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Modern therapeutic approaches to liver-related disorders

  • Author Footnotes
    ≠ These authors contributed equally to this work.
    Antoine Gardin
    Footnotes
    ≠ These authors contributed equally to this work.
    Affiliations
    Hépatologie et Transplantation Hépatique Pédiatriques, Centre de référence de l’atrésie des voies biliaires et des cholestases génétiques, FSMR FILFOIE, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Hôpital Bicêtre, AP-HP, Université Paris-Saclay, Le Kremlin-Bicêtre, France

    Genethon, 91000 Evry, France

    Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, 91000 Evry, France
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  • Author Footnotes
    ≠ These authors contributed equally to this work.
    Katharina Remih
    Footnotes
    ≠ These authors contributed equally to this work.
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Aachen, Germany
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  • Emmanuel Gonzales
    Affiliations
    Hépatologie et Transplantation Hépatique Pédiatriques, Centre de référence de l’atrésie des voies biliaires et des cholestases génétiques, FSMR FILFOIE, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Hôpital Bicêtre, AP-HP, Université Paris-Saclay, Hépatinov, Inserm U 1193, Le Kremlin-Bicêtre, France
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  • Emma Rachel Andersson
    Affiliations
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
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  • Pavel Strnad
    Correspondence
    Corresponding author. Address: Medical Clinic III, Gastroenterology, Metabolic diseases, and Intensive Care, University Hospital Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. Tel.: +49 241 80-35324.
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Aachen, Germany
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  • Author Footnotes
    ≠ These authors contributed equally to this work.

      Summary

      The liver is a key production and processing site that is essential for health. Liver dysfunction can result in both systemic and liver-specific diseases. To combat these diseases, genetic approaches have been developed that have high liver tropism and are based on gene addition/editing or gene silencing. The gene addition/editing approach has yielded encouraging clinical data on the use of viral vectors in patients with haemophilia, as well as neuromuscular diseases, and has led to trials for liver-related disorders. However, the immune response and the long-term stability of exogenous expression remain important challenges. Gene editing and mRNA therapy have yielded first in-human proof-of-concept therapeutics and vaccines, but the road to the treatment of liver-related disorders remains long. Gene silencing is accomplished primarily via antisense oligonucleotides and small-interfering RNAs (siRNAs). siRNA modification with N-acetyl galactosamine results in hepatocellular-specific targeting and catapulted the liver to the centre of siRNA research. Several siRNA drugs for liver-related disorders have recently been approved, and the pipeline of drugs under investigation is crowded. Loss-of-function mutations might also be treated with enzyme substitution therapy. This review summarises current genetic approaches as well as key enzyme substitution therapies, focusing on recently approved compounds, potential adverse effects, and future challenges. Collectively, these recent advances place the liver at the forefront of precision medicine for metabolic and genetic diseases and are expected to transform the care and treatment of patients with both liver-specific and systemic diseases.

      Keywords

      Introduction

      The liver is a central metabolic hub that plays major roles in metabolism and is particularly accessible for the development of novel therapies.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      In the past decade, numerous therapeutics with high liver tropism have been developed.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      These approaches supply genetic material, either DNA or RNA, to restore (gene addition/editing) or silence (gene silencing/editing) the expression of a gene of interest.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      (Fig. 1) These treatments extend beyond bona fide liver disorders and include targets for metabolic disorders, such as hyperlipidaemias or diseases originating in the liver, such as haemophilia,
      • Nathwani A.C.
      Gene therapy for hemophilia.
      porphyria,
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      • Sardh E.
      • Ventura P.
      • Peiró P.A.
      • Rees D.C.
      • Stölzel U.
      • et al.
      Phase 3 trial of RNAi therapeutic givosiran for acute intermittent porphyria.
      or primary hyperoxaluria.
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      • Koren M.J.
      • O’Riordan W.D.
      • Cochat P.
      • et al.
      Lumasiran, an RNAi therapeutic for primary hyperoxaluria type 1.
      These advances highlight the therapeutic potential of liver-targeted molecular approaches to treat monogenic disorders, some of which are associated with high unmet medical needs, for example, “conventional” genetic liver diseases including alpha-1 antitrypsin deficiency and Wilson disease (WD). Finally, the knowledge gained is being used to treat more common diseases, such as chronic HBV infection and non-alcoholic fatty liver disease. Although several breakthroughs have been achieved, multiple challenges remain, particularly immune responses and potential toxicity, thus necessitating the modification of drug delivery strategies.
      • Nathwani A.C.
      Gene therapy for hemophilia.
      Figure thumbnail gr1
      Fig. 1Overview of therapeutic strategies.
      AAA, poly-A tail of mRNA; ASO, antisense oligonucleotide; siRNA, small-interfering RNA; WT, wild type.
      This review provides an overview of existing liver-targeted molecular approaches, with a special focus on recently approved therapeutics. For deeper insight into their biological mechanisms, readers are referred to recent excellent reviews on these topics.
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      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      ,
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      The CRISPR tool kit for genome editing and beyond.
      Cell therapy and bioengineering/regenerative medicine have been discussed elsewhere.
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      Mesenchymal stromal cell therapy for liver diseases.
      ,
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      Understanding liver regeneration.

      Non-genetic approaches

      Although genetic strategies are the focus of this review, enzyme replacement/substitution therapies (ERT) are also used to treat many genetic diseases caused by a loss-of-function phenotype. Lysosomal and glycogen storage disorders are the prime examples. Avalglucosidase alfa-ngpt, the most recently approved member of this large family of therapeutics, is indicated for the long-term treatment of late-onset Pompe disease, which is caused by a deficiency in the lysosomal enzyme acid alpha-glucosidase leading to lysosomal accumulation of glycogen.
      • van der Ploeg A.T.
      • Reuser A.J.
      Pompe’s disease.
      Modified avalglucosidase alfa-ngpt displays more efficient uptake than its predecessor and stronger glycogen clearance from skeletal muscles.
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      • Barohn R.J.
      • Byrne B.J.
      • Desnuelle C.
      • Goker-Alpan O.
      • Ladha S.
      • et al.
      Safety, tolerability, pharmacokinetics, pharmacodynamics, and exploratory efficacy of the novel enzyme replacement therapy avalglucosidase alfa (neoGAA) in treatment-naïve and alglucosidase alfa-treated patients with late-onset Pompe disease: a phase 1, open-label, multicenter, multinational, ascending dose study.
      An overview of FDA- and/or EMA-approved ERTs can be found in Table 1.
      Because of its accessibility, the liver is an ideal target for the development of novel nucleic acid-based therapeutics.
      Table 1Overview of approved enzyme replacement therapies.
      Brand nameMarketing authorisation holderEnzymeApprovalIndication
      Prolastin-CGrifolsAlpha-1 antitrypsin inhibitorFDA: 02/1988AATD
      Aralast-NPBaxter HealthcareFDA: 05/2007
      GlassiaKamadaFDA: 07/2010
      ZemairaCSL BehringFDA: 07/2003
      RespreezaCSL BehringEMA: 08/2015
      CerezymeSanofi GenzymeImigluceraseFDA: 05/1994Gaucher disease
      EMA: 11/1997
      ElelysoPfizerTaliglucerase alfaFDA: 05/2012
      VprivTakeda ShireVelaglucerase alfaFDA: 02/2010

      EMA: 08/2010
      ReplagalTakeda ShireAgalsidase alfaEMA: 08/2001Fabry disease
      FabrazymeSanofi GenzymeAgalsidase betaFDA: 04/2003

      EMA: 08/2001
      Fabry disease
      Lumizyme

      Myozyme
      Alglucosidase alfaFDA: 05/2010

      EMA: 03/2006
      Pompe disease
      Nexviazyme

      Nexviadyme
      Avalglucosidase alfa-ngptFDA: 08/2021
      AldurazymeLaronidaseFDA: 03/2003

      EMA: 06/2003
      MPS I
      ElapraseTakeda ShireIdursulfaseFDA: 07/2006

      EMA: 01/2007
      MPS II
      VimizimBioMarin InternationalElosulfase alfaFDA: 02/2014

