Ex Vivo/In vivo Gene Editing in Hepatocytes Using “All-in-One” CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template

Abstract

SummaryAdeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.

Proceedings From the First International Workshop at Sidra Medicine: “Engineered Immune Cells in Cancer Immunotherapy (EICCI): From Discovery to Off-the-Shelf Development”, 15th–16th February 2019, Doha, Qatar

“…The MS2 bacteriophage RNA packaging mechanism has been applied to the production of non-integrating RVs, GV.MS2-CRISPR/Cas9 all-in-one particles, for efficient gene editing ( 125 , 126 ). This strategy led to efficient KO of target genes, such as TP53 in human fibroblasts or CXCR4 or CCR7 in Jurkat or T cells, respectively ( 125 – 127 ). These platforms are applicable to the generation of allogeneic CAR-T cells to prevent GvHD and/or to engineer their immune functions.…”

Delivery Approaches for Therapeutic Genome Editing and Challenges

“…Dever et al reported successful ex vivo gene editing using the AAV-based CRISPR system targeting the HBB gene in CD34 + HSPCs to correct the mutation causing sickle cell disease [ 41 ]. Recently, a single AAV vector containing SaCas9, gRNA, and donor repair template was used to correct the mutation in Fah that causes hereditary tyrosinemia-1 ex vivo in hepatocytes [ 131 ]. The study demonstrated successful repopulation of the liver by gene-modified hepatocytes, resulting in a complete rescue in mice without the detection of abnormalities.…”

In vitro Studies of Transendothelial Migration for Biological and Drug Discovery

“…In addition, these in vitro models can potentially be used to facilitate screening of quality hepatocytes for clinical transplantation. A vast interest lies in gene editing using CRISPR-Cas technology in autologous hepatocytes for gene therapy targeting inherited metabolic diseases affecting the liver, which has been effective in animal models (160)(161)(162) and is the aim for future therapies. The advantage of the gene editing approach is it eliminates the need for immunosuppression and risk of graft rejection as cells isolated from the patient's resected liver are substrates for transplantation or hepatocytes may be edited in vivo to correct a monogenetic disease.…”