The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Peter M. Glazer - One of the best experts on this subject based on the ideXlab platform.
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peptide nucleic acids and Gene Editing perspectives on structure and repair
Molecules, 2020Co-Authors: Nicholas G Economos, Elias Quijano, Adele S. Ricciardi, Stanley Oyaghire, Mark W Saltzman, Peter M. GlazerAbstract:Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic Gene Editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable Editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to Gene Editing with an emphasis on structural biochemistry and repair.
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peptide nucleic acids as a tool for site specific Gene Editing
Molecules, 2018Co-Authors: Adele S. Ricciardi, Elias Quijano, Rachael Putman, William Mark Saltzman, Peter M. GlazerAbstract:Peptide nucleic acids (PNAs) can bind duplex DNA in a sequence-targeted manner, forming a triplex structure capable of inducing DNA repair and producing specific genome modifications. Since the first description of PNA-mediated Gene Editing in cell free extracts, PNAs have been used to successfully correct human disease-causing mutations in cell culture and in vivo in preclinical mouse models. Gene correction via PNAs has resulted in clinically-relevant functional protein restoration and disease improvement, with low off-target genome effects, indicating a strong therapeutic potential for PNAs in the treatment or cure of Genetic disorders. This review discusses the progress that has been made in developing PNAs as an effective, targeted agent for Gene Editing, with an emphasis on recent in vivo, nanoparticle-based strategies.
Daniel F. Voytas - One of the best experts on this subject based on the ideXlab platform.
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multiplexed heritable Gene Editing using rna viruses and mobile single guide rnas
Nature plants, 2020Co-Authors: Evan E Ellison, Ugrappa Nagalakshmi, Maria Elena Gamo, Pinjui Huang, S P Dineshkumar, Daniel F. VoytasAbstract:An in planta Gene Editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that promote cell-to-cell mobility. Mutant progeny are recovered in the next Generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci. An efficient and multiplexed in planta Gene Editing approach is developed by infecting Cas9 transgenic plants with an RNA virus that expresses single guide RNAs carrying sequences that confer cell-to-cell mobility.
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Plant Gene Editing through de novo induction of meristems.
Nature Biotechnology, 2019Co-Authors: Michael F. Maher, Ryan A. Nasti, Macy Vollbrecht, Colby G. Starker, Matthew D. Clark, Daniel F. VoytasAbstract:Plant Gene Editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to Generate Gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and Gene-Editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and Gene edits are transmitted to the next Generation. The de novo induction of Gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant Gene Editing.
Eric B Kmiec - One of the best experts on this subject based on the ideXlab platform.
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Efficient Delivery and Nuclear Uptake Is Not Sufficient to Detect Gene Editing in CD34+ Cells Directed by a Ribonucleoprotein Complex
Elsevier, 2018Co-Authors: Shirin R. Modarai, Pawel Bialk, Dula Man, Natalia Rivera-torres, Kevin Bloh, Eric B KmiecAbstract:CD34+ cells are prime targets for therapeutic strategies for Gene Editing, because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. Although experimental advances have been reported, the associated experimental protocols have largely been less than clear or robust. As such, we evaluated the relationships among cellular delivery; nuclear uptake, often viewed as the benchmark metric of successful Gene Editing; and single base repair. We took a combinatorial approach using single-stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild-type HBB into the sickle cell genotype by evaluating conditions for two common delivery strategies of Gene Editing tools into CD34+ cells. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when the Neon Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when nucleofection is used. Despite the high efficiency of cellular transformation, and the traditional view of success in efficient nuclear uptake, neither delivery methodology enabled Gene Editing activity. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientific robustness of Gene Editing for sickle cell disease
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the position of dna cleavage by talens and cell synchronization influences the frequency of Gene Editing directed by single stranded oligonucleotides
PLOS ONE, 2014Co-Authors: Natalia Riveratorres, Bryan Strouse, Pawel Bialk, Rohina A Niamat, Eric B KmiecAbstract:With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing Gene Editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of Gene Editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible Genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP Gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of Editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting Gene Editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial Gene Editing activity. Thus, we identify novel aspects of Gene Editing that will help in the design of more effective protocols for genome modification and Gene therapy in natural Genes.
Hanspeter Kiem - One of the best experts on this subject based on the ideXlab platform.
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a nonhuman primate transplantation model to evaluate hematopoietic stem cell Gene Editing strategies for β hemoglobinopathies
Molecular therapy. Methods & clinical development, 2018Co-Authors: Olivier Humbert, Christopher W Peterson, Zachary K Norgaard, Stefan Radtke, Hanspeter KiemAbstract:Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of Genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based Gene Editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo Gene Editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.
Daniel J Siegwart - One of the best experts on this subject based on the ideXlab platform.
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selective organ targeting sort nanoparticles for tissue specific mrna delivery and crispr cas Gene Editing
Nature Nanotechnology, 2020Co-Authors: Qiang Cheng, Tuo Wei, Lukas Farbiak, Lindsay T Johnson, Sean A Dilliard, Daniel J SiegwartAbstract:CRISPR-Cas Gene Editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple Gene Editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and Gene correction therapeutics in targeted tissues.
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non viral crispr cas Gene Editing in vitro and in vivo enabled by synthetic nanoparticle co delivery of cas9 mrna and sgrna
Angewandte Chemie, 2017Co-Authors: Jason B Miller, Shuyuan Zhang, Petra Kos, Hu Xiong, Kejin Zhou, Sofya S Perelman, Hao Zhu, Daniel J SiegwartAbstract:CRISPR/Cas is a revolutionary Gene Editing technology with wide-ranging utility.[1] The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility.[1e] We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA Editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg−1). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of Gene Editing.