      EMA: 04/2014
      MPS IVA
      NaglazymeGalsulfaseFDA: 05/2005

      EMA: 01/2006
      MPS VI
      KanumaAlexionSebelipase alfaFDA: 12/2015

      EMA: 08/2015
      LAL deficiency
      This table includes only products with FDA approval and/or central EMA approval.
      AATD, alpha 1-antitrypsin deficiency; ADA, adenosine deaminase; LAL, lysosomal acid lipase; MPS, mucopolysaccharidosis.
      Although ERT significantly improves patient quality of life, numerous complications of the diseases are not improved by the therapy. Moreover, patients may develop antibodies against the supplemented proteins, thus limiting treatment efficacy. Genetic approaches are therefore an attractive alternative that might provide treatments for patients with very rare conditions.

      mRNA therapy

      mRNA therapy is an emerging therapeutic approach for diseases, which has been at the forefront of new vaccines for COVID-19.
      • Olliaro P.
      • Torreele E.
      • Vaillant M.
      COVID-19 vaccine efficacy and effectiveness—the elephant (not) in the room.
      Synthetic mRNA vaccines use the same host machinery as vaccines based on live attenuated viruses (such as measles, mumps, and rubella): the mRNA is delivered to the host’s cells and translated into viral proteins, which are recognised by the host’s immune system. The currently approved mRNA vaccines are highly efficient and very safe.
      • Olliaro P.
      • Torreele E.
      • Vaillant M.
      COVID-19 vaccine efficacy and effectiveness—the elephant (not) in the room.
      Although vaccines are an ideal application of the mRNA drug class because a few doses of transiently expressed mRNA are sufficient to generate a burst of protein expression and elicit immune memory, the promise of synthetic mRNA drugs extends to rescuing metabolic diseases through re-expression of deficient proteins, and to personalised medicine in cancer using antibody-encoding mRNA to replace protein-based monoclonal antibodies.
      Initial efforts to use synthetic mRNA as a drug were hampered by the rapid degradation of mRNA in vivo, the host response to foreign RNA,
      • Karikó K.
      • Ni H.
      • Capodici J.
      • Lamphier M.
      • Weissman D.
      mRNA is an endogenous ligand for toll-like receptor 3.
      and difficulties in mRNA delivery. Crucial developments enabling the clinical use of synthetic mRNAs as therapies included nucleoside modifications to improve protein translation and decrease the immunogenicity of the mRNA itself,
      • Anderson B.R.
      • Muramatsu H.
      • Nallagatla S.R.
      • Bevilacqua P.C.
      • Sansing L.H.
      • Weissman D.
      • et al.
      Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation.
      ,
      • Karikó K.
      • Buckstein M.
      • Ni H.
      • Weissman D.
      Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.
      as reviewed in.
      • Song J.
      • Yi C.
      Chemical modifications to RNA: a new layer of gene expression regulation.
      Furthermore, developments in lipid nanoparticle (LNP) technology, which was originally developed to deliver small-interfering RNA (siRNA) to the liver,
      • Kanasty R.
      • Dorkin J.R.
      • Vegas A.
      • Anderson D.
      Delivery materials for siRNA therapeutics.
      have been found to be ideal for protecting mRNA from degradation and mediating intracellular delivery. LNPs comprise phospholipids, sterols, polyethylene glycol (PEG)-conjugated lipids (which enable evasion from phagocytic cells), and ionizable lipids (which mediate endosomal escape after endocytosis
      • Sabnis S.
      • Kumarasinghe E.S.
      • Salerno T.
      • Mihai C.
      • Ketova T.
      • Senn J.J.
      • et al.
      A novel amino lipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates.
      ) and can mediate organ selectivity
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      • Cheng Q.
      • Wei T.
      • Yu X.
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      • Farbiak L.
      • et al.
      Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR–Cas gene editing.
      ).
      Beyond treating liver diseases, synthetic mRNA delivered to the liver can repurpose the liver as a bioreactor to replace deficient enzymes or essential proteins. Although no mRNA therapy for liver disease has been approved for use in humans, preclinical research has demonstrated success in animal models of liver diseases (Table 2). In the field of regenerative medicine, mRNA-mediated hepatic expression of hepatocyte growth factor and epidermal growth factor has been found to induce liver regeneration in mouse models.
      • Rizvi F.
      • Everton E.
      • Smith A.R.
      • Liu H.
      • Osota E.
      • Beattie M.
      • et al.
      Murine liver repair via transient activation of regenerative pathways in hepatocytes using lipid nanoparticle-complexed nucleoside-modified mRNA.
      Table 2Pre-clinical evidence of the effects of mRNA therapy.
      DiseaseModelDurabilityFunctional impactRef.
      Ornithine transcarbamylase (OTC) deficiency
      • Otcspf-ash mice
      • Human OTC mRNA, Hybrid mRNA technology delivery (LNP)
      Elevated OTC activity/protein levels up to 10 daysProlonged survival
      • Prieve M.G.
      • Harvie P.
      • Monahan S.D.
      • Roy D.
      • Li A.G.
      • Blevins T.L.
      • et al.
      Targeted mRNA therapy for ornithine transcarbamylase deficiency.
      Crigler-Najar syndrome type 1
      • Gunn rats, Gunn-UGT1a1j/BluHsdRrrc
      • Human UGT1A1 mRNA in LNP
      Cytoplasmic half-life of hUGT1A1-modRNA was 13.4 hoursDiminished bilirubin levels
      • Apgar J.F.
      • Tang J.-P.
      • Singh P.
      • Balasubramanian N.
      • Burke J.
      • Hodges M.R.
      • et al.
      Quantitative systems pharmacology model of hUGT1A1-modRNA encoding for the UGT1A1 enzyme to treat crigler-najjar syndrome type 1.
      Alpha-1 antitrypsin (AAT) deficiency
      • Primary human hepatocytes (control and AAT deficient)
      • Male NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg (SERPINA1∗E342K) Slcw/SzJ, (NSG-PiZ) mice
      • Human AAT mRNA in LNP
      AAT secretion into sinusoidal space by 24 hoursIncrease in secreted AAT protein and protease inhibitory capacity
      • Karadagi A.
      • Cavedon A.G.
      • Zemack H.
      • Nowak G.
      • Eybye M.E.
      • Zhu X.
      • et al.
      Systemic modified messenger RNA for replacement therapy in alpha 1-antitrypsin deficiency.
      Acute intermittent porphyria (AIP)
      • Phenobarbital-induced acute attack in AIP mice (crossbred T1 (C57BL/6 Pbgdtm1(neo)Uam), and T2 (C57BL/6 Pbgdtm2(neo)Uam) mice)
      • Rifampicin-induced porphyrin accumulation in rabbits.
      • Non-human primates
      • Human PBGD mRNA in LNPs
      Increased PBGD levels for up to 10 days.Reduction and normalisation of porphyrin precursors as well as the related symptoms

      Increased hepatic PBGD activity in non-human primates
      • Jiang L.
      • Berraondo P.
      • Jericó D.
      • Guey L.T.
      • Sampedro A.
      • Frassetto A.
      • et al.
      Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.
      Methylmalonic acidaemia
      • Mut-/- mice
      • Mut-/-; TgINS-MCK-Mut mice
      • Mut-/-; TgINS-CBA-G715V mice
      • hMUT mRNA in LNP
      Elevated hMUT protein levels for 5 days75%–85% reduction in plasma methylmalonic acid

