scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

2018 ◽  
Vol 115 (19) ◽  
pp. 4903-4908 ◽  
Author(s):  
Hong-Xia Wang ◽  
Ziyuan Song ◽  
Yeh-Hsing Lao ◽  
Xin Xu ◽  
Jing Gong ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Gene Activation ◽  
Cell Types ◽  
Biological Research ◽  
Knock Out ◽  
Safe Delivery ◽  
Pegylated Nanoparticles

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Siyuan Yu ◽  
Han Yang ◽  
Tingdong Li ◽  
Haifeng Pan ◽  
Shuling Ren ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Leucine Zipper ◽  
Endosomal Escape ◽  
Cell Penetrating Peptide ◽  
Macromolecular Drugs ◽  
Cell Penetrating ◽  
In Series ◽  
Factor Α

AbstractProtein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; however, cell-penetrating peptide-mediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. Here, we present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. Intravenous injection of protein phosphatase 1B fused with this system successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in a mouse model. We believe that the strategy of using multifunctional chimaeric peptides is valuable for the development of cell-penetrating peptide-based protein delivery systems, and facilitate the development of biological macromolecular drugs for use against intracellular targets.

Polymeric Vectors for Strategic Delivery of Nucleic Acids

Nano LIFE ◽  
2017 ◽  
Vol 07 (02) ◽  
pp. 1730003
Author(s):  
Andrew Dunn ◽  
Donglu Shi
Keyword(s):  
Molecular Weight ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Primary Amines ◽  
Biological Research ◽  
Specific Cell ◽  
Nucleic Acid Delivery ◽  
Biological Studies

Genomic modification through nucleic acid delivery is a frequently applied method in fundamental biological studies and offers a potent therapeutic strategy for disease treatment and biological research. Delivery of nucleic sequences is therefore an attractive facet of biological nanotechnology as highly specific, efficient, and nonantagonistic delivery is necessary for in vivo and clinical use. Previous vectors have suffered from immunogenic responses, serum dependent inactivation, and cytotoxicity, hindering their translational applicability. Current research in polymeric-based nucleotide delivery strives to offer a highly biocompatible, broad use vector through the utilization of polypeptide and polyamine conjugation that can be easily tailored for specific targeting or wide dissemination. Cross-linking low molecular weight polyamines and lipophilic derivatization for amphiphile creation has lead to improved biocompatibility and transfection efficiency compared to higher molecular weight polyamines. Derivatization of hyperbranched and dendritic polyamido- and polyamines has allowed for the formation of efficient in vivo transfection vectors; ring opening synthesis of N-carboxyanhydride amino acids have led to controlled peptide architectures for improved transfection while simultaneously providing convenient primary amines useful in functionalization. Polymer libraries of poly(ß-amino esters) have provided insights into useful architectures for in vitro and in vivo gene delivery. Grafting small molecules to polyamines, such as folate and galactose, for enhanced interaction with cell surface receptors for selective targeting of specific cell types has proven to be encouraging and remains a prominent aspect in biological nanotechnology.

scholarly journals Nanocarriers for Protein Delivery to the Cytosol: Assessing the Endosomal Escape of Poly(Lactide-co-Glycolide)-Poly(Ethylene Imine) Nanoparticles

Nanomaterials ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 652 ◽  
Author(s):  
Marianna Galliani ◽  
Chiara Tremolanti ◽  
Giovanni Signore
Keyword(s):  
Protein Delivery ◽  
Therapeutic Proteins ◽  
Endosomal Escape ◽  
Ethylene Imine ◽  
Enzyme Superoxide Dismutase ◽  
Rapid Clearance ◽  
Poly Ethylene ◽  
Enzyme Delivery

Therapeutic proteins and enzymes are a group of interesting candidates for the treatment of numerous diseases, but they often require a carrier to avoid degradation and rapid clearance in vivo. To this end, organic nanoparticles (NPs) represent an excellent choice due to their biocompatibility, and cross-linked enzyme aggregates (CLEAs)-loaded poly (lactide-co-glycolide) (PLGA) NPs have recently attracted attention as versatile tools for targeted enzyme delivery. However, PLGA NPs are taken up by cells via endocytosis and are typically trafficked into lysosomes, while many therapeutic proteins and enzymes should reach the cellular cytosol to perform their activity. Here, we designed a CLEAs-based system implemented with a cationic endosomal escape agent (poly(ethylene imine), PEI) to extend the use of CLEA NPs also to cytosolic enzymes. We demonstrated that our system can deliver protein payloads at cytoplasm level by two different mechanisms: Endosomal escape and direct translocation. Finally, we applied this system to the cytoplasmic delivery of a therapeutically relevant enzyme (superoxide dismutase, SOD) in vitro.

scholarly journals Increased PIP3 activity blocks nanoparticle mRNA delivery

2020 ◽  
Vol 6 (30) ◽  
pp. eaba5672 ◽  
Author(s):  
Kalina Paunovska ◽  
Alejandro Da Silva Sanchez ◽  
Matthew T. Foster ◽  
David Loughrey ◽  
Emmeline L. Blanchard ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Cell Metabolism ◽  
Mammalian Target Of Rapamycin ◽  
Cell Types ◽  
Endosomal Escape ◽  
Cell Uptake ◽  
Multiple Cell ◽  
Catabolic Response

