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

Abstract

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.

Keywords: gene therapy; genome editing; hepatocytes; inborn errors of metabolism; inherited metabolic disease of the liver; nucleases; therapy for rare disease.

Figure 1

Gene modification outcomes using genome editing tools. After the nuclease has located the target sequence it induces a DSB. The DSB is resolved by endogenous DNA repair machinery via the HDR or NHEJ pathway. NHEJ-mediated repair (shown on the left) involves random insertion and deletion of base pairs at the break site and results in gene disruption. Alternatively, the HDR-mediated repair in the presence of a donor template DNA, either a short ssDNA (right) or long plasmid (center), results in a desired sequence to be incorporated for gene correction or insertion of a new gene.

Figure 2

Protein-based programmable nucleases. (A) A ZFN pair consisting of a tandem array of ZFP domains, each binding to 3 nucleotides, fused to a FokI nuclease bound to adjacent DNA segments in an inverted orientation. Dimerization between the FokI domains activates a DSB at a location specified by the ZFP binding domains. (B) A TALEN pair bound to effector elements in the genome in a tail to tail orientation. Each TALEN consists of TALE repeat domains, each recognizing a single nucleotide, fused to a FokI nuclease domain. The effector domains from a TALEN pair bind to adjacent effector elements in a tail to tail orientation with optimized spacing. A DSB is induced by dimerized FokI domains upon binding of the TALE domains to the DNA target sites.

Figure 3

RNA-guided nucleases. (A) An engineered 100 nucleotide sgRNA directs the Cas9 protein to a specific 20 nucleotide target sequence, which is found adjacent to the 5’ end of the PAM sequence. The 20 nucleotides base-pair with the target strand, which correctly positions the RuvC and HNH nuclease domains to generate a DSB at the complementary target site. (B) An engineered 42 nucleotide single-guide crRNA directs the Cpf1 protein to a specific 23 nucleotide target sequence, which is located adjacent to the 3’ end of the PAM sequence. The crRNA can then base-pair with the target strand, thereby positioning the two RuvC nuclease domains to generate a DSB. The RuvC domain on the target strand will generate a cut ~19 bp down from the PAM sequence, while the RuvC domain on the nontarget strand will generate a cut ~23 bp down from the PAM sequence, creating a DSB that has 5’ overhangs of ~5 nucleotides. (C) A programmed sgRNA directs a hybrid catalytically inactive dCas9 protein with a FokI nuclease called (fCas9) to the 5’ end of the PAM sequence. After the target nucleotide sequence base-pairs with the target strand, dimerization with an identical but inverted fCas9 allows the FokI nucleases to generate a DSB between the two fCas9 monomers bound ~15 or 25 bp apart.

Figure 4

Schematic of genome editing approaches for treating metabolic liver diseases. Hepatocytes are harvested from the patient and introduced with nucleases aiming the IMD target gene with or without donor template DNA ex vivo for in situ gene correction or target gene disruption. Alternatively, iPSCs derived from the patient can be used as target cells for ex vivo gene modification and subsequently differentiated into hepatocytes in vitro. The gene corrected hepatocytes or iPSC-derived hepatocyte-like cells are then perfused through the portal vein and engraft next to untreated hepatocytes in the liver. For in vivo gene targeting, viral particles or, alternatively, nanoparticles containing nucleases with or without donor template DNA are injected into the patient and are endocytosed by hepatocytes in the liver. Once in the liver, the gene editing tools mediate correction of the IMD in the patient’s cells in vivo.