      Improved growth and survival, decreased metabolites in blood and tissue
      • An D.
      • Schneller J.L.
      • Frassetto A.
      • Liang S.
      • Zhu X.
      • Park J.-S.
      • et al.
      Systemic messenger RNA therapy as a treatment for methylmalonic acidemia.
      Thrombotic thrombocytopenic purpura
      • ADAMTS13-deficient mice
      • Human ADAMTS13 in LNPs
      Plasma hADAMTS13 mRNA detectable at 24 hoursADAMTS13 activity in plasma detectable for up to 5 days.
      • Liu-Chen S.
      • Connolly B.
      • Cheng L.
      • Subramanian R.R.
      • Han Z.
      mRNA treatment produces sustained expression of enzymatically active human ADAMTS13 in mice.
      Glycogen storage disease type 1A
      • Liver-specific G6pc-/- mouse (Albumin-Cre)
      • Human G6PC in LNPs
      Up to 14 days of expression with protein-engineered G6PC in primary hepatocytesImprovement in fasting blood glucose levels for up to 12 days.
      • Roseman D.S.
      • Khan T.
      • Rajas F.
      • Jun L.S.
      • Asrani K.H.
      • Isaacs C.
      • et al.
      G6PC mRNA therapy positively regulates fasting blood glucose and decreases liver abnormalities in a mouse model of glycogen storage disease 1a.
      Factor IX deficiency haemophilia B
      • FIX-deficient haemophilic mice
      • hFIX mRNA in lipid-enabled and unlocked nucleic acid modified RNA (LUNAR) LNPs
      Elevated FIX levels between 6-24 hours, declining after 48 hoursTherapeutically relevant improvement of FIX activity and clotting activity peaking at 24 hours.
      • Ramaswamy S.
      • Tonnu N.
      • Tachikawa K.
      • Limphong P.
      • Vega J.B.
      • Karmali P.P.
      • et al.
      Systemic delivery of factor IX messenger RNA for protein replacement therapy.
      Arginase deficiency
      • Conditional Arg1-/- mice with
      • AAV8-TBG-Cre
      • Human codon-optimised ARG1 mRNA
      hARG1 mRNA levels in liver peak at 2 hours, still elevated at 1 day.Prolonged survival by >50 days after repeated dosing, plasma metabolites improved
      • Truong B.
      • Allegri G.
      • Liu X.-B.
      • Burke K.E.
      • Zhu X.
      • Cederbaum S.D.
      • et al.
      Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.
      AAV, adeno-associated virus; AAT, alpha-1 antitrypsin; ADAMTS13, von Willebrand Factor metalloprotease; AIP, acute intermittent porphyria; ARG1, arginase 1; (h)FIX, (humanised) factor IX; G6PC, glucose-6-phosphatase; LNP, lipid nanoparticle; (h)MUT, (humanised) methylmalonyl-CoA mutase; OTC, ornithine transcarbamylase; PBGD, porphobilinogen deaminase; TBG, Thyroxine binding globulin promoter; UGT1A1, UDP-glucuronosyltransferase 1A1.
      One of the advantages of mRNA therapies is that they can be easily modified to incorporate new sequences – for example, vaccines can be adjusted to new viral strains – however, other aspects of mRNA therapeutics make them less suitable as drugs. Transient expression of mRNA is desirable in the production of antigens for a vaccine-induced immune response, but in the case of ERT, the transient expression necessitates repeated administration of mRNA. Combining synthetic mRNA with CRISPR/Cas9
      • Jinek M.
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      • Fonfara I.
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      A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity.
      ,
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • et al.
      Multiplex genome engineering using CRISPR/Cas systems.
      may circumvent these limitations. Synthetic mRNAs encoding base editors and single-guide RNAs (sgRNAs) have demonstrated in vivo efficacy for the treatment of hypercholesterolaemia in non-human primates by inducing a loss of function in proprotein convertase subtilisin kexin type 9 (PCSK9)
      • Musunuru K.
      • Chadwick A.C.
      • Mizoguchi T.
      • Garcia S.P.
      • DeNizio J.E.
      • Reiss C.W.
      • et al.
      In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.
      and more recently for the treatment of 6 patients with transthyretin amyloidosis through silencing of misfolded transthyretin (TTR).
      • Gillmore J.D.
      • Gane E.
      • Taubel J.
      • Kao J.
      • Fontana M.
      • Maitland M.L.
      • et al.
      CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis.
      Thus, recent advances in mRNA therapy coupled with CRISPR/Cas9 may leverage the advantages of both methods to treat rare liver diseases.

      Gene addition

      Different types of vectors

      In the past 20 years, gene therapy targeting the liver has become an attractive therapeutic option for monogenic disorders, and intense research and several clinical trials are ongoing. The therapeutic aim is to restore the expression of a gene of interest, either through the addition of an exogenous DNA sequence (gene addition) or through modification of the patient’s genome (gene editing) (reviewed in
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      ,
      • Kattenhorn L.M.
      • Tipper C.H.
      • Stoica L.
      • Geraghty D.S.
      • Wright T.L.
      • Clark K.R.
      • et al.
      Adeno-associated virus gene therapy for liver disease.
      and the “Gene editing” section) (Fig. 2). Of the many viral and non-viral vectors allowing nucleic acids to reach the liver, few are currently being investigated in clinical trials, and recombinant adeno-associated viral vectors (rAAVs) are the leading candidates.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      rAAVs are derived from replication-defective DNA viruses in which the viral genes have been replaced by an expression cassette of limited size (<4.7 kb). rAAV have low immunogenicity and low integration ability, and most serotypes have high liver tropism.
      • Wang D.
      • Tai P.W.L.
      • Gao G.
      Adeno-associated virus vector as a platform for gene therapy delivery.
      rAAV can persist for years as episomes in the nuclei of slowly/non-dividing cells (such as adult hepatocytes).
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      ,
      • Kotterman M.A.
      • Chalberg T.W.
      • Schaffer D v
      Viral vectors for gene therapy: translational and clinical outlook.
      Therefore, they exhibit a favourable profile for gene transfer in the adult liver but are not expected to achieve persistent transgene expression in children, because the episomes do not replicate and are diluted during the growth of the liver. Furthermore, the presence of pre-existing neutralising antibodies (NAbs) against a given rAAV serotype in approximately 40% of patients would exclude these patients from corresponding clinical trials.
      • Boutin S.
      • Monteilhet V.
      • Veron P.
      • Leborgne C.
      • Benveniste O.
      • Montus M.F.
      • et al.
      Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors.
      In addition, NAb development after rAAV infusion impedes successful reinjection and sequential use unless switching to another serotype. In contrast, an “integrative” strategy could allow for permanent transgene expression and potentially even positive selection of corrected cells; however, it carries a higher theoretical risk of genotoxicity.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      Integration can be achieved by using (i) an integrative vector such as an optimised lentivirus
      • Milani M.
      • Annoni A.
      • Moalli F.
      • Liu T.
      • Cesana D.
      • Calabria A.
      • et al.
      Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.
      or (ii) an integrative transgene dependent on a nuclease (such as CRISPR), a transposase, or a nuclease-free DNA template that integrates into the endogenous Albumin locus through homologous recombination.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      ,
      • Siew S.M.
      • Cunningham S.C.
      • Zhu E.
      • Tay S.S.
      • Venuti E.
      • Bolitho C.
      • et al.
      Prevention of cholestatic liver disease and reduced tumorigenicity in a murine model of PFIC type 3 using hybrid AAV-piggyBac gene therapy.
      ,
      • Chandler R.J.
      • Venturoni L.E.
      • Liao J.
      • Hubbard B.T.
      • Schneller J.L.
      • Hoffmann V.
      • et al.
      Promoterless, nuclease-free genome editing confers a growth advantage for corrected hepatocytes in mice with methylmalonic acidemia.
      The integrative transgene has been successfully delivered to the liver using rAAVs or LNP in preclinical studies
      • Siew S.M.
      • Cunningham S.C.
      • Zhu E.
      • Tay S.S.
      • Venuti E.
      • Bolitho C.
      • et al.
      Prevention of cholestatic liver disease and reduced tumorigenicity in a murine model of PFIC type 3 using hybrid AAV-piggyBac gene therapy.
      • Chandler R.J.
      • Venturoni L.E.
      • Liao J.
      • Hubbard B.T.
      • Schneller J.L.
      • Hoffmann V.
      • et al.
      Promoterless, nuclease-free genome editing confers a growth advantage for corrected hepatocytes in mice with methylmalonic acidemia.
      • Li N.
      • Gou S.
      • Wang J.
      • Zhang Q.
      • Huang X.
      • Xie J.
      • et al.
      CRISPR/Cas9-Mediated gene correction in newborn rabbits with hereditary tyrosinemia type I.
      • Yin H.
      • Song C.-Q.
      • Dorkin J.R.
      • Zhu L.J.
      • Li Y.
      • Wu Q.
      • et al.
      Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.
      ; however, studies in humans are limited. Although promising, the use of viral vectors carries certain risks, as discussed below.
      Figure thumbnail gr2
      Fig. 2Overview of strategies used for gene addition and gene editing.
      In a gene addition strategy, the transgene either is episomal in the nucleus, if delivered through rAAV or adenovirus, or integrates in the genome. For the latter, an integrative vector (lentivirus), a self-integrating transgene (often in the albumin locus) using homologous recombination, or a transposase that is co-delivered with the transgene can be applied. In gene editing, a DNA template is co-delivered with Cas9 and the guide RNA by using rAAV or lipid nanoparticles. The guide RNA targets Cas9 to the specific locus and induces a DNA double strand break, which is then repaired through either HDR or NHEJ. In NHEJ, the gene is often silenced because of insertions and/or deletions, whereas in HDR, the co-delivered DNA fragment serves as a template to introduce the desired modification and often restores gene expression. In base editing and prime editing, fusion of Cas9 to nucleotide deaminases or reverse transcriptase directs non-nuclease mediated single nucleotide edits or precise editing of DNA, respectively. AAV, adeno-associated virus; dsDNA, double strand DNA; HDR, homology-directed repair; NHEJ, non-homologous end-joining; RT, reverse transcriptase; ssDNA, single-stranded DNA; ssRNA, single strand RNA; TALEN, transcription activator-like effector nuclease; TP, transposase; ZFN, zinc finger nuclease.

      Immune response and liver toxicity

      Despite showing significant efficacy, early clinical trials have highlighted some limitations of rAAV gene therapy. The first is the immune response, specifically the T-cell response against the capsid and NAb production.
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      ,
      • Kotterman M.A.
      • Chalberg T.W.
      • Schaffer D v
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      ,
      • Nathwani A.C.
      • Tuddenham E.G.D.
      • Rangarajan S.
      • Rosales C.
      • McIntosh J.
      • Linch D.C.
      • et al.
      Adenovirus-associated virus vector–mediated gene transfer in hemophilia B.
      The T cell-mediated immune response usually occurs between 6 and 10 weeks after infusion; it manifests as an increase in the liver enzyme alanine aminotransferase with a possible loss of transduced hepatocytes and is proportional to the vector dose administered.
      • Nathwani A.C.
      Gene therapy for hemophilia.
      ,
      • Nathwani A.C.
      • Tuddenham E.G.D.
      • Rangarajan S.
      • Rosales C.
      • McIntosh J.
      • Linch D.C.
      • et al.
      Adenovirus-associated virus vector–mediated gene transfer in hemophilia B.
      Early steroid treatment decreases this immune response, which does not recur during follow-up, because it is directed mainly against capsid antigens that are transiently present in the initial phase.
      • Nathwani A.C.
      Gene therapy for hemophilia.
      ,
      • Nathwani A.C.
      • Tuddenham E.G.D.
      • Rangarajan S.
      • Rosales C.
      • McIntosh J.
      • Linch D.C.
      • et al.
      Adenovirus-associated virus vector–mediated gene transfer in hemophilia B.
      In contrast, no immune responses have been observed against the transgene in haemophilia trials.
      • Nathwani A.C.
      • Reiss U.
      • Tuddenham E.
      • Chowdary P.
      • McIntosh J.
      • Riddell A.
      • et al.
      “Adeno-Associated mediated gene transfer for hemophilia B: 8 Year follow up and impact of removing ‘empty viral particles’ on safety and efficacy of gene transfer” [abstract]. 60th ASH annual meeting.
      ,
      • Pasi K.J.
      • Rangarajan S.
      • Mitchell N.
      • Lester W.
      • Symington E.
      • Madan B.
      • et al.
      Multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia A.
      Finally, these trials have also revealed the importance of innate immune responses to CpG contained in the expression cassette, which was not anticipated on the basis of preclinical studies. Two haemophilia B trials were stopped after complete loss of transgene expression occurred 6–10 weeks after the infusion of vectors with high CpG content, despite the use of steroids.
      • Konkle B.A.
      • Walsh C.E.
      • Escobar M.A.
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      • Young G.
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      • et al.
      BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.
      ,
      • Pipe S.
      • Stine K.
      • Rajasekhar A.
      • Everington T.
      • Poma A.
      • Crombez E.
      • et al.
      “101HEMB01 is a phase 1/2 open-label, single ascending dose-finding trial of DTX101 (AAVrh10FIX) in patients with moderate/severe hemophilia B that demonstrated meaningful but transient expression of human factor IX (hFIX)” [abstract]. 59th ASH annual meeting.
      Haemophilia research paved the way for liver-targeted gene therapy, highlighting both the efficacy and limitations of this approach.
      A second limitation is genotoxicity. The Rep proteins of wild type (WT) AAV enable integration of viral DNA into the host genome.
      • Kotterman M.A.
      • Chalberg T.W.
      • Schaffer D v
      Viral vectors for gene therapy: translational and clinical outlook.
      Clonal insertions of WT AAV2 genomes have been identified in hepatocellular carcinoma (HCC) in patients without known risk factors, thus suggesting an oncogenic role in humans.
      • Nault J.-C.
      • Datta S.
      • Imbeaud S.
      • Franconi A.
      • Mallet M.
      • Couchy G.
      • et al.
      Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.
      ,
      • la Bella T.
      • Imbeaud S.
      • Peneau C.
      • Mami I.
      • Datta S.
      • Bayard Q.
      • et al.
      Adeno-associated virus in the liver: natural history and consequences in tumour development.
      The 3´-inverted terminal repeats of AAV2, which were present in most of these insertions, can transactivate nearby oncogenes through a liver-specific enhancer sequence.
      • Logan G.J.
      • Dane A.P.
      • Hallwirth C v
      • Smyth C.M.
      • Wilkie E.E.
      • Amaya A.K.
      • et al.
      Identification of liver-specific enhancer–promoter activity in the 3′ untranslated region of the wild-type AAV2 genome.
      In contrast to that with WT AAV, the risk of HCC after rAAV infusion appears low, because all coding sequences including the Rep genes have been removed. Nonetheless, random genomic integration has been identified at a frequency of 0.1 to 1% in preclinical models.
      • Dalwadi D.A.
      • Calabria A.
      • Tiyaboonchai A.
      • Posey J.
      • Naugler W.E.
      • Montini E.
      • et al.
      AAV integration in human hepatocytes.
      ,
      • Nguyen G.N.
      • Everett J.K.
      • Kafle S.
      • Roche A.M.
      • Raymond H.E.
      • Leiby J.
      • et al.
      A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.
      Most studies in adult mice have not found an increased risk of HCC or gene expression dysregulation after rAAV infusion.
      • Bell P.
      • Wang L.
      • Lebherz C.
      • Flieder D.B.
      • Bove M.S.
      • Wu D.
      • et al.
      No evidence for tumorigenesis of AAV vectors in a large-scale study in mice.
      ,
      • Li H.
      • Malani N.
      • Hamilton S.R.
      • Schlachterman A.
      • Bussadori G.
      • Edmonson S.E.
      • et al.
      Assessing the potential for AAV vector genotoxicity in a murine model.
      In contrast, rAAV infusion in neonatal mice has been associated with an incidence of HCC exceeding 50% in some studies, because of the insertion of the vector into the Rian locus, which is expressed during the neonatal period, inside the Mir341 gene.
      • Donsante A.
      • Miller D.G.
      • Li Y.
      • Vogler C.
      • Brunt E.M.
      • Russell D.W.
      • et al.
      AAV vector integration sites in mouse hepatocellular carcinoma.
      • Chandler R.J.
      • LaFave M.C.
      • Varshney G.K.
      • Trivedi N.S.
      • Carrillo-Carrasco N.
      • Senac J.S.
      • et al.
      Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy.
      • Wang P.-R.
      • Xu M.
      • Toffanin S.
      • Li Y.
      • Llovet J.M.
      • Russell D.W.
      Induction of hepatocellular carcinoma by in vivo gene targeting.
      An orthologue of this locus exists in most species including humans, but without the rodent-specific Mir341 gene, and is not an integration hotspot. No HCC has been identified after rAAV infusion at 8 years of follow-up in cats,
      • Ferla R.
      • Alliegro M.
      • Dell’Anno M.
      • Nusco E.
      • Cullen J.M.
      • Smith S.N.
      • et al.
      Low incidence of hepatocellular carcinoma in mice and cats treated with systemic adeno-associated viral vectors.
      nor have genotoxic insertions been observed in non-human primates or humans, although insertions have been found throughout the genome.
      • Gil-Farina I.
      • Fronza R.
      • Kaeppel C.
      • Lopez-Franco E.
      • Ferreira V.
      • D’Avola D.
      • et al.
      Recombinant AAV integration is not associated with hepatic genotoxicity in nonhuman primates and patients.
      A recent study has reported rAAV insertion within oncogenes in the livers of 2 dogs with haemophilia, with clonal expansion but no HCC after 6–10 years of follow-up.
      • Nguyen G.N.
      • Everett J.K.
      • Kafle S.
      • Roche A.M.
      • Raymond H.E.
      • Leiby J.
      • et al.
      A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.
      Recently, 1 patient from a trial of Uniqure for haemophilia A (AMT-061) has been reported to have developed HCC 1 year after infusion. However, this patient had a history of chronic hepatitis B and C virus infection as well as non-alcoholic fatty liver disease. Molecular analysis of this tumour revealed low random rAAV insertions without clonal expansion, thus suggesting that the rAAV did not contribute to oncogenesis.
      • Kaiser J.
      Experimental gene therapy for hemophilia probably did not cause patient’s liver tumor.
      Normal alpha-fetoprotein levels and no cases of tumourigenesis were reported after long-term follow-up (12–15 years) of 4 patients with severe haemophilia B included in one of the first clinical trials of rAAV-mediated gene transfer.
      • George L.A.
      • Ragni M.v.
      • Rasko J.E.J.
      • Raffini L.J.
      • Samelson-Jones B.J.
      • Ozelo M.
      • et al.
      Long-term follow-up of the first in human intravascular delivery of AAV for gene transfer: AAV2-hFIX16 for severe hemophilia B.
      In summary, despite the random integration of rAAV and clonal expansion observed in preclinical studies, clinical evidence suggests a relatively safe profile over periods as long as 15 years, although meticulous long-term monitoring is mandatory. Removing the 3´-inverted terminal repeat liver-specific enhancer or avoiding the use of a promoter with strong transactivation potential
      • Chandler R.J.
      • LaFave M.C.
      • Varshney G.K.
      • Trivedi N.S.
      • Carrillo-Carrasco N.
      • Senac J.S.
      • et al.
      Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy.
      could further decrease this risk.
      Beyond genotoxicity, clinical trials for spinal muscular atrophy or myotubular myopathy (MTM) using much higher vector doses than those used for haemophilia to enable muscle targeting (>1014 vector genome (vg)/kg) have reported acute liver injury.
      • Chand D.
      • Mohr F.
      • McMillan H.
      • Tukov F.F.
      • Montgomery K.
      • Kleyn A.
      • et al.
      Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy.
      • Wilson J.M.
      • Flotte T.R.
      Moving forward after two deaths in a gene therapy trial of myotubular myopathy.
      • Shieh P.B.
      • Bönnemann C.G.
      • Müller-Felber W.
      • Blaschek A.
      • Dowling J.J.
      • Kuntz N.L.
      • et al.
      Re: “moving forward after two deaths in a gene therapy trial of myotubular myopathy” by Wilson and flotte.
      Approximately 90% of the patients included in the spinal muscular atrophy trials exhibited liver enzyme elevation within 1 week after rAAV infusion, probably because of unwanted rAAV liver targeting.
      • Chand D.
      • Mohr F.
      • McMillan H.
      • Tukov F.F.
      • Montgomery K.
      • Kleyn A.
      • et al.
      Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy.
      For MTM, at least 3 patients with pre-existing liver disease died from liver failure after receiving high vector doses (3.1014 vg/kg).
      • Wilson J.M.
      • Flotte T.R.
      Moving forward after two deaths in a gene therapy trial of myotubular myopathy.
      ,
      • Shieh P.B.
      • Bönnemann C.G.
      • Müller-Felber W.
      • Blaschek A.
      • Dowling J.J.
      • Kuntz N.L.
      • et al.
      Re: “moving forward after two deaths in a gene therapy trial of myotubular myopathy” by Wilson and flotte.
      These findings have important implications for patients with chronic liver diseases, such as WD or progressive familial intrahepatic cholestasis (PFIC), because preclinical studies have suggested that higher vector doses may be required to correct these phenotypes.
      • Siew S.M.
      • Cunningham S.C.
      • Zhu E.
      • Tay S.S.
      • Venuti E.
      • Bolitho C.
      • et al.
      Prevention of cholestatic liver disease and reduced tumorigenicity in a murine model of PFIC type 3 using hybrid AAV-piggyBac gene therapy.
      ,
      • Aronson S.J.
      • Bakker R.S.
      • Shi X.
      • Duijst S.
      • ten Bloemendaal L.
      • de Waart D.R.
      • et al.
      Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice.
      ,
      • Murillo O.
      • Moreno D.
      • Gazquez C.
      • Barberia M.
      • Cenzano I.
      • Navarro I.
      • et al.
      Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.
      A tight balance must be achieved between an appropriate vector dose to correct the phenotype and the adverse effects of potential liver toxicity and the immune response, which increase with vector dose; this balance is likely to be disease specific.

