Medical history and prior medications (5.2) 
● 
 
 
 
 
 
 
Vital signs (SBP/DBP, pulse, body temperature, height, 
weighte) (5.5.2) 
● 
● 
 
● 
 
 
● 
Physical examination (5.2) 
● 
 
 
 
 
 
 
FSH test (postmenopausal women only) (5.5.3) 
● 
 
 
 
 
 
 
Pregnancy test (WOCBP only)f (5.5.3) 
● 
● 
 
● 
 
 
● 
Record concomitant medications (4.3.4) 
● 
● 
● 
● 
● 
● 
● 
Adverse event reporting (5.5.1) 
● 
● 
● 
● 
● 
● 
● 
Weekly contact by site personnel (telephone, text, email) 
 
 
● 
● 
● 
● 
● 
Randomization (8.11) 
 
 
 
● 
 
 
 
Blood Pheg and Tyr (5.4.1) 
● 
● 
● 
● 
● 
● 
● 
3-Day dietary intake assessmenth (3.3) 
 
● 
● 
● 
● 
● 
● 
Administer PPI once per day (4.3.4.1) 
●i 
● 
● 
● 
● 
● 
● 
IMP immediately after mealsj (3.2) 
 
● 
● 
● 
● 
● 
● 
Laboratory tests (hematology/CBC with differential, CRP, 
serum chemistry, urinalysis) (5.5.3) 
● 
● 
 
● 
 
 
● 
Electrocardiogram (supine for 5 minutes)(5.5.2) 
● 
 
 
 
 
 
 
Immunogenicity samplek (5.2)  
 
● 
 
 
 
 
 
Optional fecal sample for microbiome analysisl (5.2) 
● 
 
 
 
 
 
● 
 
AE = adverse event; CBC = complete blood count; CRP = C-reactive protein; DEP = dose-escalating period; DBP = diastolic blood pressure; FSH = follicle-
stimulating hormone; IMP = investigational medicinal product; iTD = individually titrated dose; Phe = phenylalanine; PPI = proton pump inhibitor; RWP = 
randomized withdrawal period; SBP = systolic blood pressure; Term = termination; Tyr = tyrosine; WOCBP = women of childbearing potential. 
a Weeks are relative to the study period (i.e., each part begins with Week 1). All assessments/visits may be conducted within a 3-day window. 


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b All visits/assessments may be performed in the clinic or by a home healthcare professional at an alternate location (e.g., home, office, etc.), except for screening 
and end of DEP which must either be in person or via a virtual platform capable of completing all scheduled events including examination. 
c Participants who discontinue prematurely during the DEP will complete an early termination visit 30 days after the last dose of IMP as described in Table 6 of 
the study protocol. Participants who discontinue prematurely during RWP will complete RWP study measures as scheduled prior to the early termination visit. 
Early termination visits are described in Section 5.6 of the study protocol. 
d Participants may titrate down to the previous dose level during the iTD period if there is difficulty tolerating a higher dose level or at the investigator’s 
discretion. Visits and blood draws occur after a participant has been at a dose level for 3 weeks (including the ramp of that dose level as shown in Table 4 of the 
study protocol). Participants must remain on their iTD for 3 weeks before proceeding to Part 2. Participants cannot up-titrate again until the OLE.  
e Height and weight measured only at screening. 
f Serum pregnancy test at screening; urine pregnancy test at other visits. 
g All blood Phe should be drawn in duplicate. Baseline samples in DEP and RWP must be taken prior to the first doses in those periods. In Part 1, blood Phe will 
be measured every 3 weeks after a change in dose level during the iTD-finding period. In Part 2, blood Phe will be measured at Weeks 1, 3, and 4. See 
Section 5.4.1 of the study protocol of the protocol for more information. Patients enrolled in the optional DEP weekly substudy will have weekly blood Phe 
during the DEP. See Section 5.4.2 of the study protocol for more information. 
h A 3-day dietary intake assessment will occur for 3 consecutive days prior to scheduled study visits and blood Phe draws. See Diet Manual for more 
information.  
i The first dose of a PPI will be taken on or before Day –7 and continue daily as described in Section 4.3.4.1 of the study protocol.  
j Investigational medicinal product should be taken within 30 minutes of finishing a meal. See the Diet Manual for further details. 
k Samples will be used for monitoring anti-drug antibodies as needed (see Section 6.3 of the study protocol). 
l Fecal samples can be done at any time during the screening period, no less than 5 days prior to baseline visit. 
 
 
 


 
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Table 12:Schedule of Events: Part 3, Open-Label Extension Period and Follow-
up 
 
Quarterly Visits 
End of Study 
Early Term. 
 
Every 90 days 
 ± 5 days 
at least 30 days 
after last dose 
at least 30 days 
after last dose 
Assessmenta (Protocol Section) 
 
 
 
Vital signs (SBP/DBP, pulse, body temperature) 
(5.5.2) 
● 
● 
 
Pregnancy test (WOCBP only)b (5.5.3) 
● 
● 
 
Record concomitant medications (4.3.4) 
● 
● 
● 
Adverse event reporting (5.5.1) 
● 
● 
● 
Blood Phec and Tyr (5.4.1) 
● 
 
 
3-Day dietary intake assessmentd (3.3) 
● 
● 
 
Administer PPI once per day (4.3.4.1) 
● 
 
 
IMP immediately after meals (TID) (3.2.3) 
● 
 
 
Laboratory tests (hematology/CBC w/differential, 
CRP, serum chemistry) (5.5.3) 
● 
● 
 
Optional fecal sample for microbiome analysis (5.2) 
 
● 
 
AE = adverse event; CBC = complete blood count; CRP = C-reactive protein; DEP = dose-escalating 
period; DBP = diastolic blood pressure; IMP = investigational medicinal product; Phe = phenylalanine; 
PPI = proton pump inhibitor; RWP = randomized withdrawal period; SBP = systolic blood pressure; 
Term = termination; TID = 3 times per day; Tyr = tyrosine; WOCBP = women of childbearing 
potential.  
a All visits/assessments may be performed in the clinic or by a home healthcare professional at an 
alternate location (e.g., home, office). 
b Urine pregnancy test at quarterly visits.  
c All blood Phe should be drawn in duplicate.  
d A 3-day dietary intake assessment will occur immediately before all scheduled study visits. 
 
 
 
 


 
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6 APPENDIX 2: CLINICAL LABORATORY TESTS 
Hematology 
(CBC with 
differential) 
Basophils% 
Basophils 
Eosinophils% 
Eosinophils 
Hematocrit 
Hemoglobin 
Lymphocytes% 
Lymphocytes 
Mean corpuscular 
hemoglobin 
Mean corpuscular volume 
Monocytes% 
Monocytes 
Neutrophils% 
Neutrophils 
Platelet count 
Red blood cells 
White blood cells 
Serum 
chemistry 
Glucose 
BUN 
Creatinine with eGFR 
Sodium 
Potassium 
Chloride 
Calcium 
Total protein 
Albumin 
Fractionated bilirubin (total 
direct and indirect) 
Follicle-stimulating hormone 
(for postmenopausal women 
only)a 
Alkaline phosphatase 
Aspartate aminotransferase 
Alanine aminotransferase 
Pregnancy (for WOBCP only) 
Glucose 
C-reactive protein 
Urinalysis 
Specific gravity 
pH 
Glucose 
Bilirubin 
Ketones 
Occult blood 
Protein 
Nitrite 
Leukocyte esterase 
Pregnancy (for WOBCP 
only) 
Abbreviations: BUN = blood urea nitrogen; eGFR = estimated glomerular filtration rate; WOBCP = 
women of childbearing potential. 
a Performed at screening only. 
 


 
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7 APPENDIX 3: DMC Deliverables 
The analysis methodology for the DMC deliverables will be the methods as outlined 
in this SAP. The set of tables, figures and listings to be provided to the DMC at each 
of the scheduled meetings will be outlined in a separate DMC SAP document, and 
will follow the table, figure and listing shells document supporting this SAP. Note 
that some of the tables, figures, and listings may not be provided at all meetings due 
to a lack of data for a specific study period (e.g., there may be limited data in the OLE 
study period at the initial DMC meetings).