Developing and validating a podocyte cell-based diagnostic assay for identifying recurrent focal and segmental glomerulosclerosis patients

Diagnosing glomerular diseases accurately and in timely fashion is key to developing a successful treatment plan and thereby prevent their progression to ESRDend stage renal diseaseHowevertheir successful diagnosis remains challenging due to the complex diagnostic procedures used including invasive renal biopsiesThusthere is a compelling need to develop simplified procedures to detect glomerular disease patients with high accuracy and efficiencyThe primary goal of this STTR proposal is to develop a novel cell based assay that will serve as a non invasive diagnostic clinical tool to detect a form of glomerular disease commonly known as recurrent focal and segmental glomerulosclerosisrFSGSImportantlythis revolutionary concept can be extended to develop similar assays for other glomerular diseases and thereforereduce or eliminate the need for renal biopsiesThis will be the long term goal of our company InDepth pharmaceuticalsAlthough there are multiple etiologies for FSGSdysfunction of podocytes leading to cell death and proteinuria are primary outcomes of all forms of FSGSThis Phase I study involves development of an assay that will specifically diagnose rFSGS patients in whichFSGS recurs following renal transplant within hours to weeks and affects more than a third of FSGS patientsThustimely diagnosis of rFSGS patients will prevent ineffective renal transplants that are destined to failSince FSGS targets podocyte damage and deathour unique approach involved mRNA profiling of cultured podocytes treated with rFSGS patient plasma to reveal upregulated genes involved in cellular damageBased on maximal upregulation from the profiling datawe selected three proapoptotic candidate rFSGS responsive genes ILBMFand IGFBPthat were specifically elevated in rFSGS patient plasma treated podocytestheir promoter regions were identified and cloned into a luciferase based reporter vector and transfected into podocytes HEKto generate stable cell linesStrikinglywhen these cell lines were exposed to plasma from rFSGS patientsincreased reporter activity was notedin contrastno reporter activity was noted with all the other glomerular disease patients testedRemarkablythe statistical analysis showed more thanspecificity in detecting rFSGS patientsTo further test our concept and enhance the specificity and robustness of our assayin the Specific Aimusing the identified gene promoters we will construct cell lines that show lower variability and enhanced responsiveness to rFSGS plasmaby employing two parallel approachesincluding a CRSPR Cas system for directed insertion of promoter reporter construct at the Safe Harbor site AAVSlocus PPP RCin cellular genome and an alternate approachthat involves conventional transfection followed by clonal selection to identify clone s with highest magnitude of inductionIn the Specific Aimwe will conduct initial validation of constructed cell lines by measuring their responses towards plasma from various nephropathy patientsSeveral rFSGS and non rFSGS patient plasma are available through various indigenous and collaborative efforts between MUSC and InDepth pharmaceuticals to power this study Project Narrative This study uses a novel approach to develop diagnostic kit for detecting recurrent FSGS patientsthereby preventing immediate loss of renal graft following renal transplant in FSGS patientsImportantlythis revolutionary approach can widely be used in developing similar assays for several other glomerular diseases