      Key clinical trials for haemophilia and liver diseases

      The first disease for which a liver-targeted gene therapy approach has been successful is haemophilia B (reviewed in
      • Nathwani A.C.
      Gene therapy for hemophilia.
      ); major insights have been obtained from these clinical trials regarding both efficacy and several of the limitations described above. Through the use of rAAV vectors (rAAV8 and rAAV5) injected into a peripheral vein, a high activity variant of the coagulation factor IX (FIX) gene has been stably expressed by hepatocytes, thus increasing circulating FIX activity to 30–40% of normal. Patients showed a significant decrease in bleeding episodes, and more than 90% of patients were able to stop using recombinant factor IX.
      • Nathwani A.C.
      Gene therapy for hemophilia.
      ,
      • George L.A.
      • Sullivan S.K.
      • Giermasz A.
      • Rasko J.E.J.
      • Samelson-Jones B.J.
      • Ducore J.
      • et al.
      Hemophilia B gene therapy with a high-specific-activity factor IX variant.
      ,
      • Pipe S.W.
      • Recht M.
      • Key N.S.
      • Leebeek F.W.G.
      • Castaman G.
      • Lattimore S.U.
      • et al.
      First data from the phase 3 HOPE-B gene therapy trial: efficacy and safety of etranacogene dezaparvovec (AAV5-Padua hFIX variant; AMT-061) in adults with severe or moderate-severe hemophilia B treated irrespective of pre-existing anti-capsid neutralizing antibodies.
      Although the duration of transgene expression remains unclear, several studies have reported expression for more than 8 years.
      • Nathwani A.C.
      • Reiss U.
      • Tuddenham E.
      • Chowdary P.
      • McIntosh J.
      • Riddell A.
      • et al.
      “Adeno-Associated mediated gene transfer for hemophilia B: 8 Year follow up and impact of removing ‘empty viral particles’ on safety and efficacy of gene transfer” [abstract]. 60th ASH annual meeting.
      For haemophilia A, the clotting factor VIII (FVIII) coding sequence is too long (7 kb) to be packaged into a single rAAV vector. Therefore, a truncated mutant (FVIII-SQ) has been used in phase I/II clinical trials with 2 to 3 years of follow-up; this treatment has yielded an impressive decrease in bleeding episodes (>90%) and FVIII concentrate infusions (>95%).
      • Nathwani A.C.
      Gene therapy for hemophilia.
      ,
      • Pasi K.J.
      • Rangarajan S.
      • Mitchell N.
      • Lester W.
      • Symington E.
      • Madan B.
      • et al.
      Multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia A.
      ,
      • Rangarajan S.
      • Walsh L.
      • Lester W.
      • Perry D.
      • Madan B.
      • Laffan M.
      • et al.
      AAV5–Factor VIII gene transfer in severe hemophilia A.
      An immune response requiring transient steroid use was observed in most patients treated with the highest doses of vectors. These trials have demonstrated that a single liver-targeted rAAV infusion induces prolonged expression of the transgene in undamaged liver, as well as near-complete correction of the phenotype without significant toxicity.
      Several clinical trials are currently ongoing for genetic liver metabolic diseases involving a specific enzyme deficiency in patients with no or mild fibrosis (Table 3 and complete list in
      • Kuzmin D.A.
      • Shutova M.v.
      • Johnston N.R.
      • Smith O.P.
      • Fedorin V.v.
      • Kukushkin Y.S.
      • et al.
      The clinical landscape for AAV gene therapies.
      ).
      Table 3Ongoing clinical trials for liver-targeted gene therapy.
      Sponsor (product)Vector (serotype-promoter-Tg)TrialStatus
      Haemophilia B
      St-Jude/UCLscAAV2/8-LP1-hFIXco WTPhase I/II (NCT00979238)Closed
      Spark Therapeutics (Fidanacogene elaparvovec)ssAAV Spark100-ApoE/hAAT-hFIXco PaduaPhase III (NCT03861273)Ongoing
      uniQure (Etranagogene dezaparvovec)ssAAV5-LP1-hFIXco PaduaPhase III (NCT03569891)Ongoing
      Shire/Takeda (BAX335)scAAV2/8-TTR-hFIXco PaduaPhase I/II (NCT01687608)Stopped (loss of expression)
      Ultragenyx (DTX101)AAVrh10-LP-hFIXco WTPhase I (NCT02618915)Stopped (loss of expression)
      Freeline (FLT180a)AAVS3-FRE1-hFIXco PaduaPhase I/II (NCT03369444)Ongoing
      Haemophilia A
      Pfizer/Sangamo (Giroctocogene fitelparvovec)ssAAV2/6-hFVIII-SQPhase I/II (NCT03061201)Ongoing
      BioMarin Pharmaceutical (Valoctocogene roxaparvovec)ssAAV5-ApoE-hAAT-hFVIII-SQPhase III (NCT03370913)Ongoing
      Sparks Therapeutics (SPK-8011)ssAAV LK03–LP–hFVIII-SQPhase I/II (NCT03003533)Ongoing
      St-Jude (GO-8 study)rAAV2/8-HLP-hFVIII-V3Phase I/II (NCT03001830)Ongoing
      Urea cycle disorder
      Ultragenyx (Avalotcagene ontaparvovec)scAAV8-TBG/AMBP-hOTCcoPhase I/II (NCT02991144)Ongoing
      Porphyria
      uniQure/University of NavarrassAAV2/5–Albe/hAAT-hPBGDPhase I (NCT02082860)Closed
      Crigler-Najjar
      Genethon (GNT-003)ssAAV8-ApoE/hAAT-hUGT1A1Phase I/II (NCT03466463)Ongoing
      Glycogen storage disease type Ia
      Ultragenyx (DTX401)AAV8-hGPE-hG6PC1Phase I/II (NCT03517085)Ongoing
      Phenylketonuria
      BioMarin Pharmaceutical (BMN-307)AAV5-LP-hPAHPhase I/II NCT04480567Ongoing (Clinical hold Sept 2021)
      Homology Medicines (HMI-102)AAVHSC15-ApoE/hAAT-hPAHPhase I/II NCT03952156Ongoing
      Wilson disease
      Vivet Therapeutics (VTX801)AAV8-hAAT-miniAtp7bPhase I/II (NCT04537377)Ongoing
      Ultragenyx (UX701)AAV9-LP-hATP7BPhase I/II/III (NCT04884815)Ongoing
      Methylmalonic acidaemia
      LogicBio Therapeutics (hLB-001)AAV LK03

