electroporation-mediated-delivery-cas9-ribonucleoproteins-mrna-into

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Clemson University

Hemoglobinopathies are genetic disorders caused by aberrant hemoglobin expression or structure changes, resulting in severe mortality and health disparities worldwide. Sickle cell disease (SCD) and β-thalassemia, the most common forms of hemoglobinopathies, are typically treated using transfusions and pharmacological agents. Allogeneic hematopoietic stem cell transplantation is the only curative therapy, but has limited clinical applicability. Although gene therapy approaches have been proposed based on the insertion and forced expression of wild-type or anti-sickling β-globin variants, safety concerns may impede their clinical application. A novel curative approach is nuclease-based gene correction, which involves the application of precision genome-editing tools to correct the disease-causing mutation. This review describes the development and potential application of gene therapy and precision genome-editing approaches for treating SCD and β-thalassemia. The opportunities and challenges in advancing a curative therapy for hemoglobinopathies are also discussed.

Treating hemoglobinopathies using gene-correction approaches: promises and challenges.

Inherited metabolic diseases (IMDs) of the liver represent a vast and diverse group of rare genetic diseases characterized by the loss or dysfunction of enzymes or proteins essential for metabolic pathways in the liver. Conventional gene therapy involving adeno-associated virus (AAV) serotype 8 vectors provide therapeutically high levels of hepatic transgene expression facilitating the correction of the disease phenotype in pre-clinical studies and are currently being evaluated in clinical trials for multiple IMDs. However, insertional mutagenesis and immunogenicity risks as well as efficacy limitations represent major drawbacks for the AAV system. Genome editing tools, particularly the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system, offer multiple advantages over conventional gene transfer and have the potential to further advance the promises of gene therapy. Here, we provide a critical assessment of conventional gene therapy and genome editing approaches for therapeutic correction of the most investigated metabolic liver disorders, namely familial hypercholesterolemia, hemophilia, ornithine transcarbamylase deficiency, hereditary tyrosinemia type 1, and alpha-1 antitrypsin deficiency. In addition, we elaborate on the barriers and future directions for advancing novel nuclease mediated gene therapies for IMDs.

Nuclease-Mediated Gene Therapies for Inherited Metabolic Diseases of the Liver.

Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.

Delivery Approaches for Therapeutic Genome Editing and Challenges.

Inflammatory diseases and cancer metastases lack concrete pharmaceuticals for their effective treatment despite great strides in advancing our understanding of disease progression. One feature of these disease pathogeneses that remains to be fully explored, both biologically and pharmaceutically, is the passage of cancer and immune cells from the blood to the underlying tissue in the process of extravasation. Regardless of migratory cell type, all steps in extravasation involve molecular interactions that serve as a rich landscape of targets for pharmaceutical inhibition or promotion. Transendothelial migration (TEM), or the migration of the cell through the vascular endothelium, is a particularly promising area of interest as it constitutes the final and most involved step in the extravasation cascade. While  models of cancer metastasis and inflammatory diseases have contributed to our current understanding of TEM, the knowledge surrounding this phenomenon would be significantly lacking without the use of  platforms. In addition to the ease of use, low cost, and high controllability,  platforms permit the use of human cell lines to represent certain features of disease pathology better, as seen in the clinic. These benefits over traditional pre-clinical models for efficacy and toxicity testing are especially important in the modern pursuit of novel drug candidates. Here, we review the cellular and molecular events involved in leukocyte and cancer cell extravasation, with a keen focus on TEM, as discovered by seminal and progressive  platforms.  studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of  platforms for biological discovery. This success shows the potential for applying these platforms for pharmaceutical compound screening. In addition to immune and cancer cell TEM, we discuss the promise of hepatocyte transplantation, a process in which systemically delivered hepatocytes must transmigrate across the liver sinusoidal endothelium to successfully engraft and restore liver function. Lastly, we concisely summarize the evolving field of porous membranes for the study of TEM.

 Studies of Transendothelial Migration for Biological and Drug Discovery.

Adeno-associated virus vectors are the most used delivery method for liver-directed gene editing. Still, they are associated with significant disadvantages that can compromise the safety and efficacy of therapies. Here, we investigate the effects of electroporating CRISPR-Cas9 as mRNA and ribonucleoproteins (RNPs) into primary hepatocytes regarding on-target activity, specificity, and cell viability. We observed a transfection efficiency of >60% and on-target insertions/deletions (indels) of up to 95% in primary mouse hepatocytes electroporated with Cas9 RNPs targeting , the gene encoding hydroxyphenylpyruvate dioxygenase. In primary human hepatocytes, we observed on-target indels of 52.4% with Cas9 RNPs and >65% viability after electroporation. These results establish the impact of using electroporation to deliver Cas9 RNPs into primary hepatocytes as a highly efficient and potentially safe approach for therapeutic liver-directed gene editing and the production of liver disease models.

Article	Year
Treating hemoglobinopathies using gene-correction approaches: promises and challenges. Human genetics\| PubMed ID: 27314256
Nuclease-Mediated Gene Therapies for Inherited Metabolic Diseases of the Liver. The Yale journal of biology and medicine\| PubMed ID: 29259521
Delivery Approaches for Therapeutic Genome Editing and Challenges. Genes\| PubMed ID: 32977396
Studies of Transendothelial Migration for Biological and Drug Discovery. Frontiers in medical technology\| PubMed ID: 35047883	2020
Electroporation-mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes. The CRISPR journal\| PubMed ID: 35238624	2022

Renee N. Cottle

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