Autism spectrum disorder (ASD) is a neurodevelopmental condition with core deficits in social communication and behavior, often accompanied by gastrointestinal (GI) and metabolic dysfunction. Emerging evidence supports the role of neuroinflammation (including autoimmune components), gut-brain axis disruption, and metabolic dysregulation in ASD pathophysiology. Multimodal interventions targeting these pathways-using autologous cSVF, cryopreserved MSCs, FMT, and dietary modulation-intent is that these multimodal interventions may offer synergistic benefits for adolescents and adults with ASD.

This refined protocol integrates the latest evidence for a multimodal intervention-autologous cSVF, cryopreserved autologous MSCs, FMT, and a structured ketogenic protocol program-for adolescent and adult ASD management.

1. Protocol Summary

   Title:

   A Phase 1-2 Randomized Controlled Trial Evaluating the Safety and Efficacy of Multimodal Therapy (Autologous cSVF + Cryopreserved Autologous MSCs, Fecal Microbiota Transplantation, Ketogenic Program Reset, and OT/PT Training) in Adolescents and Adults with Autism Spectrum Disorder (ASD-Multi-level scoring)

   Sponsor: Blacktie Principal Investigator: Robert W. Alexander, MD, FICS Study Phase: Phase I/II Study Centers: 1-3 Clinical Applied Research Laboratories/academic centers with expertise in ASD, stem cell processing and utilization, plus gastroenterology/PMD consultants available for FMT in patient's locale

   Compliance:

   NIH and IRB oversight Informed consent for all participants

   ________________________________________

2. Background and Rationale Autism spectrum disorder (ASD) is a neurodevelopmental condition with core deficits in social communication and behavior, often accompanied by gastrointestinal (GI) and metabolic dysfunction. Emerging evidence supports the role of neuroinflammation (including autoimmune components), gut-brain axis disruption, and metabolic dysregulation in ASD pathophysiology. Multimodal interventions targeting these pathways-using autologous cSVF, cryopreserved MSCs, FMT, and dietary modulation-may offer synergistic benefits for adolescents and adults with ASD.

   ________________________________________

3. Objectives and Endpoints

   Primary Objective:

   • Evaluate the safety and tolerability of the multimodal interventions compared to current standard of care (SOC) for supportive therapy.

   Secondary Objectives:

   • Assess changes in ASD core symptoms, GI function, behavioral and social outcomes, and quality of life.

   Endpoints:

   • Incidence of adverse events (AEs) and serious adverse events (SAEs)
   • Change in validated ASD and GI symptom scales (CARS, VABS-II, GSRS, SRS, ABC)
   • Microbiome diversity and metabolic biomarker changes
   • Assessed performance and QoL evaluations

4. Study Design and Methodology

   • Design: Multi-disciplined, open labelled, controlled, parallel-group (1:1:1), tracked for cellular and biologics used, open-label, including tracking of diet/OT-PT
   • Sample Size: 50 participants (range ages 10-90, DSM-5 ASD diagnosis)
   • Randomization: Stratified by age, sex, and baseline GI symptoms, Socialization
   • Interim Analysis: Futility analysis at 50% enrollment

5. Study Population

   Inclusion Criteria:

   • Age 10-90 years, DSM-5 ASD diagnosis
   • Stable on current medications (if so treated at time of selection)
   • With/without comorbid GI symptoms

   Exclusion Criteria:

   • Recent antibiotics/probiotics
   • Severe GI disease or malnutrition
   • Recent major surgery
   • Other Medical/Surgery issues that preclude the treatment with the protocol; including spectrum limitations for patient tolerance or compliance.

