Safety and Efficacy of Klotho and Follistatin Gene Therapy

Serum α-Klotho protein concentration will be quantified using a validated ELISA. Results will be determined from picograms per milliliter (pg/mL) for each participant at each time point. Higher or lower values have no inherent directionality and will be interpreted in study context.

Serum follistatin concentration will be quantified using a validated ELISA. Results will be reported as picograms per milliliter (pg/mL) for each participant at each time point. Higher or lower values have no inherent directionality and will be interpreted in study context.

Assessed through a checklist version of the PRO-CTCAE with each symptom options being none, mild, moderate, or severe. Items will be scored with 0, 1, 2, 3 respectively. Item responses will be summarized as number and percentage of participants experiencing each adverse event by system/organ class. High scores indicate highest severity of symptoms and low scores indicate no symptoms.

The WHOQOL-BREF is a self-report questionnaire that includes four domains: Physical Health, Psychological, Social Relationships, and Environment. Each domain score is transformed to a 0-100 scale, with higher scores indicating better quality of life. Changes from baseline will be examined per domain.

The Pattern Comparison Processing Speed Test measures processing speed using age-adjusted T-scores (mean 50, SD 10). Higher scores reflect faster cognitive processing. Changes from baseline will be analyzed per participant and time point.

Picture Sequence Memory Test evaluates episodic memory. Each form (A and B) yields an age-adjusted T-score (mean 50, SD 10). Higher scores indicate better memory performance. Forms A and B will be averaged.

The Flanker Inhibitory Control and Attention Test measures inhibitory control and attention. Scores are age-adjusted T-scores (mean 50, SD 10). Higher scores indicate better performance. Changes from baseline will be analyzed per participant and time point.

The Dimensional Change Card Sort Test assesses cognitive flexibility. Scores are age-adjusted T-scores (mean 50, SD 10). Higher scores indicate better executive function. Changes from baseline will be analyzed per participant and time point.

Picture Vocabulary Test measures receptive vocabulary. Results are expressed as age-adjusted T-scores (mean 50, SD 10), with higher scores indicating better performance. Change from baseline will be analyzed per participant and time point.

Whole-blood DNA methylation will be measured using bisulfite sequencing performed by Generation Lab. Epigenetic biological age will be computed from these methylation data using validated algorithms. Age estimates will be expressed in years. Lower epigenetic age relative to chronological age indicates younger biological status.

Duration (seconds) that the participant can maintain an unsupported one-leg stance on the left and right leg separately, measured from the point of foot lift until loss of balance or foot contact with the ground.

Total number of full squats the participant can complete with proper form within a continuous, unassisted trial, measured from the start of movement until the participant stops or is unable to continue.

Total number of standard push-ups completed with proper form in a continuous trial, counted from the first repetition until the participant stops or is unable to continue.

Total number of sit-ups the participant can complete with proper form in a continuous trial, measured from the first repetition until the participant stops or is unable to continue.

hs-CRP will be measured using an immunoturbidimetric assay and expressed in mg/L. hs-CRP is a marker of systemic inflammation and cardiovascular risk.

Ionized calcium will be measured using an ion-selective electrode analyzer. Results will be reported in millimoles per liter (mmol/L) and represent physiologically active calcium fraction.

Serum Cystatin C will be measured using a standardized immunoassay and reported in mg/L for each participant and time point. Cystatin C is a biomarker of glomerular filtration rate and provides a mechanistic link between α-Klotho activity and renal function.

Serum inorganic phosphorus will be measured on a standard clinical chemistry analyzer. Results will be reported in mg/dL for each participant and time point. Phosphorus levels reflect systemic phosphate homeostasis influenced by α-Klotho and FGF23 activity.

Urinary phosphate will be assessed using a validated chemistry assay. Will be expressed either as total phosphate excretion in milligrams per 24 hours (mg/24 h) or as the phosphate-to-creatinine ratio (mg phosphate per g creatinine). This measure reflects renal handling of phosphate and functional effects of α-Klotho on phosphate excretion.

Intact Parathyroid Hormone (PTH) will be measured in serum using a two-site immunoassay that detects the full-length molecule. Results will be reported in picograms per milliliter (pg/mL) per participant and time point. PTH reflects parathyroid activity within the α-Klotho-FGF23-vitamin D feedback pathway.

Serum 1,25-dihydroxyvitamin D (calcitriol) will be quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations will be expressed in picograms per milliliter (pg/mL). This hormone regulates calcium and phosphate balance and is a downstream marker of α-Klotho-FGF23-PTH axis modulation.

A standard comprehensive metabolic panel (CMP) will be collected to evaluate general metabolic and organ function for safety monitoring. The standard panel includes 14 markers: Glucose, calcium, sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatinine (serum), albumin, total protein, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and total bilirubin. Individual components will be assessed clinically to ensure no major changes to health stability but these will not analyzed as specific study endpoints.

Platelet Factor 4 (PF4) will be measured using a validated ELISA and reported as nanograms per milliliter (ng/mL) or optical density (OD) units. PF4 is a marker of platelet activation and potential immune-mediated coagulation effects.

Fibrinogen antigen concentration will be measured by nephelometry using the Cardio IQ® assay. Reported in milligrams per deciliter (mg/dL).

Hemoglobin A1c (HbA1c) will be measured by standardized assay methods and reported as percent (%). Estimated average glucose (eAG) will be reported alongside HbA1c for interpretability only and will be derived from the HbA1c value using a validated conversion formula. eAG is not independently measured. Change from baseline HbA1c will be reported as a percentage and the corresponding eAG value will be included for interpretability.

Plasma homocysteine concentration will be measured by immunoassay or chromatography-based methods and reported in micromoles per liter (µmol/L).

Plasma ADMA and SDMA will be quantified by mass spectrometry-based assay and reported in micromoles per liter (µmol/L).

Serum IL-6 levels will be measured by high-sensitivity immunoassay and reported in picograms per milliliter (pg/mL).

Serum TNF-α concentration will be measured by immunoassay and reported in picograms per milliliter (pg/mL).

Serum IL-1β levels will be quantified using a cytokine immunoassay and reported in picograms per milliliter (pg/mL).

Plasma IFN-γ concentration will be measured using an immunoassay and reported in picograms per milliliter (pg/mL).

Serum IL-10 levels will be measured by cytokine immunoassay and reported in picograms per milliliter (pg/mL).

Serum myostatin concentration will be measured using a validated immunoassay and reported in nanograms per milliliter (ng/mL).

Serum activin A will be quantified by immunoassay and reported in picograms per milliliter (pg/mL).

Serum IGF-1 will be measured using a standardized immunoassay with age-adjusted calibration and reported in nanograms per milliliter (ng/mL).