Abstract: FR-PO230

CRISPR-Cas9-Induced Expression of Endogenous APOL1-G0 Reduced Cytotoxicity of Renal Risk Variant APOL1-G1

Session Information

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Authors

  • Olabisi, Opeyemi A., Massachusetts General Hospital, Boston, Massachusetts, United States
  • Moore, Savannah, Massachusetts General Hospital, Brookline, Massachusetts, United States
  • Pollak, Martin R., Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, United States
Background

Two protein coding mutations in the APOL1 gene account for much of the excess risk of non-diabetic end stage kidney disease (ESRD) among individuals of recent African Ancestry. These 2 common mutations (named G1- missense mutation, and G2- 2 amino acid deletion), collectively referred to as renal risk variant (RRV) APOL1 increase the risk of FSGS, HIVAN and hypertension-associated ESRD. The pattern of inheritance of APOL1-nephropathy is recessive--the disease is most consistently seen in African Americans with 2 copies of RRV APOL1 relative to those with zero or 1 copy of the risk allele. Yet, strong evidence suggests that cytoxicity of RRV APOL1 is due to gain of function. Expression of both G1 or G2 APOL1 result in toxicity in HEK-293 cells, human and mouse podocytes. We predict that induced cellular expression of wildtype APOL1 (G0) would compete with and reduce cytotoxicity of RRV APOL1 in HEK-293 cells.

Methods

We generated HEK-293 cell line that stably express APOL1-G1 in the presence of exogenous tetracycline. Transient transfection of these stable HEK-293 cells with CRISPR-transcription activator complex(dCas9-Vp64, p65-HSF1, and guide RNA specific for APOL1 promoter) induced robust expression of endogenous APOL1-G0. We then measured cytotoxicity of APOL1-G1 in the presence or abscence of these induced APOL1-G0.

Results

In the presence of APOL1-G0, the cytotoxicity of APOL1-G1 is significantly reduced up to 50%.

Conclusion

This result suggests that the cytoxicity of renal risk variants APOL1-G1 could be competitively be reduced by wild type APOL1-G0. This may explain in part why the risk of APOL1-nephropathy is negligible among individuals who carry at least one copy of wild type APOL1-G0, but high among individuals with 2 copies of RRV. Also, this finding suggests that differential upregulation of APOL1-G0 or downregulation of RRV APOL1 in podocytes may be a useful have therapeutic intervention.

Funding

  • Private Foundation Support