6. Intervention Protocols 6.1 Autologous Cellular Stromal Vascular Fraction (cSVF)

   • Collection: Small volume Lipoaspirate via sterile microcannula harvesting of small aggregate tSVF - under local or supplemental anesthesia as dictated by patient ability to tolerate.
   • Processing: Enzymatic digestion for cSVF and cellular isolation/concentration of heterogeneous cell groups within the cSVF (via collagenase, centrifugation, and possible undesignated cell characterization (CD34+, CD90+, CD105+)
   • Dosing: =/> 10 x 106 - 5 × 10^7 cells delivered by standard Normal Saline IV infusion
   • Administration: Intravenous (IV) route with in-line sterile 150u filtration
   • Applied LED Red Light Exposure cSVF throughout deployment
   • Standard American Cell Technology (ACT) protocol harvesting, testing and overnight shipping on day of Harvest for Cryopreservation Protocol for storage of autologous Mesenchymal Stem Cells (MSCs characterized as above) for future interval delivery
   • Safety: Monitored for infusion reactions, infection, and thromboembolic events] 6.2 Cryopreserved Autologous MSCs
   • Source: Autologous microcannula harvested tSVF or bone marrow (BMAC)
   • Cryopreservation: Slow freezing (-1°C/min to -80°C, then liquid nitrogen), 10% DMSO, clinical-grade solutions. ACT Standard and monitored storage per FDA requirement
   • Viability: ≥70% post-thaw, confirmed by ORFLO Flow Cytometry
   • Dosing: 1-30 × 10^6 cells within sterile Normal Saline (balanced) IV infusion minimum
   • Administration: Peripheral IV route with in-line 150 u micron filtration and Red Light Therapy
   • Quality Control: Surface marker analysis, sterility, [endotoxin, karyotype on record with FDA certified Cell Banking Laboratory- ACT] 6.3 Fecal Microbiota Transplantation (FMT)
   • Donor Screening: Pathogen, parasite, and virus screening; FDA GMP processing
   • Preparation: Standardized, frozen human gut microbiota, delivered on dry ice
   • Pre-treatment: 14 days oral vancomycin, bowel cleanse (MoviPrep), acid suppression (Prilosec)
   • Dosing: High initial dose (oral/rectal), followed by daily oral maintenance for 7-8 weeks
   • Monitoring: GI and behavioral symptoms (GSRS, CARS, SRS, VABS-II)14-17]] 6.4 Ketogenic/Carnivore Diet (Carnivore RESET Program)
   • Diet: High-fat, low-carbohydrate, animal-based reset; macronutrient ratios to induce a state of ketosis (indicative of fat burning and reduction of insulin resistance.
   • Baseline Blood Testing: FBS, Fasting Insulin Levels, Hemoglobin A1c, Lipid Panel, Fasting Ketones, CBC, CRP, and other appropriate testing
   • Monitoring: Dietary logs, blood ketones, nutritional assessments at least quarterly
   • Support: PMC, Gastroenterologist,etc Provider or Dietitian, compliance checks near patient's home
   • Repeat testing of Blood levels repeated at 90-180 days
   • Safety: Monitor for metabolic derangements, GI side effects 6.5 OT/PT Training
   • Program: Standardized occupational and physical therapy modules
   • Continuation of Verbal, Socialization, and Educational Efforts
   • Frequency: 2-3 sessions/week, individualized goals (may be home monitored)

7. Safety Monitoring Plan

   • Adverse Event Monitoring: Continuous, with prompt reporting of SAEs
   • Infusion Reactions: Pre- and post-infusion monitoring for autologous cSVF/MSCs
   • Dietary Safety: Regular metabolic and nutritional assessments and tracking
   • FMT Safety: Exclusion of high-risk participants, close monitoring for infection

8. Outcome Measures and Biomarker Assessments Domain Assessment Tools/Markers ASD Symptoms CARS, SRS, ABC, VABS-II; observational behavior, socialization changes, communication abilities, GI Symptoms Bristol Stool Scale; Management testing of bowel flora bacteria Metabolic Blood glucose, ketones, vitamin B6, dopamine Inflammation Fecal calprotectin, serum IgA, ESR/CRP Safety AE/SAE logs, laboratory monitoring

   ________________________________________

9. Statistical Analysis Plan

   • Primary Analysis: Safety (AEs/SAEs) by intervention Provider
   • Secondary Analysis: Change in symptom scales and biomarkers (ANCOVA, mixed models)
   • Interim Analysis: Futility and safety at 50% enrollment check

10. Regulatory and Ethical Considerations

    • FDA/NIH Clinical Trial Submission: For Trial approval for use cSVF, MSCs, and FMT
    • IRB Approval: NIH/FDA Approval or equivalent recognized IRB Board (GARM International Review Board)
    • Informed Consent: All participants/guardians
    • Data Privacy: HIPAA-compliant data management

Inclusion Criteria:

• ADS
• Stable on current medications or therapy
• With/wthout comorbid GI (gut/Axis) symptoms

Exclusion Criteria:

• Recent antiobiotics or probiotic therapy within 30 days
• Severe GI disease or malnutrition
• Recent major surgery that may interfere with study course
• Other Med/Surgery issues that may preclude treatment with this protocol
• Inability to perform the monitoring of FMT without supervision by qualfied Provider