Development of First-in-Class Gene Therapies to Treat Non-Small Cell Lung Cancer (NSCLC)
Abstract
Rationale: Gene therapy transfers genetic material into cells, enabling them to ‘manufacture’ proteins of interest to treat diseases, like cystic fibrosis, but also non-genetic diseases, such as age-related macular degeneration; whereby cells are instructed to manufacture and secrete anti-inflammatory proteins. Gene therapy approaches can be used to convert cells into manufacturing units for therapeutic peptides. Peptides with good drug-like properties but shortcomings in absorption, posology, and dose-scheduling would significantly benefit from this approach. We developed peptide drugs (PEPDRGs) which target the novel, untouched, intracellular protein TMBIM6. TMBIM6 plays an integral role in many diseases; in oncology, TMBIM6 is overexpressed in many cancers with its knockdown inducing rapid cancerous cell death. Prior assessment of PEPDRG on treating cancers showed >50% paraptotic cell death in 24 hours of treatment, rising to >92% in 96 hours across all cancer cell lines tested. PEPDRGs are highly soluble in solvents; blood and urine and do not precipitate/aggregate even at high concentrations (>100 mg/ml) or under physiological stress conditions. PEPDRGs follow a two-compartment pharmacokinetic model and have excellent in vivo stability (85-93% remaining in the body after 24 hours), low clearance (4.1-9.3 ml/min) and efficacy. In vivo breast and ovarian cancer models showed PEPDRG induces >90% tumor shrinkage within 25 days with no measurable/observable toxicity or immunogenicity. However, the distribution phase in the two-compartment PK model reduces PEPDRG blood concentrations below its EC50, requiring frequent, daily dosing to reach an effective concentration. We propose using gene therapy approaches to convert epithelial/muscle cells, via direct DNA transfer, into PEPDRG manufacturing and secreting cells. We piloted this approach by transfecting cultured epithelial cells (IEC-6) with a PEPDRG gene therapy vector, which showed PEPDRGs could indeed be manufactured and secreted, without toxicity or deleterious effect; furthermore, secreted PEPDRG transferred to wells containing cancerous cells (MCF-7)-induced paraptotic cell death. We believe this novel approach would generate breakthrough technology for NSCLCs, which overexpress TMBIM6, and TMBIM6 is shown to be important in NSCLC survival, growth, and spread. This approach could negate burdensome multi-dosing, intravenous treatment schedules; minimize hospitalization; improve survivability; and enable round-the-clock cancer treatment. Objectives: Aim 1 (1 month): Produce 20 PEPDRG constructs. Aim 2 (1.5 months): Assess PEPDRG production, secretion, and impact on cell viability. Transfect PEPDRG constructs into epithelial or muscle cells to assess levels of PEPDRG production, secretion, extra-cellular stability, and impact on manufacturing cell viability. Aim 3 (3 months): In vitro assessment of secreted PEPDRG on NSCLC cell viability over a 7-day period. Identify secreted PEPDRGs that induce cell death in NSCLC cell lines and assess MOA for paraptosis. Methods: Aim 1: Manufacture 20 gene therapy constructs with different PEPDRGs and N-terminal secretion signals for delivery using non-viral vector approaches. Potential Pitfalls: None expected. Aim 2: PEPDRG constructs will be transfected into epithelial (bronchial/tracheal ATCC: PCS-300-010) and intestinal (ATCC: CCD 841 CoN and HIEC-6) or skeletal muscle (ATCC: HSkMC) cells, and a time series analysis (7 days) will be performed to determine PEPDRG production (secreted vs. un-secreted), extra-cellular stability, and cytotoxicity. Potential Pitfalls: Secretion levels may be too low or production too high, resulting in aggregation; both can be overcome by modifications to the promoter region or secretion signal. Pilot data indicated cytotoxicity was not an issue. Aim 3: ATCC lung cancer panel TCP-1016 will be seeded grown until 70-80% confluent. PEPDRG effi
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310001
Entities
People
- Keith Robinson
Organizations
- United States Army