Safety and Efficacy of Injectable Klotho Plasmid Gene Therapy in Humans

Serum α-Klotho protein concentration will be quantified using a validated enzyme-linked immunosorbent assay (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.

Fibroblast Growth Factor 23 (FGF23) concentration will be quantified in serum using a validated enzyme-linked immunosorbent assay (ELISA). Results will be expressed in picograms per milliliter (pg/mL) for each participant and time point. FGF23 is a downstream effector of α-Klotho signaling and reflects activity of the phosphate-vitamin D regulatory axis.

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.

Serum inorganic phosphorus will be measured on a standard clinical chemistry analyzer. Results will be reported in milligrams per deciliter (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. Results 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.

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

The World Health Organization Quality of Life Brief Version (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 reported for each domain separately.

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.

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.

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.

The Reynolds Intellectual Assessment Scales-Second Edition (RIAS-2) measures verbal, nonverbal, and composite intelligence. Each index yields a standard score (mean 100, SD 15) and subtests yield scaled scores (mean 10, SD 3). Higher scores indicate better cognitive performance. Changes from baseline will be reported for each selected index separately.

Serum glucose will be measured using a standard clinical chemistry analyzer and expressed in milligrams per deciliter (mg/dL). Elevated glucose may indicate altered carbohydrate metabolism or metabolic stress.

Total calcium will be quantified using a colorimetric method on a clinical chemistry analyzer. Results will be expressed in milligrams per deciliter (mg/dL). Calcium levels reflect bone-mineral balance and parathyroid function.

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.

Sodium concentration will be measured using an ion-selective electrode analyzer and expressed in millimoles per liter (mmol/L). Sodium reflects hydration and electrolyte balance.

Serum potassium will be quantified using an ion-selective electrode analyzer and expressed in millimoles per liter (mmol/L). Potassium levels reflect renal and electrolyte homeostasis.

Serum chloride will be measured using an ion-selective electrode analyzer. Results will be expressed in millimoles per liter (mmol/L) and reflect acid-base and hydration status.

Serum bicarbonate will be measured as total CO₂ on a clinical chemistry analyzer. Results will be expressed in millimoles per liter (mmol/L) and indicate systemic acid-base balance.

Blood urea nitrogen (BUN) will be measured using a standard enzymatic assay and expressed in milligrams per deciliter (mg/dL). BUN reflects renal function and nitrogen metabolism.

Serum creatinine will be measured using a validated enzymatic method on a clinical chemistry analyzer. Results will be reported in milligrams per deciliter (mg/dL) and serve as an indicator of renal filtration.

Serum albumin will be quantified using a bromocresol green dye-binding method and expressed in grams per deciliter (g/dL). Albumin reflects nutritional status and hepatic synthetic function.

Total protein will be measured using the biuret colorimetric assay and expressed in grams per deciliter (g/dL). This parameter provides an overview of plasma protein balance.

Serum alkaline phosphatase will be quantified by an enzymatic colorimetric method and expressed in international units per liter (IU/L). ALP reflects hepatobiliary and bone function.

Serum ALT activity will be measured enzymatically and expressed in international units per liter (IU/L). ALT serves as a marker of hepatocellular injury.

Serum AST activity will be determined by an enzymatic method and expressed in international units per liter (IU/L). AST reflects hepatic and, to a lesser extent, cardiac or muscle integrity.

Total bilirubin will be measured using a diazo-based colorimetric assay and expressed in milligrams per deciliter (mg/dL). Bilirubin serves as an indicator of liver function and hemolysis.

High-sensitivity C-reactive protein will be measured using an immunoturbidimetric assay and expressed in milligrams per liter (mg/L). hs-CRP serves as a marker of systemic inflammation and cardiovascular risk.

Asymmetric dimethylarginine (ADMA) will be quantified using LC-MS/MS and expressed in micromoles per liter (µmol/L). ADMA reflects endothelial function and nitric oxide metabolism.

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

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.

Resting-state cerebral hemodynamic activity will be measured using the Kernel Flow headset, which integrates time-domain functional near-infrared spectroscopy (TD-fNIRS) and behavioral data. Kernel's proprietary machine learning algorithms will process whole-brain optical signals to generate an Estimated Brain Age, expressed in years. The metric represents the predicted biological brain age relative to chronological age, derived from composite models trained on resting-state fNIRS and behavioral features. Lower Estimated Brain Age values indicate more youthful brain function. Data will be reported per participant at baseline and after treatment.

Cognitive performance will be assessed using Kernel Flow's composite metrics derived from time-domain functional near-infrared spectroscopy (TD-fNIRS) and brief behavioral tasks. Domain-specific percentile scores will be generated for Complex Attention, Executive Function, Learning and Memory, Language, and Perceptual-Motor Control, as well as an overall Cognitive Performance Index. Each score reflects age-normed percentile performance, integrating brain and behavioral features using Kernel's proprietary machine learning models. Higher percentiles indicate better cognitive performance relative to age-matched norms. Metrics will be reported per participant at baseline and after treatment.