      Integrative Tg into Albumin locus
      Phase I/II NCT04581785Ongoing
      TTR-mutated amyloidosis
      Intellia Therapeutics (NTLA-2001)LNP-encapsulating Cas9 mRNA and TTR-specific sgRNAPhase I NCT04601051Ongoing
      AAV, adeno-associated virus; Albe, albumin enhancer; ApoE, apolipoprotein E enhancer; AMBP, Alpha1-Microglobulin/Bikunin Precursor promoter; hAAT, human alpha1-antitrypsin promoter; hATP7B, human copper transporter ATP7B; hFIXco, human codon optimized Factor IX wild-type (WT) or harboring the Padua mutation (Padua); hFVIII, human Factor VIII harboring a deletion of the B-domain without (SQ) or with a specific peptide insertion (V3); hG6PC1, human glucose-6-phosphatase; hOTCco, human codon optimized ornithine transcarbamylase; hPAH, human phenylalanine hydroxylase; hPBGD, human porphobilinogen deaminase; hUGT1A1, human UDP-glucuronosyltransferase 1A1; LNP, lipid nanoparticle; LP, liver promoter without further specification; LP1, liver promoter 1; miniAtp7b, truncated version of the human ATP7B gene; scAAV, self-complementary AAV, sgRNA, single-guide RNA; ssAAV, single-stranded AAV; TBG, thyroxine binding globulin promoter; Tg, transgene; TTR, transthyretin.
      For urea cycle disorders, a phase I/II trial (NCT02991144) using an AAV8 encoding ornithine transcarbamylase is ongoing in adult patients. Interim results after 1 to 3 years of follow-up indicated that among the 9 patients treated, 3 had complete correction, defined as an ability to discontinue the otherwise required specific diet and nitrogen scavengers; 3 had a partial response; and no response was observed in the 3 remaining patients.
      • Harding C.
      • Luz Couce M.
      • Geberhiwot T.
      • Tan W.-H.
      • Khan A.
      • Aldamiz-Echevarria L.
      • et al.
      “AAV8 gene therapy as a potential treatment in adults with late-onset ornithine transcarbamylase (OTC) deficiency: updated results from a phase 1/2 clinical trial” [abstract]. In ASGCT International congress 2021.
      For glycogen storage disease type Ia, a phase I/II trial (NCT03517085) using AAV8 encoding glucose-6-phosphatase is ongoing in adult patients. Interim results showed an improvement in patients that allowed for decreased dietetic support.
      • Rodriguez-Buritica D.
      • Ahmad A.
      • Couce Pico M.L.
      • Derks T.
      • Mitchell J.
      • Riba-Wolman R.
      • et al.
      “AAV8-Mediated liver-directed gene therapy as a potential therapeutic option in adults with glycogen storage disease type Ia (GSDIa): updated phase 1/2 clinical trial results” [abstract]. In ASGCT internation congress 2021.
      For both diseases, phase III trials are expected to begin shortly. For Crigler-Najjar syndrome, a phase I/II trial (NCT03466463) is also ongoing and has yielded positive interim results for the highest dose.
      • D’Antiga L.
      • Beuers U.
      • Brunetti-Pierri N.
      • Baumann U.
      • di Giorgio A.
      • Aronson S.J.
      • et al.
      “Adeno-associated virus vector mediated gene therapy for Crigler Najjar syndrome: preliminary report of safety and efficacy from the CareCN clinical trial” [Abstract]. In EASL International Congress 2021.
      These promising results suggest that a single rAAV infusion sustains expression of the transgene for as long as 2–3 years and provides significant clinical benefits in some patients.
      The immune response, transgene loss in children during liver growth, and potential toxicity are major challenges in the gene therapy field.

      Considerations regarding liver gene transfer in children

      Although clinical trials have primarily enrolled adult patients, the treatment of children is of considerable interest. Two major challenges in treating children are rAAV episome dilution in growing livers and NAb development, which precludes reinjection of the same vector. The rate of episome dilution in humans is difficult to predict because few clinical data are available and humans have slower liver growth than model animals such as rodents.
      • Coppoletta J.M.
      • Wolbach S.B.
      Body length and organ weights of infants and children: a study of the body length and normal weights of the more important vital organs of the body between birth and twelve years of age.
      Notably, interim results from a clinical trial using rAAV to express the missing enzyme arylsulfatase B in the livers of 5–10-year-old patients with mucopolysaccharidosis type VI have reported stable arylsulfatase B levels (25–30% of normal) for at least 1 year.
      • Brunetti-Pierri N.
      • Ferla R.
      • Ginocchio V.
      • Rossi A.
      • Fecarotta S.
      • Romano R.
      • et al.
      Safety and efficacy of liver-directed gene therapy in patients with mucopolysaccharidosis type VI [Abstract]. In ASGCT international congress 2021.
      One approach to overcome episome dilution involves an integrative strategy,
      • Chandler R.J.
      • Venturoni L.E.
      • Liao J.
      • Hubbard B.T.
      • Schneller J.L.
      • Hoffmann V.
      • et al.
      Promoterless, nuclease-free genome editing confers a growth advantage for corrected hepatocytes in mice with methylmalonic acidemia.
      as used in the clinical trial for methylmalonic acidaemia (NCT04581785), or a gene editing strategy (described below). Strategies to prevent the formation of NAbs after rAAV infusion – via the use of mTOR inhibitors encapsulated in LNPs co-administered with the vector,
      • Ilyinskii P.O.
      • Michaud A.M.
      • Roy C.J.
      • Rizzo G.L.
      • Elkins S.L.
      • Capela T.
      • et al.
      Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles.
      • Ilyinskii P.O.
      • Michaud A.M.
      • Rizzo G.L.
      • Roy C.J.
      • Leung S.S.
      • Elkins S.L.
      • et al.
      ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.
      • Weber N.
      • Salas-Gomez D.
      • Odriozola L.
      • Ros-Ganan I.
      • Hommel M.
      • Kishimoto T.K.
      • et al.
      “Coadministration of AAV expressing MDR3 (VTX-803) and ImmTOR allows for vector Re-administration to treat progressive familial intrahepatic cholestasis type 3 (PFIC3) in juvenile Abcb4-/- mice” [abstract]. In ASGCT international congress 2021.
      or by removing extant NAb via immunoadsorption
      • Salas D.
      • Kwikkers K.L.
      • Zabaleta N.
      • Bazo A.
      • Petry H.
      • van Deventer S.J.
      • et al.
      Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates.
      or systemic administration of IgG-cleaving endopeptidase
      • Leborgne C.
      • Barbon E.
      • Alexander J.M.
      • Hanby H.
      • Delignat S.
      • Cohen D.M.
      • et al.
      IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies.
      – have enabled successful re-administration of the vector in pre-clinical models. However, their utility in clinical settings remains to be demonstrated.
      The first clinical trials to assess safety and dose optimisation in children will probably be performed in diseases not involving liver damage, such as haemophilia or metabolic diseases, in which the toxicity is anticipated to be low, and biochemical endpoints are relatively easy to evaluate. Although greater benefit can be expected from early treatment – particularly to prevent neurologic sequelae in metabolic diseases (urea cycle disorders, phenylketonuria, or methylmalonic acidaemia) or significant liver injury (PFIC) – gene therapy in young children is associated with a higher risk of losing transgene expression due to episome dilution as well as an elevated theoretical risk of tumorigenesis because of physiological liver cell proliferation during the first years of life. The latter risk must be balanced against the severity of the underlying disease as well as the high risk of spontaneous HCC in several of the “targetable” diseases (e.g., PFIC2, type I tyrosinaemia [HT1]).
      • Davit-Spraul A.
      • Fabre M.
      • Branchereau S.
      • Baussan C.
      • Gonzales E.
      • Stieger B.
      • et al.
      ATP8B1 and ABCB11 analysis in 62 children with normal gamma-glutamyl transferase progressive familial intrahepatic cholestasis (PFIC): phenotypic differences between PFIC1 and PFIC2 and natural history.
      ,
      • Sniderman King L.
      • Trahms C.
      • Scott C.R.
      Tyrosinemia type I.
      Moreover, the seroprevalence of anti-AAV NAbs is low in infants and increases with age.
      • Li C.
      • Narkbunnam N.
      • Samulski R.J.
      • Asokan A.
      • Hu G.
      • Jacobson L.J.
      • et al.
      Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia.
      Chronic liver diseases, such as PFIC, warrant specific considerations (reviewed in
      • Bosma P.J.
      • Wits M.
      • Oude-Elferink R.P.J.
      Gene therapy for progressive familial intrahepatic cholestasis: current progress and future prospects.
      ). Preclinical studies have suggested that advanced liver disease could decrease rAAV-based liver transduction, thus requiring higher doses of vectors for phenotype correction.
      • Siew S.M.
      • Cunningham S.C.
      • Zhu E.
      • Tay S.S.
      • Venuti E.
      • Bolitho C.
      • et al.
      Prevention of cholestatic liver disease and reduced tumorigenicity in a murine model of PFIC type 3 using hybrid AAV-piggyBac gene therapy.
      ,
      • Murillo O.
      • Moreno D.
      • Gazquez C.
      • Barberia M.
      • Cenzano I.
      • Navarro I.
      • et al.
      Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.
      In this setting, higher liver toxicity may be expected, as observed in the MTM trial.
      • Wilson J.M.
      • Flotte T.R.
      Moving forward after two deaths in a gene therapy trial of myotubular myopathy.
      ,
      • Shieh P.B.
      • Bönnemann C.G.
      • Müller-Felber W.
      • Blaschek A.
      • Dowling J.J.
      • Kuntz N.L.
      • et al.
      Re: “moving forward after two deaths in a gene therapy trial of myotubular myopathy” by Wilson and flotte.
      Inclusion of patients with limited fibrosis, such as those with PFIC with mild phenotypes, or patients who are stable under standard of care, such as those receiving copper chelation for WD or NTBC ((2-(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexanedione) for HT1, could mitigate this risk. This strategy is being used in ongoing clinical trials in adult patients with WD (Table 3). In patients with advanced liver diseases, gene therapy may be used as a bridge to LT. However, because paediatric genetic fibrogenic diseases lead to substantial liver fibrosis early in life, young infants would be the best candidates for gene therapy. In setting the minimal age limit to include children in clinical trials, not only theoretical safety concerns but also high unmet medical needs should be considered, as well as the difficulty in identifying a sufficient number of eligible patients, given the rarity of the disease. Thus, the first gene therapy trials in children with genetic fibrogenic diseases are anticipated to be international phase I/II studies focused on young children (ideally below the age of 10 years) – with a mild phenotype (and genotype), limited fibrosis, and moderately abnormal liver tests – who are receiving standard of care. Evaluation of efficacy will be more challenging than that of metabolic diseases and will probably require liver biopsies and bile sampling to evaluate transgene expression and phenotype correction. Further challenges include the identification of safe and efficient vector doses with long-term transgene expression and the possibility of treating patients as early as possible in life, before the development of substantial fibrosis.
      In conclusion, although many advances have been made with gene therapy, the treatment of paediatric patients remains challenging.

