ENGINEERING GENOME-BASED THERAPEUTICS FOR SELECTIVE ELIMINATION OF SENESCENT HUMAN CELLS

Abstract

Background: Cellular senescence plays a major role in aging-related diseases by irreversible cell-cycle arrest as well as secretion of pro-inflammatory senescence-related secretory phenotype (SASP) factors. Current senolytic therapies are often non-specific and damaging to healthy proliferating cells.

Objective: To develop a genome-based therapeutic platform to selectively detect and kill senescent human cells employing programmable CRISPR-associated genetic circuits.

Methodology: Human fibroblast and epithelial cell models of senescence were induced by replicative exhaustion and doxorubicin-mediated DNA damage. Biomarkers of senescence including p16INK4a and p21CIP1 were profiled by qRT-PCR and RNA-seq . Here, we have engineered a synthetic CRISPR-Cas therapeutic circuit coupled to senescence-responsive promoters to achieve selective induction of apoptosis in senescent cells. Therapeutic efficacy and specificity were tested by functional assays, flow cytometry and cytokine profiling.

Results: The engineered system efficiently and selectively removed approximately 82% of senescent cells while maintaining viability in non-senescent controls at over 93%. Treatment reduced the levels of SASP-associated cytokines IL-6 and IL-8 by 72% and 65%, respectively. There was negligible off-target genome editing activity.

Conclusion: Genome-engineered senolytic therapeutics are highly promising for the specific targeting of senescent human cells and may offer a promising therapeutic approach for the treatment of aging-associated diseases and the enhancement of tissue regeneration.