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COMPETITIVENESS OPERATIONAL PROGRAMME 2014-2020, PRIORITY AXIS 1, ACTION 1.1.4

CiPA3: Development of automated patch-clamp technologies for pro-arrhythmogenic risk testing of drug candidates

PROJECT    P_37_675   MySMIS code: 106926    BENEFICIARY: UNIVERSITY OF BUCHAREST, FACULTY OF BIOLOGY


The general objective of our project is to transform the „CiPA paradigm” into a robust highly reliable and reproducible industrial standard.

Specific objectives:

– development of methods to overcome hiPSC-CM preparations immaturity in Ca2+ handling and limit effects of L-type Ca2+ current inactivation, via transient overexpression of SERCA/PMCA Ca2+ pumps and/or improved cytoplasmic Ca2+ buffering with soluble calmodulin subunits, or other methods

– use of these enhanced hiPSC-CM preparations to overcome the problems encountered with cardiac ion channel screening in heterologous cell lines, at least for some important ion current components (peak and late Na+ current, L-type Ca2+ current, fast transient outward K+ current, myocardial inward rectifier K+ current (IK1), „funny” hyperpolarization-activated current)

– development of assays for measuring pharmacological effects for an extended panel of ion channels and transporters, in natural conditions and an environment whereby all auxiliary subunits and regulatory mechanisms are present

– development and refinement of a proprietary (patented) assay on hiPSC-CM combining current-clamp and voltage-clamp protocols aimed to measure as many as possible distinct ion current components in physiological conditions, with physiological internal and external solutions, in the same cell, without pharmacological interventions, and in addition to study complex cell electrophysiology properties, such as the range of externally applied steady current that elicits pacemaking or APD restitution properties

– development of an advanced humanized hiPSC-CM electrophysiology model suitable for pharmacology assays (using Markov-type kinetic models for several ion current components)

– development of suitable parallel/distributed computing algorithms for fast numeric integration of the model

– development of optimization algorithms to use data obtained in real experiments on hiPSC-CM with the CytoPatch automated patch-clamp platform to quickly tune model parameters such as to match the experimentally recorded AP shape and additional features in (almost) real time

– use within the same experiment of dynamic clamp protocols to validate the model and assess pharmacological effects of drug candidates via restoration of AP shape with electronically generated currents applied at the current clamp input of the CytoPatch amplifier.

Some preliminary results are presented in the accompanying images.


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