Cardiotoxicity screening methods

• US 10,048,275 B2
• Filed: 03/11/2016
• Issued: 08/14/2018
• Est. Priority Date: 03/13/2015
• Status: Active Grant

• Current Assignee: Quelltx Incorporated
• Original Assignee: Q-State Biosciences, Inc.
• Inventors: Kralj, Joel, Dempsey, Graham, Werley, Christopher, Cohen, Adam
• Primary Examiner(s): Singh, Satyendra K
• Application Number:
  US15/067,610
• Publication Number:
  US 20160266144A1
• Time in Patent Office: 886 Days
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2800/326 Arrhythmias, e.g. ventricular fibrillation, tachycardia, atrioventricular block, torsade de pointes
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

Screening compounds by exposing a plurality of cardiomyocytes to a compound, wherein the cardiomyocytes express an optogenetic reporter of membrane potential and an optogenetic reporter of calcium level; receiving light from the optogenetic reporter of membrane potential; creating an AP waveform using the received light; and analyzing the AP waveform to determine the presence or absence of a risk for arrhythmia associated with the compound.

Claims

1. A cardiomyocyte expressing:

at least one microbial rhodopsin as an optogenetic reporter of voltage activity, wherein the microbial rhodopsin is one selected from the list consisting of QuasAr1 and QuasAr2;

a light-gated ion channel as an optogenetic actuator of electrical activity; and

a genetically encoded calcium indicator as an optogenetic reporter of calcium level.

2. The cardiomyocyte of claim 1, wherein the genetically encoded calcium indicator comprises a GCaMP variant.

3. The cardiomyocyte of claim 2, wherein the genetically encoded calcium indicator is one selected from the list consisting of jRCaMP1a, jRGECO1a and RCaMP2.

4. The cardiomyocyte of claim 3, wherein the light-gated ion channel comprises a blue-shifted actuator and the genetically encoded calcium indicator comprises a red-shifted calcium indicator.

5. The cardiomyocyte of claim 4, wherein the light-gated ion channel comprises an algal channelrhodopsin.

6. The cardiomyocyte of claim 5, wherein the light-gated ion channel comprises a blue-shifted actuator that is one selected from the list consisting of CheRiff, TsChR, and PsChR.

7. The cardiomyocyte of claim 6, wherein the microbial rhodopsin and the genetically encoded calcium indicator are expressed together as part of a fusion protein.