Novel vectors and approaches for gene therapy in liver diseases

Abstract

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Keywords: AAT, α1-antitrypsin; AAV, adeno-associated virus; AHP, acute hepatic porphyrias; AIP, acute intermittent porphyria; ALAS1, aminolevulic synthase 1; APCs, antigen-presenting cells; ASGCT, American Society of Gene and Cell Therapy; ASGPR, asialoglycoprotein receptor; ASOs, antisense oligonucleotides; Ad, adenovirus; CBS, cystathionine β-synthase; CN, Crigel-Najjar; CRISPR, clustered regularly interspaced short palindromic repeats; CRISPR/Cas9, CRISPR associated protein 9; DSBs, double-strand breaks; ERT, enzyme replacement therapy; FH, familial hypercholesterolemia; FSP27, fat-specific protein 27; GO, glycolate oxidase; GSD1a, glycogen storage disorder 1a; GT, gene therapy; GUSB, β-glucuronidase; GalNAc, N-acetyl-D-galactosamine; HDAd, helper-dependent adenovirus; HDR, homology-directed repair; HT, hereditary tyrosinemia; HemA/B, haemophilia A/B; IDS, iduronate 2-sulfatase; IDUA, α-L-iduronidase; IMLD, inherited metabolic liver diseases; ITR, inverted terminal repetition; LDH, lactate dehydrogenase; LDLR, low-density lipoprotein receptor; LNP, Lipid nanoparticles; LTR, long terminal repeat; LV, lentivirus; MMA, methylmalonic acidemia; MPR, metabolic pathway reprograming; MPS type I, MPSI; MPS type VII, MPSVII; MPS, mucopolysaccharidosis; NASH, non-alcoholic steatohepatitis; NHEJ, non-homologous end joining; NHPs, non-human primates; Non-viral vectors; OLT, orthotopic liver transplantation; OTC, ornithine transcarbamylase; PA, propionic acidemia; PB, piggyBac; PCSK9, proprotein convertase subtilisin/kexin type 9; PEG, polyethylene glycol; PEI, polyethyleneimine; PFIC3, progressive familial cholestasis type 3; PH1, Primary hyperoxaluria type 1; PKU, phenylketonuria; RV, retrovirus; S/MAR, scaffold matrix attachment regions; SB, Sleeping Beauty; SRT, substrate reduction therapy; STK25, serine/threonine protein kinase 25; TALEN, transcription activator-like effector nucleases; TTR, transthyretin; UCD, urea cycle disorders; VLDLR, very-low-density lipoprotein receptor; WD, Wilson’s disease; ZFN, zinc finger nucleases; apoB/E, apolipoprotein B/E; dCas9, dead Cas9; efficacy; gene addition; gene editing; gene silencing; hepatocytes; immune response; lncRNA, long non-coding RNA; miRNAs, microRNAs; siRNA, small-interfering RNA; toxicity; viral vectors.

Fig. 1

Gene therapy strategies currently being used for the treatment of liver diseases can be divided into 3 main categories. Gene repair involves the correction of an existing mutation to restore the expression of the correct version of the protein. Gene supplementation (or addition) is simply the delivery of the correct version of the gene expressing the missing or defective protein. Gene silencing requires the elimination or reduction of the expression of a protein to reduce toxic metabolites.

Fig. 2

Gene therapy strategies. Ex vivo gene therapy starts with an extraction of cells that are transduced with the vector carrying the therapeutic gene and then reintroduced into the body. In vivo gene therapy is based on the direct administration of the gene delivery vector or genetic material to the organism and can utilise different types of vectors, including non-viral and viral vectors. AAV, adeno-associated virus.

Fig. 3

Non-viral and viral vectors used most frequently in liver-directed gene therapy. Each possesses varying characteristics, benefits, and limitations, which are essential for their selection in a wide variety of gene therapy applications.

Fig. 4

Examples of several strategies that have advanced the field of gene therapy. Naked plasmid DNA, plasmid DNA containing S/MAR sequences, transposons, and gene editing nucleases (e.g. CRISPR/Cas9). dsDNA, double-stranded DNA; S/MAR, scaffold matrix attachment regions; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA.