Efficacy and Safety of NTI164 in Children and Young Adults With Rett Syndrome

A validated, FDA-accepted 45-item caregiver-assessed tool to assess a variety of behavioural features impaired in RTT. The caregiver rates items as 0 = not true, 1 = somewhat true or sometimes true, or 2 = very true. Symptoms assessed include maladaptive behaviours, mood disruption, repetitive movements, fear/anxiety, breathing abnormalities, hand behaviours, and gross motor skills. A higher score indicates greater impairment/disease severity.

7-point scale rating a clinician's impression of disease severity at a given time point, a higher score indicates more severe illness.

A clinician-rated tool to assess how much an individual's symptoms improve or worsen following an intervention on a 7-point scale. 1-3 = improvement, 4 = no change, 5-7 = worsening. It is anticipated the CGI-I score will be <4 following NTI164 treatment, indicating moderate-substantial improvement.

An 8-point scale assessing areas of functional impairment (0 = normal function, 7 = most severe impairment).

Rates the effect of 4 health problems (inattentiveness, impulsivity, or mood; ability to think and remember; neurologic or physical limitations; epilepsy) on 11 aspects of the child and/or family's life (overall health, relationships with parents, relationships with siblings, relationship between your spouse/partner, relationships with child's friends/peers, social life - acceptability by others, social life - number of activities, school - academics, child's self-esteem, loss of original hopes for child/self, and family activities. A higher score indicates a lower level of disability. QoL component rates the QoL of the patient on a 6-point scale (1 = poor, 6 = excellent).

Assesses 4 aspects of burden (physical, emotional, and social burden, and time dependence) on the primary caregiver of the patient. Subdomains include general caregiver tasks, emotional and social impact of caregiving, and financial burden. A higher score indicates greater impairment and caregiver burden.

A descriptive scale covering 5 dimensions of health (mobility, looking after myself, doing usual activities, having pain or discomfort, and feeling worried, sad, or unhappy). This version of the scale has 5 levels to each answer (5L) and is worded so as to be more child friendly (Y). A higher score indicates greater impairment/worse health status.

A caregiver rated scale often used as the first step in routine screening to determine if a developmental evaluation is needed. In the context of RTT, it can be used to assess functional communication. A higher score indicates a better communication ability.

To further clarify the mechanism of action of NTI164, both bulk and single-cell RNA sequencing will be performed on peripheral blood mononuclear cells (PBMCs) from patients at specified time points. This will provide additional information regarding which genes are being expressed in patients at either abnormally low or high levels compared to a non-RTT population, and the effects NTI164 has on gene expression. This workflow includes RNA extraction, depletion of ribosomal RNA via hybrid capture, library preparation, and sequencing. Cleaned sequence reads will then be aligned against the Homo sapiens genome (Build version hg38) and STAR aligner (v2.5.3a) will be used to map unique reads to the genomic sequences. This is exploratory so genes of interest cannot be identified in advance.

To further clarify the mechanism of action of NTI164, proteomics and phosphoproteomics will be performed on peripheral blood mononuclear cells (PBMCs) from patients at specified time points. This will provide additional information regarding which proteins are being expressed at either abnormally low or high levels compared to a non-ASD population, as well as how extensively these proteins are phosphorylated compared to a non-ASD population, and the effects NTI164 has on protein expression and phosphorylation. Samples will be tagged using the TMTpro system and hydrophilic ion liquid chromatography fractionation performed. This is exploratory so proteins of interest cannot be identified in advance.

To further clarify the mechanism of action of NTI164, methylomics will be performed on whole blood from patients at specified time points. This will provide additional information on methylation patterns (e.g. addition of a methyl group to histones or DNA) in patients compared to non-patients, and how NTI164 effects this type of epigenetic modification and how genes of interest may be regulated. This is exploratory so patterns of interest cannot be identified in advance. Genome-wide methylation will be assessed using the Illumina MethylationEPIC v2.0 BeadChip (or the latest version if a new version is available to the study team at the time of analysis).

To further clarify the mechanism of action of NTI164, a cytokine assay investigating a panel of inflammatory markers will be performed on plasma from patients at specified time points. The cytokine assay will likely be the LEGENDplex Human Neuroinflammation Panel 1 (13 plex). Cytokines investigated with this panel include VILIP-1, CCL2, sTREM-2, b-NGF, TGF-b1, TNF-a, IL-6, s-TREM-1, sRAGE, CX3CL1, VEGF, BDNF, and IL-18.