Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction.

Gene editing for the cure of inborn errors of metabolism (IEMs) has been limited by inefficiency of adult hepatocyte targeting. Here, we demonstrate that in utero CRISPR/Cas9-mediated gene editing in a mouse model of hereditary tyrosinemia type 1 provides stable cure of the disease. Following this, we performed an extensive gene expression analysis to explore the inherent characteristics of fetal/neonatal hepatocytes that make them more susceptible to efficient gene editing than adult hepatocytes. We showed that fetal and neonatal livers are comprised of proliferative hepatocytes with abundant expression of genes involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs), key for efficient gene editing by CRISPR/Cas9. We demonstrated the same is true of hepatocytes after undergoing a regenerative stimulus (partial hepatectomy), where post-hepatectomy cells show a higher efficiency of HDR and correction. Specifically, we demonstrated that HDR-related genome correction is most effective in the replicative phase, or S-phase, of an actively proliferating cell. In conclusion, this study shows that taking advantage of or triggering cell proliferation, specifically DNA replication in S-phase, may serve as an important tool to improve efficiency of CRISPR/Cas9-mediated genome editing in the liver and provide a curative therapy for IEMs in both children and adults.

Use of an adeno-associated virus serotype Anc80 to provide durable cure of phenylketonuria in a mouse model.

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pahenu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pahenu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 1012 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

In vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.

Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing.

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.

The future of gene-targeted therapy for hereditary tyrosinemia type 1 as a lead indication among the inborn errors of metabolism.

INTRODUCTION: Inborn errors of metabolism (IEMs) often result from single-gene mutations and collectively cause liver dysfunction in neonates leading to chronic liver and systemic disease. Current treatments for many IEMs are limited to maintenance therapies that may still require orthotropic liver transplantation. Gene therapies offer a potentially superior approach by correcting or replacing defective genes with functional isoforms; however, they face unique challenges from complexities presented by individual diseases and their diverse etiology, presentation, and pathophysiology. Furthermore, immune responses, off-target gene disruption, and tumorigenesis are major concerns that need to be addressed before clinical application of gene therapy. AREAS COVERED: The current treatments for IEMs are reviewed as well as the advances in, and barriers to, gene therapy for IEMs. Attention is then given to ex vivo and in vivo gene therapy approaches for hereditary tyrosinemia type 1 (HT1). Of all IEMs, HT1 is particularly amenable to gene therapy because of a selective growth advantage conferred to corrected cells, thereby lowering the initial transduction threshold for phenotypic relevance. EXPERT OPINION: It is proposed that not only is HT1 a safe indication for gene therapy, its unique characteristics position it to be an ideal IEM to develop for clinical investigation.

Ex Vivo Cell Therapy by Ectopic Hepatocyte Transplantation Treats the Porcine Tyrosinemia Model of Acute Liver Failure.

The effectiveness of cell-based therapies to treat liver failure is often limited by the diseased liver environment. Here, we provide preclinical proof of concept for hepatocyte transplantation into lymph nodes as a cure for liver failure in a large-animal model with hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. Autologous porcine hepatocytes were transduced ex vivo with a lentiviral vector carrying the pig Fah gene and transplanted into mesenteric lymph nodes. Hepatocytes showed early (6 h) and durable (8 months) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated sufficient liver mass to clinically ameliorate the acute liver failure and HT1 disease as early as 97 days post-transplantation. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes from lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Therefore, ectopic transplantation of healthy hepatocytes cures this pig model of liver failure and presents a promising approach for the development of cures for liver disease in patients.

Ex Vivo Hepatocyte Reprograming Promotes Homology-Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation.

Ex vivo CRISPR/Cas9-mediated gene editing in hepatocytes using homology-directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end-joining is the predominant DNA repair pathway for double-strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9-mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down-regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo. To assess functionality long-term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single-point mutation were transduced ex vivo with two adeno-associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2-kb homology template. Adeno-associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte-directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector's ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.