CD46 knock-out using CRISPR/Cas9 editing of hTERT immortalized human cells modulates complement activation

Abstract

The kidney is especially sensitive to diseases associated with overactivation of the complement system. While most of these diseases affect kidney glomeruli and the microvasculature, there is also evidence for tubulointerstitial deposition of complement factors. Complement inactivating factors on cell membranes comprise CD55, CD59 and CD46, which is also termed membrane cofactor protein (MCP). CD46 has been described as localized to glomeruli, but especially also to proximal tubular epithelial cells (RPTECs). However, human cell culture models to assess CD46 function on RPTECs are still missing. Therefore, we here performed gene editing of RPTEC/TERT1 cells generating a monoclonal CD46 -/- cell line that did not show changes of the primary cell like characteristics. In addition, factor I and CD46-mediated cleavage of C4b into soluble C4c and membrane deposited C4d was clearly reduced in the knock-out cell line as compared to the maternal cells. Thus, human CD46 -/- proximal tubular epithelial cells will be of interest to dissect the roles of the epithelium and the kidney in various complement activation mediated tubulointerstitial pathologies or in studying CD46 mediated uropathogenic internalization of bacteria. In addition, this gives proof-of-principle, that telomerized cells can be used in the generation of knock-out, knock-in or any kind of reporter cell lines without losing the primary cell characteristics of the maternal cells.

Gene targeting as a therapeutic avenue in diseases mediated by the complement alternative pathway

“…Application of CRISPR technology to the complement field is still in early development, although reports have been published on a human CD46, CD55, and CD59 knockout HAP1 cell line, a CD46 knockout hTERT cell line, and a C3‐deficient pig generated using CRISPR/Cas9. 37 , 38 , 39 In June 2021, Apellis Pharmaceuticals and Beam Therapeutics announced a partnership to develop up to six gene editing programs targeting C3 and other complement proteins for the treatment of diseases of the eye, liver, and brain (Press release: Apellis and Beam Therapeutics Enter Exclusive Research Collaboration to Apply Base Editing to Discover Novel Therapies for Complement‐Driven Diseases. Published June 30, 2021, on www.apellis.com ).…”

Complement C4, Infections, and Autoimmune Diseases

“…protease factor I together with co-factor CD46 (14,15). iC4b is further cleaved to thioester linked C4d (45 kDa) and soluble C4c (146 kDa), which can be used as a biomarker for complement activation from classical and lectin pathways.…”

Complement Biosensors Identify a Classical Pathway Stimulus in Complement-Mediated Hemolytic Uremic Syndrome

“…Our study highlights many important observations on the role of complement and IgM in disease pathophysiology. Although most literature focuses on their role as AP regulators, there is also evidence that FH and CD46 mitigate classical pathway activity [34][35][36][37][38][73][74][75] . Our data suggest that in CM-HUS the role of CD46 in regulating classical activity is more important than its role in controlling the amplification loop.…”