Abstract 528: Towards Therapeutic Genome Editing in Vascular Endothelium by Novel Nanoparticle Delivery of Crispr Plasmid Dna

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Xianming Zhang ◽  
You-yang Zhao
Keyword(s):  
Protein Expression ◽  
Genome Editing ◽  
Vascular Endothelium ◽  
Plasmid Dna ◽  
Adult Life ◽  
Therapeutic Delivery ◽  
Crispr System ◽  
Highly Efficient ◽  
Adult Mice

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal and adult life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can therapeutically deliver the CRISPR system to selectively target cardiovascular endothelium in adult mice. Methods: We developed novel PLGA-based nanoparticles which was for the first time shown to be uptaken efficiently by the vascular endothelium without specific liver accumulation following i.v. administration. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter (EC-specific) and gRNA by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including heart, lung, and aorta in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in ECs. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively targeting the vascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

Abstract 17091: High Efficient Therapeutic Genome Editing in Cardiovascular Endotheliumby Nanoparticle Delivery of Crispr Plasmid

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Hua Jin ◽  
Xiaojia Huang ◽  
Xianming Zhang ◽  
Youyang Zhao
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Genome Editing ◽  
Therapeutic Delivery ◽  
Crispr System ◽  
Highly Efficient ◽  
Adult Mice ◽  
High Efficient ◽  
System Components

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient in vivo genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can deliver CRISPR system components in vivo to selectively target cardiovascular endothelium in adult mice. Methods: We developed a novel nanoparticles. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter and gRNA driven by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including lung, heart, aorta, and peripheral blood vessels in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in cardiovascular endothelial cells. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively in the cardiovascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

scholarly journals A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1862-1862
Author(s):  
Gregory J. Cost ◽  
Morayma Temoche-Diaz ◽  
Janet Mei ◽  
Cristina N. Butterfield ◽  
Christopher T. Brown ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Attractive Alternative ◽  
Vitro Assay ◽  
Crispr System ◽  
Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing

2019 ◽  
Vol 12 ◽  
pp. 111-122 ◽  
Author(s):  
Ang Li ◽  
Ciaran M. Lee ◽  
Ayrea E. Hurley ◽  
Kelsey E. Jarrett ◽  
Marco De Giorgi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Crispr System

scholarly journals Prime editing primarily induces undesired outcomes in mice

2020 ◽  
Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Mouse Embryos ◽  
Biomedical Science ◽  
New Approach ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Large Deletions

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

scholarly journals Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

2016 ◽  
Vol 34 (3) ◽  
pp. 328-333 ◽  
Author(s):  
Hao Yin ◽  
Chun-Qing Song ◽  
Joseph R Dorkin ◽  
Lihua J Zhu ◽  
Yingxiang Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Crispr System ◽  
System Components

scholarly journals Non-viral Delivery of Zinc Finger Nuclease mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets

2019 ◽  
Vol 27 (4) ◽  
pp. 866-877 ◽  
Author(s):  
Anthony Conway ◽  
Matthew Mendel ◽  
Kenneth Kim ◽  
Kyle McGovern ◽  
Alisa Boyko ◽  
...  
Keyword(s):  
Genome Editing ◽  
Zinc Finger ◽  
Zinc Finger Nuclease ◽  
Therapeutic Gene ◽  
Highly Efficient

scholarly journals Genome editing in plants using CRISPR type I-D nuclease

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Keishi Osakabe ◽  
Naoki Wada ◽  
Tomoko Miyaji ◽  
Emi Murakami ◽  
Kazuya Marui ◽  
...  
Keyword(s):  
Genome Editing ◽  
First Generation ◽  
Genome Engineering ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Type I ◽  
Crispr System ◽  
Target Dna ◽  
Short Indels

Abstract Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

scholarly journals Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA

Cell Reports ◽  
2022 ◽  
Vol 38 (1) ◽  
pp. 110196
Author(s):  
Xianming Zhang ◽  
Hua Jin ◽  
Xiaojia Huang ◽  
Birendra Chaurasiya ◽  
Daoyin Dong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Vascular Endothelium ◽  
Plasmid Dna ◽  
Nanoparticle Delivery

scholarly journals Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pengpeng Liu ◽  
Shun-Qing Liang ◽  
Chunwei Zheng ◽  
Esther Mintzer ◽  
Yan G. Zhao ◽  
...  
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Tumor Formation ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Modification System ◽  
Adult Mice ◽  
Genome Modification ◽  
Split Intein

AbstractPrime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.

scholarly journals Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

2020 ◽  
Author(s):  
Pengpeng Liu ◽  
Shun-Qing Liang ◽  
Chunwei Zheng ◽  
Esther Mintzer ◽  
Yan G. Zhao ◽  
...  
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Tumor Formation ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Modification System ◽  
Adult Mice ◽  
Genome Modification ◽  
Split Intein

AbstractPrime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.

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