Engineered stem cells and cellular products produced and secreted by such cells, methods of preparing, and uses thereof
Inventors
VIGNEAULT, Patrick • Davis, Darryl • Nattel, Stanley • PARENT, Sandrine
Assignees
L'institut De Cardiologie De Montreal • University of Ottawa
Publication Number
US-11795434-B2
Publication Date
2023-10-24
Expiration Date
2039-11-08
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Abstract
There is disclosed a cardiac explant-derived stem cell (EDC), the cell comprising a gene encoding an intermediate-conductance Ca2+-activated K+ channel, and wherein the gene causes an overexpression of the intermediate-conductance Ca2+-activated K+ channel, and methods of producing same. There is also disclosed a method of producing engineered EDCs having a modulated bioelectric property, the method comprising: obtaining EDCs; introducing a KCNN4 gene into the EDCs to increase the expression of KCa3.1 channels, to produce engineered EDCs. There is also disclosed a composition for treating or ameliorating a damaged myocardium in a subject, the composition comprising extracellular vesicles isolated from cultures of engineered EDCs. There is also disclosed a method for treating or ameliorating a damaged myocardium in a subject, comprising administering the engineered EDCs or the extracellular vesicles isolated from cultures of engineered EDCs to the damaged myocardium of the subject.
Core Innovation
The invention discloses engineered cardiac explant-derived stem cells (EDCs) comprising a gene encoding an intermediate-conductance Ca2+-activated K+ channel, specifically the KCNN4 gene encoding the KCa3.1 channel, causing overexpression of this channel. This overexpression modulates the bioelectric properties of the EDCs, leading to a hyperpolarized transmembrane potential, enhanced Ca2+ signaling, increased intracellular Ca2+ concentration, and expanded paracrine repertoire. These properties direct stem cell functions including proliferation, differentiation, cytokine secretion, and extracellular vesicle production.
The problem addressed is the modest and inconsistent improvement in cardiac function following adult stem cell therapy for heart failure, due to limited reparative potential of transplanted cells. There is a need for improved methods and compositions that enhance repair and cardiac function post-injury, such as from myocardial infarction. The invention proposes modulation of ionic flows and bioelectric properties in stem cells to overcome decreased expression levels of the intermediate-conductance Ca2+-activated K+ channel, improving therapeutic efficacy through increased cell proliferation, paracrine signalling, and myocardial repair.
Claims Coverage
The patent contains multiple independent claims focusing on engineered adult cardiac explant-derived stem cells (EDCs), methods of producing these cells, methods of producing extracellular vesicles, and medicaments for treating damaged myocardium. The main inventive features center around the genetic modification to overexpress the intermediate-conductance Ca2+-activated K+ channel (KCNN4/KCa3.1) and the resulting functional benefits.
Engineered adult cardiac explant-derived stem cell with overexpressed intermediate-conductance Ca2+-activated K+ channel
An adult cardiac explant-derived stem cell (EDC), specifically CD90−, comprising a gene encoding the intermediate-conductance Ca2+-activated K+ channel (KCNN4/KCa3.1), wherein the gene causes overexpression of this channel.
Functional modulation by KCNN4 gene expression in EDCs
The gene is configured to drive expression of the KCa3.1 channel to hyperpolarize EDC membrane, enhance Ca2+ signaling, increase intracellular Ca2+ concentration, expand the paracrine repertoire relative to controls, decrease resting membrane potential, maintain electrical gradient for Ca2+ influx, increase relevant miRNA, increase extracellular vesicle production, and increase secretion of cytokines such as VEGF, angiogenin, IGFBP3, SDF-1α, and ICAM-1.
Method of treating damaged myocardium by administering engineered EDCs
Administering the engineered adult cardiac explant-derived stem cells of the invention to the damaged myocardium of a subject to promote angiogenesis, cytokine production, post-infarct healing, immunomodulation, cardiomyocyte proliferation/salvage, oxidative stress protection, fibrosis reduction, and cell engraftment.
Method of producing engineered EDCs with modulated bioelectric properties
Obtaining adult cardiac explant-derived stem cells, introducing the gene encoding the intermediate-conductance Ca2+-activated K+ channel (KCNN4/KCa3.1) to increase its expression, and selecting for CD90− cells, resulting in engineered EDCs with hyperpolarized membrane and enhanced properties including increased proliferation, cytokine expression, extracellular vesicle number, miRNA associated with myocardial repair, and relevant extracellular vesicle contents.
Method of producing extracellular vesicles for treatment
Culturing engineered adult cardiac explant-derived stem cells in ischemic-like conditions (e.g., low oxygen and absence of growth factors) to produce extracellular vesicles enriched with cytokines and miRNAs implicated in myocardial repair, followed by isolation of these extracellular vesicles for therapeutic administration.
Pharmaceutical compositions and medicaments comprising engineered cells or extracellular vesicles
Compositions comprising the engineered EDCs, extracellular vesicles produced by these cells, or conditioned cell culture media, optionally with pharmaceutically acceptable carriers, for therapeutic use in damaged myocardium.
The claims collectively define engineered CD90− adult cardiac explant-derived stem cells overexpressing the KCNN4 gene encoding KCa3.1 channels, methods to produce these engineered cells with enhanced bioelectric and paracrine properties, methods to produce extracellular vesicles with therapeutic factors, and therapeutic compositions and methods for treating damaged myocardium with such cells or their secreted products.
Stated Advantages
Engineered EDCs with overexpressed KCa3.1 show increased proliferation and enhanced paracrine profiles leading to improved therapeutic potential.
Improved cardiac function through enhanced angiogenesis, cardiomyogenesis, myocardial salvage, and immunomodulation without increasing the risk of malignant cardiac arrhythmias.
Increased long-term engraftment and retention of transplanted cells in the myocardium.
Broadening of cytokine secretion and increased extracellular vesicle production containing beneficial cytokines and miRNAs.
Documented Applications
Treatment or amelioration of damaged myocardium, such as post-myocardial infarction, via administration of engineered adult cardiac explant-derived stem cells or extracellular vesicles derived therefrom.
Therapeutic use in heart failure, coronary artery disease, ischemic heart disease, and related cardiovascular conditions.
Administration routes include intra-myocardial injection, myocardial infusion (intra-arterial or intra-venous), or parenteral delivery.
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