Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

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 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 × 106 cells per liver and cell viability of >85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of >35% in mouse hepatocytes. To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes.

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 Hpd, 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.

Delivery Approaches for Therapeutic Genome Editing and Challenges.

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.

In vitro Studies of Transendothelial Migration for Biological and Drug Discovery.

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 in vivo 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 in vitro platforms. In addition to the ease of use, low cost, and high controllability, in vitro 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 in vitro platforms. In vitro studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of in vitro 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.

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

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.

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

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 ß-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 ß-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 ß-thalassemia. The opportunities and challenges in advancing a curative therapy for hemoglobinopathies are also discussed.

Controlled delivery of ß-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection.

Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human ß-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with ß-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents.