taVNS for FRNS in Children

Study Design A parallel, double blinded, randomized placebo controlled trial comparing daily taVNS use with sham therapy will be conducted in children 3 to17 years of age with FRNS. Thirty participants with FRNS (defined as having 2 or more relapses in 6 months within 6 months of diagnosis or 4 or more relapses in any 12 month period) will be randomized 1 to 1 to taVNS or sham therapy. Participants will be enrolled at two pediatric tertiary hospitals over a two year time period, with completion of the study by year three. All participants will perform daily taVNS therapy (active for taVNS arm or inactive for sham arm) for 5 minutes each day for a total of 26 weeks. Participants will monitor heart rate with each treatment and log home urine results. Participants will be monitored monthly with in person study visits at Weeks 8, 16 and 26 alternating with virtual remote video visits at Weeks 4, 12 and 20. Biosample specimens will be collected at baseline, 8 weeks, 16 weeks and 26 weeks. There will be a follow up period of an additional 26 weeks. All participants will be given the option to receive the active taVNS treatment at the end of the randomized period.

Intervention The device is the Roscoe Medical Transcutaneous Electrical Nerve Stimulation (TENS) 7000 unit, a FDA approved commercially available handheld electrical pulse generator. A custom-produced silicone-equivalent electrode ear clip manufactured at Feinstein will be placed at the cymba concha of the left ear with electrode gel to provide stimulation (or sham) to the auricular branch of the vagus nerve. taVNS will be performed on the left side because there is less of a risk of cardiac side effects. Maximum power density limit calculations were performed based on data from the FDA as a function of pulse width, current amplitude, electrode size, and effective impedance, suggesting an electrode size of greater than 58 mm2 must be used. The custom-made electrodes for this study are larger than this limit, thus ensuring safety. The device will be set to a frequency of 30 Hz and pulse width of 300 μs based on previous literature. All participants in both trials will perform the intervention therapy for 5 minutes each day. The timing and duration of treatment are based upon the known half-life of the biological cytokine response, previous literature demonstrating efficacy of daily 5 minute treatments and our prior work. The study period will be 26 weeks. This time period was chosen based on the definition of FRNS using a 6-month period, prior clinical trials and ethical concerns for enrolling placebo controls for a longer period of time. All participants will be told that they may or may not feel stimulation from the device. Participants will be asked not to mention any aspects of the stimulation procedures to study investigators/physicians (except to the trainer) to maintain blinding. The device has a Patient Compliance Meter that can record the number of times it was used. This will be used to monitor compliance.

taVNS Group: The intensity setting for pulse amplitude will be adjusted to the participant's tolerance (if a sensation is felt) to a maximum level of 2.5. In patients who are under the age at which they can verbalize perceptual threshold and tolerability (<5 years), the investigators will initiate intensity at the lowest setting and incrementally increase the value until the level at which perceptual threshold, defined by changes in facial expression, patient becomes fidgety, or displays behaviors which denote sensation is being perceived. Then, the intensity level will be decreased till the perceptual indicators are no longer present, and the resultant level will be used for the treatment. A maximum power density of 150 milliwatts (mW)/cm2 will be utilized for patients under the age of 5 years, a level that is substantially below the 250 mW/cm2 maximum level defined by the FDA to prevent injury. Participants and guardians will be instructed to adjust intensity to highest level of tolerance each time the device is used and level will be logged.

Sham group: The sham device will be altered internally so that electrical stimulation is not delivered, but the device will appear to function (Feinstein Bioengineering). Externally, the sham device will look identical to the taVNS device. The participant will be told to increase the intensity until tolerated if a sensation is felt, but will be asked to stop at a maximum level of 3. This inactive sham method was chosen because previous studies have shown that stimulation with placement of the ear clip on other parts of the ear such as the earlobe, although not innervated by the vagus nerve, results in some vagus nerve activity. Inactive sham methodology has been used in previous studies.

Study Phases The study will consist of three parts. Part 1 - Screening Period- up to 8 weeks. Informed consent/assent will be obtained at screening prior to the conduct of any study-related procedures. Participants will be screened to confirm inclusion/exclusion criteria are met. Participants must be off steroid treatment for 14 days prior to Day 1 and the participant must be in remission (negative urine protein creatinine (UPC) on first morning urine) on Day 1.

Part 2 - Randomized Control Period - 26 weeks: Thirty participants with FRNS who meet all of the eligibility criteria will be randomized 1:1 to either taVNS or sham treatment. A trainer will instruct the parent/guardian on use of the device at the randomization visit. Participants will use the intervention device as directed for 5 minutes per day for 26 weeks. Participants will be monitored monthly with in-person study visits at Weeks 8, 16 and 26 alternating with virtual remote video visits at Weeks 4, 12 and 20. The visit window will be +/- 7 days. At each in person visit, the following will be conducted:

• Vital signs and physical examination
• Assessment for nephrotic syndrome relapses. Home urine protein logs will be reviewed.
• Blood and urine samples will be collected at each in person visit.
• Assessment of study intervention adherence. Parents/guardians will meet with the trainer and will be reoriented on taVNS device use at each visit as a safety measure. The device counter number will be recorded as a measure of adherence.
• Monitor for adverse events and tolerability: Parents/guardians will share a study log with investigators, which describes daily taVNS use, side effects, and any changes in heart rate.

At each virtual remote video visit, we will observe the participant while doing the intervention procedure, assess for nephrotic syndrome relapses, and monitor for any adverse events.

There will be a 4 week acclimation phase. If the participant relapses during this period, they will be treated with standard of care steroids and continue the intervention. If the participant relapses a second time or relapses after the acclimation phase, they will be terminated from the randomization phase and entered into the follow up period.

Part 3 - Follow Up Period - 26 weeks: At the completion of the randomized period, participants will be followed for an additional 26 weeks to assess clinical status. For those who stop the intervention, study visits will occur in-person during regularly scheduled clinical visits or via telehealth visit every 8 weeks, whichever is sooner. Participants will be assessed for the number of nephrotic syndrome relapses and home urine protein logs will be reviewed. First morning UPC will be recorded. All participants will be given the option to receive the active taVNS treatment at the end of the randomized period. Unblinding of treatment assignment from the randomized phase will not occur prior to consent for open-label use. As open-label extension trials may introduce significant bias, data obtained from these participants will not be considered part of this research and no statistical analysis will be carried out. However, clinical status and safety will continue to be monitored. In person study visits will occur every 8 weeks for those continuing use of taVNS.

Participant Discontinuation/Withdrawal from the Study Participation is completely voluntary and participants may decide to withdraw at any time. If a participant becomes steroid dependent at any point, they will be withdrawn from the study by investigators and offered alternative immunosuppressive therapy as per standard of care. If a participant in the SRNS group has deterioration in clinical status (e.g. worsening edema, rising creatinine), the treating physician may recommend withdrawal from the study at any point.

The pilot trial is not powered to determine statistically significant differences in efficacy endpoints comparing taVNS with sham therapy. Efficacy analyses will be reserved for the subsequent main trial. Rather, sample sizes were chosen to appropriately assess the feasibility, tolerability and variance effect size endpoints of the pilot trials.