scholarly journals Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jared Carlson-Stevermer ◽  
Amritava Das ◽  
Amr A. Abdeen ◽  
David Fiflis ◽  
Benjamin I Grindel ◽  
...  
Keyword(s):  
Cell Model ◽  
Induced Pluripotent Stem Cell ◽  
Human Infant ◽  
Design Parameters ◽  
Compound Heterozygous ◽  
Gene Correction ◽  
Multiple Alleles ◽  
Induced Pluripotent ◽  
Polygenic Diseases

AbstractCompound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

scholarly journals Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases

2020 ◽  
Author(s):  
Jared Carlson-Stevemer ◽  
Amritava Das ◽  
Amr Abdeen ◽  
David Fiflis ◽  
Benjamin Grindel ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Human Infant ◽  
Design Parameters ◽  
Compound Heterozygous ◽  
Gene Correction ◽  
Multiple Alleles ◽  
Cell Genome ◽  
Polygenic Diseases

Abstract Gene correction of multiple alleles for compound heterozygous recessive or polygenic diseases is a promising therapeutic strategy. However, the targeting of multiple alleles using genome editors in a single cell could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that SpyCas9-based S1mplex genome editors can be designed and developed to correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in a patient-derived, induced pluripotent stem cell (iPSC) model of Pompe disease robustly expressed the corrected transcript from both corrected alleles. The translated protein from the gene-corrected cells was properly processed after translation and was able to enzymatically cross-correct diseased cells at levels equivalent to standard-of-care, enzyme replacement therapy (ERT). Using a novel in silico model for the in vivo delivery of these and many other genome editors into a developing liver of a human infant, we identify progenitor cell targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters controlling the potency and efficacy of in vivo somatic cell genome editing. Both single and double gene correction are efficacious for in vivo somatic cell editing strategies, while double gene correction is more effective than single-gene correction for autologous cell therapy with ex vivo gene-corrected cells. This work establishes that precise gene correction using genome editors to correct multiple distinct gene variants could be efficacious in the treatment of recessive and polygenic disorders.

scholarly journals Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC

2021 ◽  
Vol 10 (14) ◽  
pp. 3061
Author(s):  
Robert N. Hawthorne ◽  
Adriana Blazeski ◽  
Justin Lowenthal ◽  
Suraj Kannan ◽  
Roald Teuben ◽  
...  
Keyword(s):  
Stem Cell ◽  
Structural Changes ◽  
Cell Model ◽  
Arrhythmogenic Right Ventricular Cardiomyopathy ◽  
Induced Pluripotent Stem Cell ◽  
Cytokine Expression ◽  
Calcium Handling ◽  
Disease Manifestation ◽  
Time To Peak ◽  
Induced Pluripotent

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive heart condition which causes fibro-fatty myocardial scarring, ventricular arrhythmias, and sudden cardiac death. Most cases of ARVC can be linked to pathogenic mutations in the cardiac desmosome, but the pathophysiology is not well understood, particularly in early phases when arrhythmias can develop prior to structural changes. Here, we created a novel human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of ARVC from a patient with a c.2358delA variant in desmoglein-2 (DSG2). These DSG2-mutant (DSG2Mut) hiPSC-CMs were compared against two wildtype hiPSC-CM lines via immunostaining, RT-qPCR, Western blot, RNA-Seq, cytokine expression and optical mapping. Mutant cells expressed reduced DSG2 mRNA and had altered localization of desmoglein-2 protein alongside thinner, more disorganized myofibrils. No major changes in other desmosomal proteins were noted. There was increased pro-inflammatory cytokine expression that may be linked to canonical and non-canonical NFκB signaling. Action potentials in DSG2Mut CMs were shorter with increased upstroke heterogeneity, while time-to-peak calcium and calcium decay rate were reduced. These were accompanied by changes in ion channel and calcium handling gene expression. Lastly, suppressing DSG2 in control lines via siRNA allowed partial recapitulation of electrical anomalies noted in DSG2Mut cells. In conclusion, the aberrant cytoskeletal organization, cytokine expression, and electrophysiology found DSG2Mut hiPSC-CMs could underlie early mechanisms of disease manifestation in ARVC patients.

