Controlling CRISPR with Small Molecule Regulation for Somatic Cell Genome Editing

AbstractSomatic cell genome editing (SCGE) is highly promising for therapeutic innovation. Multifold financial and academic incentives exist for the quickest possible translation from preclinical to clinical studies. This study demonstrates that the majority of 46 preclinical SCGE studies discussed in expert reviews as particularly promising for clinical translation do not report on seven key elements for robust and confirmatory research practices: (1) randomization, (2) blinding, (3) sample size calculation, (4) data handling, (5) pre-registration, (6) multi-centric study design, and (7) independent confirmation. Against the background of the high incentives for clinical translation and recent concerns about the reproducibility of published preclinical evidence, we present the here examined reporting standards (1-4) and the new NIH funding criteria for SCGE research (6-7) as a viable solution to protect this promising field from backlashes. We argue that the implementation of the novel methodological standards, e.g. “confirmation” and “pre-registration”, is promising for preclinical SCGE research and provides an opportunity to become a lighthouse example for trust-worthy and useful translational research.

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

10.21203/rs.3.rs-90150/v1 ◽

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.

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The NIH Somatic Cell Genome Editing program

Nature ◽

10.1038/s41586-021-03191-1 ◽

2021 ◽

Vol 592 (7853) ◽

pp. 195-204

Author(s):

Krishanu Saha ◽

◽

Erik J. Sontheimer ◽

P. J. Brooks ◽

Melinda R. Dwinell ◽

...

Keyword(s):

Somatic Cell ◽

Genome Editing ◽

The United States ◽

Third Party ◽

Wide Range ◽

Functional Consequences ◽

Cell Genome ◽

Large Animals ◽

Genome Modifications

AbstractThe move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium’s plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled—along with validated datasets—into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit—and the knowledge generated by its applications—as a means to accelerate the clinical development of new therapies for a wide range of conditions.

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194 EFFECT OF SMALL MOLECULE INHIBITORS ON Oct-4, Nanog, AND Sox-2 EXPRESSION IN BOVINE FETAL FIBROBLAST CELLS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab194 ◽

2017 ◽

Vol 29 (1) ◽

pp. 206

Author(s):

L. W. C. Gaspard ◽

K. R. Bondioli

Keyword(s):

Stem Cell ◽

Small Molecules ◽

Somatic Cell ◽

Culture Medium ◽

Target Genes ◽

Embryonic Stem ◽

Fibroblast Cells ◽

Fetal Fibroblast ◽

Transcript Levels ◽

Cell Genome

Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. However, the majority of cells that undergo reprogramming become trapped in a partially reprogrammed state that is characterised by the down-regulation of somatic cell marker genes, incomplete reactivation of pluripotency genes, maintenance of viral expression, and the inability to form chimeras. Several small molecules, which act on specific signaling pathways or chromatin modifications, have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the roadblocks encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and reactivate the embryonic stem cell genome. Chemical treatment of cells before reprogramming can remodel the epigenetic landscape to be more like that of embryonic stem cell by removing the repressive epigenetic marks and relaxing chromatin structure to allow the reprogramming factors easier access to target genes. In the present study, we assessed the effect of pretreatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen-activated protein kinase inhibitor; CHIR99021, a glycogen synthase kinase-3 inhibitor; and NuP0178 (NuPotential, Baton Rouge, LA, USA), a G9a histone methyltransferase inhibitor. Cells were seeded at a density of 0.7 × 106 and expanded before being divided evenly into 2 groups. Control cells were cultured in complete culture medium (DMEM with 10% fetal bovine serum), whereas treatment cells were cultured in complete culture medium containing 0.5 mM PD0325901, 3 mM CHIR99021, and 1.8 mM NuP0178. Messenger RNA was isolated from cell cultures using Dynabeads® mRNA DIRECT™ Kit (Dynal Inc., Lake Success, NY, USA) on Day 7 and 14, and the resulting RNA products were transcribed into cDNA using Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative RT–PCR was performed to measure transcript levels of Oct-4, Nanog, Sox-2, and poly adenylate polymerase in treated and untreated cells at Days 7 and 14. Transcript levels were quantified by relative quantification using the ΔΔCt method and expressed as ratios of target genes (Oct-4, Nanog, Sox-2) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine fetal fibroblast cells. No difference in expression levels between untreated and treated cells was detected at either Day 7 or 14. Currently, we are utilising chromatin immunoprecipitation to examine chromatin and DNA methylation patterns around the promoters of Oct-4, Nanog, and Sox-2 to further elucidate the effects of treatment with this combination of small molecule inhibitors.

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31 EFFICIENT GENERATION OF klotho MUTATIONS IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS USING A DELIVERY OF Cas9 RIBONUCLEOPROTEINS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab31 ◽

2017 ◽

Vol 29 (1) ◽

pp. 123

Author(s):

S. Lee ◽

M. H. Jung ◽

H. J. Oh ◽

O.-J. Koo ◽

B. C. Lee

Keyword(s):

Somatic Cell ◽

Genome Editing ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Premature Aging ◽

Human Diseases ◽

Limited Information ◽

Transcription Activator ◽

Klotho Gene ◽

Donor Cells

Pigs are useful models for studying human diseases because of the similarity of their anatomy and physiology. Recent advances in genome editing techniques such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat-associated Cas9 system (CRISPR/Cas9) have made it possible to produce animals for specific purposes. Especially, recent application of the CRISPR/Cas9 system improved the efficiency of genome editing in pigs with higher targeting efficiency or percentage of desired mutation compared to other meganucleases (ZFNs and TALENs). The klotho deficiency in small animals such as mice is characterised by an extremely shortened life span with multiple aging-like phenotypes similar to human premature-aging syndromes. However, limited information is available on the function of klotho in large animals such as pigs. The objective of this study was to determine whether the use of non-selected porcine fibroblasts electroporated with Cas9/sgRNA ribonucleoproteins, targeting the klotho gene, for somatic cell nuclear transfer (SCNT) results in high mutation rates in embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and type 2 Cas9 RNPs (total 36 μg, 1:4 ratio, respectively) were transfected into porcine fibroblasts via Neon (Life Technologies) with a single DC pulse of 1400 V for 30 ms. Then, transfected fibroblasts were cultured for 1 day and used randomly for SCNT without selection. SCNT was performed by enucleation of in vitro-matured porcine oocyte, followed by injection of non-selected donor cells, fusion with a single DC pulse of 200 V/mm for 30 μs using an electro cell fusion generator (LF101; Nepa Gene Co.), and electrical activation with a single DC pulse of 150 V/mm for 60 μs using a BTX Electro-Cell Manipulator 2001 (BTX Inc.). The SCNT embryos were cultured in PZM5 culture medium to Day 7 and analysed for the presence of modifications to the klotho gene. Blastocysts were classified as modified if they contained an INDEL as measured by both T7E1 assay and deep sequencing of PCR amplicons spanning the targeted exon. The klotho modification rate was 65% (n = 13), of which 38.5% (n = 5) of the embryos contained biallelic modifications. In conclusion, SCNT with non-selected donor cells transfected with Cas9/sgRNA RNPs might be an efficient and simple tool to produce klotho deficient pigs as models for human diseases. Further studies are required to generate klotho deficient pigs by performing embryo transfer to the recipients. This study was supported by Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry and fisheries (#311011–05–5-SB010, #114059–03–2-SB010), Research Institute for Veterinary Science, TS Corporation and the BK21 plus program.

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