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

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.

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Xianming Zhang ◽  
You-yang Zhao

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.


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

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 ◽  
...  

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.


Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Juan Castor ◽  
Darijana Horvat ◽  
Walter E Cromer ◽  
Thomas J Kuehl ◽  
David C Zawieja ◽  
...  

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na + /K + ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture. Methods: LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1μM before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of β-catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure β-catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1μM L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The β-catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VE-cadherin and LYVE-1 protein expression, but MBG did not. Conclusions: We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the β-catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.


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

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Lauren A. Bailey ◽  
Azemat Jamshidi-Parsian ◽  
Tulsi Patel ◽  
Nathan A. Koonce ◽  
Alan B. Diekman ◽  
...  

AbstractBackground Despite aggressive treatment for glioblastoma multiforme (GBM), including surgical resection, radiotherapy and temozolomide (TMZ) chemotherapy, over 90% of patients experience tumor recurrence. Galectins are carbohydrate-binding proteins that are overexpressed in the stroma of GBM tumors, and are potent modulators of GBM cell migration and angiogenesis. The objective of this study was to analyze glioma and endothelial cell galectin expression in response to combined chemoradiation. Methodology The effects of TMZ, ionizing radiation, or combined chemoradiation on galectin protein secretion and expression were assessed in U87 orthotopically grown GBM tumors in mice, as well as in vitro in U87 human glioma cells and human umbilical vein endothelial cells (HUVECs). Results We found that combination chemoradiation increased galectin-1 and galectin-3 protein expression in U87 glioma cells. In response to radiation alone, U87 cells secreted significant levels of galectin-1 and galectin-3 into the microenvironment. HUVEC co-culture increased U87 galectin-1 and galectin-3 protein expression 14 - 20% following chemoradiation, and conferred a radioprotective benefit to U87 glioma cells. In vivo, radiation alone and combination chemoradiation significantly increased tumor galectin-1 expression in an orthotopic murine model of GBM. Conclusions Glioma cell galectin expression increased following combined chemoradiation, both in vitro and in vivo. The presence of endothelial cells further increased glioma cell galectin expression and survival, suggesting that crosstalk between tumor and endothelial cells in response to standard chemoradiation may be an important factor in mediating glioma recurrence, potentially via galectin upregulation.


2022 ◽  
Author(s):  
Steinunn Sara Helgudóttir ◽  
Kasper Bendix Johnsen ◽  
Lisa Juul Routhe ◽  
Charlotte L.M. Rasmussen ◽  
Azra Karamehmedovic ◽  
...  

Abstract BackgroundThe objectives of the present study were to investigate whether the expression of transferrin receptor 1 (TfR1), glucose transporter 1 (Glut1), or Cluster of Differentiation 98 Heavy Chain (CD98hc) is epigenetically regulated in brain capillary endothelial cells (BCECs) denoting the blood-brain barrier (BBB).MethodsThe expression of these targets was investigated both in vitro and in vivo following treatment with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Mice were injected intraperitoneally with VPA followed by analysis of isolated brain capillaries, and the capillary depleted brain samples. Brain tissue, isolated brain capillaries, and cultured primary endothelial cells were analyzed by RT-qPCR, immunolabeling and ELISA for expression of TfR1, Glut1 and CD98hc. We also studied the vascular targeting in VPA-treated mice injected with monoclonal anti-transferrin receptor (Ri7) conjugated with 1.4 nm gold nanoparticles. ResultsValidating the effects of VPA on gene transcription in BCECs, transcriptomic analysis identified 24,371 expressed genes, of which 305 were differentially expressed with 192 upregulated and 113 downregulated genes. In vitro using BCECs co-cultured with glial cells, the mRNA expression of Tfrc was significantly higher after VPA treatment for 6 h with its expression returning to baseline after 24 h. Conversely, the mRNA expression of Glut1 and Cd98hc was unaffected by VPA treatment. In vivo, the TfR1 protein expression in brain capillaries increased significantly after treatment with both 100 mg/kg and 400 mg/kg VPA. Conversely, VPA treatment did not increase GLUT1 or CD98hc. Using ICP-MS-based quantification, the brain uptake of nanogold conjugated anti-TfR1 antibodies was non-significant in spite of increased expression of TfR1. ConclusionsWe report that VPA treatment upregulates TfR1 at the BBB both in vivo and in vitro in isolated primary endothelial cells. In contrast, VPA treatment does not influence the expression of GLUT1 and CD98hc. The increase in the overall TfR1 protein expression however does not increase transport of TfR-targeted monoclonal antibody and indicates that targeted delivery using the transferrin receptor should aim for increased mobilization of already available transferrin receptor molecules to improve trafficking through the BBB.


Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  

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.


2008 ◽  
Vol 295 (5) ◽  
pp. C1292-C1301 ◽  
Author(s):  
Anke C. Webler ◽  
U. Ruth Michaelis ◽  
Rüdiger Popp ◽  
Eduardo Barbosa-Sicard ◽  
Andiappan Murugan ◽  
...  

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.


2019 ◽  
Vol 27 (4) ◽  
pp. 866-877 ◽  
Author(s):  
Anthony Conway ◽  
Matthew Mendel ◽  
Kenneth Kim ◽  
Kyle McGovern ◽  
Alisa Boyko ◽  
...  

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