scholarly journals Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Peinkofer ◽  
Martina Maass ◽  
Kurt Pfannkuche ◽  
Agapios Sachinidis ◽  
Stephan Baldus ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Developmental Stage ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

scholarly journals hiPSC-derived 3D Bioprinted Skeletal Muscle Tissue Implants Regenerate Skeletal Muscle Following Volumetric Muscle Loss

2021 ◽  
Author(s):  
Yasamin A. Jodat ◽  
Ting Zhang ◽  
Ziad Al Tanoury ◽  
Tom Kamperman ◽  
Kun Shi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
De Novo ◽  
Induced Pluripotent Stem Cell ◽  
Muscle Loss ◽  
Repair Capacity ◽  
Volumetric Muscle Loss ◽  
Induced Pluripotent ◽  
Long Fibers ◽  
Regenerative Therapies

Abstract Engineering of biomimetic tissue implants provides an opportunity for repairing volumetric muscle loss (VML), beyond a tissue’s innate repair capacity. Here, we present thick, suturable, and pre-vascularized 3D muscle implants containing human induced pluripotent stem cell-derived myogenic precursor cells (hiPSC-MPCs), which can differentiate into skeletal muscle cells while maintaining a self-renewing pool. The formation of contractile myotubes and millimeter-long fibers from hiPSC-MPCs is achieved in chemically, mechanically, and structurally tailored extracellular matrix-based hydrogels, which can serve as scaffolds to ultimately organize the linear fusion of myoblasts. Embedded multi-material bioprinting is used to deposit complex patterns of perfusable vasculatures and aligned hiPSC-MPC channels within an endomysium-like supporting gel to recapitulate muscle architectural integrity in a facile yet highly rapid manner. Moreover, we demonstrate successful graft-host integration and de novo muscle formation upon in vivo implantation of pre-vascularized constructs within a VML model. This work pioneers the engineering of large pre-vascularized hiPSC-derived muscle tissues toward next generation VML regenerative therapies.

Abstract 14908: Anatomic Validation of Induced Pluripotent Stem Cell-derived Aortic Smooth Muscle Cell Model of Loeys Dietz Syndrome

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Albert J Pedroza ◽  
Samantha Churovich ◽  
Nobu Yokoyama ◽  
Ken Nakamura ◽  
Cristiana Iosef Husted ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Stem Cell ◽  
Aortic Root ◽  
Pluripotent Stem Cell ◽  
Cell Model ◽  
Expression Profiles ◽  
Induced Pluripotent Stem Cell ◽  
Aortic Root Aneurysm ◽  
Ligand Expression ◽  
Induced Pluripotent

Introduction: Mutations in TGF-beta (TGF-ß) signaling genes lead to aortic root aneurysm in Loeys Dietz syndrome (LDS). Smooth muscle cells (SMCs) in the proximal aorta develop from two embryologic origins: second heart field (SHF) and neural crest (NC). Induced pluripotent stem cell (iPSC) models simulate these lineages, but direct correlation to clinical disease is lacking. Hypothesis: iPSC-derived SMCs accurately model lineage-specific aortopathy in LDS. Methods: We generated SMC lines from root and ascending aortic surgical tissue and iPSC-derived SMCs through SHF and NC-specific pathways from an LDS patient ( TGFBR1 mutation). Lineage-specific TGF-ß responses were determined by western blot/ELISA. RNA sequencing and RT-PCR identified SMC transcriptomes. Results: Aortic root SMCs showed greater canonical TGF-ß activation (p-SMAD2/3) versus ascending at baseline and with TGF-ß stimulation ( Figure ). Synonymous results were seen in SHF versus NC SMCs from the iPSC pathway. RNAseq identified 1,600 differentially expressed genes between iPSC lineages, including altered TGF-ß receptor and ligand expression profiles. Primary aortic lines validated iPSC data: root SMCs showed enriched TGF-ß receptor 1/2/3 expression (1.7-, 3.9- and 5.9-fold) while ascending SMCs overexpressed TGFB1 and TGFB2 ligands (1.8- and 3.5-fold). Despite discordant TGF-ß activation, SMC contractile gene expression was similar between lineages in aortic and iPSC-SMCs, suggesting alternative downstream effects in LDS aneurysm. Conclusion: iPSC-derived SMCs effectively model lineage-specific aortic root aneurysm pathology, validating this model as a tool for mechanistic testing and therapy discovery.

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