The biological pathways that affect drug delivery in vivo remain poorly understood. We hypothesized that altering cell metabolism with phosphatidylinositol (3,4,5)-triphosphate (PIP3), a bioactive lipid upstream of the metabolic pathway PI3K (phosphatidylinositol 3-kinase)/AKT/ mTOR (mammalian target of rapamycin) would transiently increase protein translated by nanoparticle-delivered messenger RNA (mRNA) since these pathways increase growth and proliferation. Instead, we found that PIP3 blocked delivery of clinically-relevant lipid nanoparticles (LNPs) across multiple cell types in vitro and in vivo. PIP3-driven reductions in LNP delivery were not caused by toxicity, cell uptake, or endosomal escape. Interestingly, RNA sequencing and metabolomics analyses suggested an increase in basal metabolic rate. Higher transcriptional activity and mitochondrial expansion led us to formulate two competing hypotheses that explain the reductions in LNP-mediated mRNA delivery. First, PIP3 induced consumption of limited cellular resources, “drowning out” exogenously-delivered mRNA. Second, PIP3 triggers a catabolic response that leads to protein degradation and decreased translation.

Programmable DNA cleavage in vitro by Cas9

2013 ◽  
Vol 41 (6) ◽  
pp. 1401-1406 ◽  
Author(s):  
Tautvydas Karvelis ◽  
Giedrius Gasiunas ◽  
Virginijus Siksnys
Keyword(s):  
Dna Cleavage ◽  
Gene Editing ◽  
Streptococcus Thermophilus ◽  
Cell Types ◽  
Strand Break ◽  
Eukaryotic Cell ◽  
Modular Organization ◽  
Cleavage Reaction

The ternary Cas9–crRNA–tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system functions as an Mg2+-dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA–crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. By altering the crRNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a gene-editing tool in various eukaryotic cell types. Using Streptococcus thermophilus Cas9t as a model system, we demonstrate the feasibility of Cas9t as a programmable molecular tool for in vitro DNA manipulations.

scholarly journals Efficient in vivo genome editing mediated by stem cells-derived extracellular vesicles carrying designer nucleases

2021 ◽  
Author(s):  
Sylwia Bobis-Wozowicz ◽  
Karolina Kania ◽  
Kinga Nit ◽  
Natalia Blazowska ◽  
Katarzyna Kmiotek-Wasylewska ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Extracellular Vesicles ◽  
Gene Knockout ◽  
Marker Gene ◽  
Cell Types ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Precise genome editing using designer nucleases (DNs), such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system, has become a method of choice in a variety of biological and biomedical applications in recent years. Notably, efficacy of these systems is currently under scrutiny in about 50 clinical trials. Although high DNs activity in various cell types in vitro has already been achieved, efficient in vivo genome editing remains a challenge. To solve this problem, we employed stem cells-derived extracellular vesicles (EVs) as carriers of DNs. We used umbilical cord-derived mesenchymal stem cells (UC-MSCs) and induced pluripotent stem cells (iPSCs) as EV-producer cells, since they are both applied in regenerative medicine. In our proof-of-concept studies, we achieved up to 50% of EGFP marker gene knockout in vivo using EVs carrying either ZFN, TALEN or the CRISPR/Cas9 system, particularly in the liver. Importantly, we obtained almost 50% of modified alleles in the liver of the experimental animals, when targeting the Pcsk9 gene, whose overexpression is implicated in hypercholesterolemia. Taken together, our data provide strong evidence that stem cells-derived EVs constitute a robust tool in delivering DNs in vivo, which may be harnessed to clinical practice in the future.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

scholarly journals Heme Oxygenase-1 Deficiency and Oxidative Stress: A Review of 9 Independent Human Cases and Animal Models

2021 ◽  
Vol 22 (4) ◽  
pp. 1514 ◽  
Author(s):  
Akihiro Yachie
Keyword(s):  
Oxidative Stress ◽  
Animal Models ◽  
Heme Oxygenase ◽  
Inflammatory Diseases ◽  
Cell Types ◽  
Pharmacological Intervention ◽  
Heme Oxygenase 1 ◽  
Inflammatory Reactions

Since Yachie et al. reported the first description of human heme oxygenase (HO)-1 deficiency more than 20 years ago, few additional human cases have been reported in the literature. A detailed analysis of the first human case of HO-1 deficiency revealed that HO-1 is involved in the protection of multiple tissues and organs from oxidative stress and excessive inflammatory reactions, through the release of multiple molecules with anti-oxidative stress and anti-inflammatory functions. HO-1 production is induced in vivo within selected cell types, including renal tubular epithelium, hepatic Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-1 plays critical roles in these cells. In vivo and in vitro studies have indicated that impaired HO-1 production results in progressive monocyte dysfunction, unregulated macrophage activation and endothelial cell dysfunction, leading to catastrophic systemic inflammatory response syndrome. Data from reported human cases of HO-1 deficiency and numerous studies using animal models suggest that HO-1 plays critical roles in various clinical settings involving excessive oxidative stress and inflammation. In this regard, therapy to induce HO-1 production by pharmacological intervention represents a promising novel strategy to control inflammatory diseases.

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