      Genomic and epigenomic editing

      Disorders in which genes are aberrantly over-expressed, or in which genes direct production of misfolded proteins are ideal candidates for genomic or epigenomic editing. However, recent advances in the field have demonstrated that gene addition can also benefit from genome-editing approaches. Genome editing entails direct modification of a patient’s genome at a specific locus with a nuclease such as CRISPR-Cas9 (reviewed in
      • Maestro S.
      • Weber N.D.
      • Zabaleta N.
      • Aldabe R.
      • Gonzalez-Aseguinolaza G.
      Novel vectors and approaches for gene therapy in liver diseases.
      ,
      • Wang L.
      • Zheng W.
      • Liu S.
      • Li B.
      • Jiang X.
      Delivery of CRISPR/Cas9 by novel strategies for gene therapy.
      ,
      • Wang H.
      • la Russa M.
      • Qi L.S.
      CRISPR/Cas9 in genome editing and beyond.
      ), or editing with nuclease-impaired Cas base editors
      • Komor A.C.
      • Kim Y.B.
      • Packer M.S.
      • Zuris J.A.
      • Liu D.R.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      • Gaudelli N.M.
      • Komor A.C.
      • Rees H.A.
      • Packer M.S.
      • Badran A.H.
      • Bryson D.I.
      • et al.
      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
      • Nishida K.
      • Arazoe T.
      • Yachie N.
      • Banno S.
      • Kakimoto M.
      • Tabata M.
      • et al.
      Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.
      or prime editors.
      • Anzalone A.v.
      • Randolph P.B.
      • Davis J.R.
      • Sousa A.A.
      • Koblan L.W.
      • Levy J.M.
      • et al.
      Search-and-replace genome editing without double-strand breaks or donor DNA.
      Epigenomic editing uses a nuclease-null Cas protein fused to various effector proteins to mediate genomic re-organisation, chromatin looping, or biochemical modification of histones and DNA (reviewed in
      • Goell J.H.
      • Hilton I.B.
      CRISPR/Cas-Based epigenome editing: advances, applications, and clinical utility.
      ).
      In CRISPR nuclease editing, an sgRNA enables targeting of Cas9 to the locus, and the nuclease induces a double-strand DNA break (DSB) which is repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ).
      • Wang H.
      • la Russa M.
      • Qi L.S.
      CRISPR/Cas9 in genome editing and beyond.
      In HDR, a co-delivered DNA fragment containing regions with sequence similarity to the locus provides a template to introduce the desired modification into the genome. Because HDR is often coupled to replication, it is inefficient in slowly dividing cells. In NHEJ, the 2 newly generated DNA ends are ligated with the insertion or deletion of several base pairs (indels), thus leading to gene silencing if the DSB occurs in the coding sequence and is repaired out-of-frame. Base editing and prime editing (reviewed in
      • Newby G.A.
      • Liu D.R.
      In vivo somatic cell base editing and prime editing.
      ) avoid DSBs and instead use impaired Cas9 fused to cytidine deaminase (for C·G to T·A base editing),
      • Komor A.C.
      • Kim Y.B.
      • Packer M.S.
      • Zuris J.A.
      • Liu D.R.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ,
      • Nishida K.
      • Arazoe T.
      • Yachie N.
      • Banno S.
      • Kakimoto M.
      • Tabata M.
      • et al.
      Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.
      deoxyadenosine deaminase (for A·T to G·C editing),
      • Gaudelli N.M.
      • Komor A.C.
      • Rees H.A.
      • Packer M.S.
      • Badran A.H.
      • Bryson D.I.
      • et al.
      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
      or reverse transcriptase (for base-to-base, insertion, or deletion editing).
      • Anzalone A.v.
      • Randolph P.B.
      • Davis J.R.
      • Sousa A.A.
      • Koblan L.W.
      • Levy J.M.
      • et al.
      Search-and-replace genome editing without double-strand breaks or donor DNA.
      The efficiency of prime editing has been further improved with the inhibition of the DNA mismatch repair machinery by an engineered protein
      • Chen P.J.
      • Hussmann J.A.
      • Yan J.
      • Knipping F.
      • Ravisankar P.
      • Chen P.-F.
      • et al.
      Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.
      ; nuclear localisation signal architecture and optimised codon usage
      • Anzalone A.v.
      • Randolph P.B.
      • Davis J.R.
      • Sousa A.A.
      • Koblan L.W.
      • Levy J.M.
      • et al.
      Search-and-replace genome editing without double-strand breaks or donor DNA.
      ,
      • Liu P.
      • Liang S.-Q.
      • Zheng C.
      • Mintzer E.
      • Zhao Y.G.
      • Ponnienselvan K.
      • et al.
      Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice.
      ; and Cas9 fused to a chromatin-modulating peptide.
      • Park S.-J.
      • Jeong T.Y.
      • Shin S.K.
      • Yoon D.E.
      • Lim S.-Y.
      • Kim S.P.
      • et al.
      Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor.
      Use of dual or twin prime editing guide RNAs to direct transcription of complementary sequences also improves editing outcomes.
      • Anzalone A.
      • Gao X.
      • Podracky C.
      • Nelson A.
      • Koblan L.
      • Raguram A.
      • et al.
      Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases.
      • Choi J.
      • Chen W.
      • Suiter C.C.
      • Lee C.
      • Chardon F.M.
      • Yang W.
      • et al.
      Precise genomic deletions using paired prime editing.
      • Lin Q.
      • Jin S.
      • Zong Y.
      • Yu H.
      • Zhu Z.
      • Liu G.
      • et al.
      High-efficiency prime editing with optimized, paired pegRNAs in plants.
      Prime editing is thus a rapidly developing field, and this versatile approach could enable gene therapy for more than 89% of known genetic diseases.
      • Newby G.A.
      • Liu D.R.
      In vivo somatic cell base editing and prime editing.
      The first successful clinical trials using CRISPR have adopted an ex vivo approach in sickle cell disease; correction of haematopoietic stem cell disease has been shown to occur through autologous transplantation of gene-edited cells, in which NHEJ disruption of BCL11A, encoding an essential transcription factor repressing γ-globin and foetal haemoglobin expression in erythroid cells, leads to transfusion independence, owing to durable engraftment of cells expressing high levels of foetal haemoglobin.
      • Frangoul H.
      • Altshuler D.
      • Cappellini M.D.
      • Chen Y.-S.
      • Domm J.
      • Eustace B.K.
      • et al.
      CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia.
      A recent in vivo approach for TTR-mediated amyloidosis using LNPs to deliver the Cas9 mRNA and the guide RNA to the liver led to a >90% decrease in circulating TTR levels.
      • Gillmore J.D.
      • Gane E.
      • Taubel J.
      • Kao J.
      • Fontana M.
      • Maitland M.L.
      • et al.
      CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis.
      If its efficacy is demonstrated, this method could provide a valuable alternative to the approved repeated siRNA injections. The use of gene editing with HDR in vivo will be more challenging because of its low efficiency in the adult liver. However, this method could be of interest for the treatment of children, particularly if the corrected cells have a selective advantage, as has been demonstrated for some liver diseases in preclinical studies.
      • Li N.
      • Gou S.
      • Wang J.
      • Zhang Q.
      • Huang X.
      • Xie J.
      • et al.
      CRISPR/Cas9-Mediated gene correction in newborn rabbits with hereditary tyrosinemia type I.
      Finally, the major caveats are potential genotoxicity, possible off-target effects, and large-scale chromosomal deletions.
      • Cullot G.
      • Boutin J.
      • Toutain J.
      • Prat F.
      • Pennamen P.
      • Rooryck C.
      • et al.
      CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.
      ,
      • Boutin J.
      • Rosier J.
      • Cappellen D.
      • Prat F.
      • Toutain J.
      • Pennamen P.
      • et al.
      CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.
      Safety data are scarce in human patients, and toxicity will need to be monitored closely.
      The risks of off-target effects and indels may be largely mitigated using base editing or prime editing, which have shown efficacy and low off-target editing in animal models. Tyrosinaemia in mice has been addressed by restoring fumarylacetoacetate hydrolase (Fah) expression by correcting a splice-site mutation,
      • Song C.-Q.
      • Jiang T.
      • Richter M.
      • Rhym L.H.
      • Koblan L.W.
      • Zafra M.P.
      • et al.
      Adenine base editing in an adult mouse model of tyrosinaemia.
      • Jang H.
      • Shin J.
      • Jo D.
      • Seo J.
      • Yu G.
      • Gopalappa R.
      • et al.
      Prime editing enables precise genome editing in mouse liver and retina.
      • Jiang T.
      • Henderson J.M.
      • Coote K.
      • Cheng Y.
      • Valley H.C.
      • Zhang X.-O.
      • et al.
      Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.
      and mouse and macaque models of hypercholesterolaemia have been treated with base editors to mutate PCSK9.
      • Musunuru K.
      • Chadwick A.C.
      • Mizoguchi T.
      • Garcia S.P.
      • DeNizio J.E.
      • Reiss C.W.
      • et al.
      In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.
      ,
      • Chadwick A.C.
      • Wang X.
      • Musunuru K.
      In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing.
      • Carreras A.
      • Pane L.S.
      • Nitsch R.
      • Madeyski-Bengtson K.
      • Porritt M.
      • Akcakaya P.
      • et al.
      In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model.
      • Rothgangl T.
      • Dennis M.K.
      • Lin P.J.C.
      • Oka R.
      • Witzigmann D.
      • Villiger L.
      • et al.
      In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.
      • Wang L.
      • Xue W.
      • Zhang H.
      • Gao R.
      • Qiu H.
      • Wei J.
      • et al.
      Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations.
      PCSK9 expression has also been successfully repressed through epigenome editing with Cas9 fused to a Krüppel-associated box epigenetic repressor motif (KRAB, dCas9KRAB): PCSK9 levels have been repressed for as many as 24 weeks in mice.
      • Thakore P.I.
      • Kwon J.B.
      • Nelson C.E.
      • Rouse D.C.
      • Gemberling M.P.
      • Oliver M.L.
      • et al.
      RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.
      Tyrosinaemia has been corrected through a modified prime editing approach termed PE-Cas9-based deletion and repair to remove a 1.38-kb pathogenic insertion in Fah and repair the deletion junction.
      • Jiang T.
      • Zhang X.-O.
      • Weng Z.
      • Xue W.
      Deletion and replacement of long genomic sequences using prime editing.
      Phenylketonuria has been treated in mice through both base editing
      • Villiger L.
      • Rothgangl T.
      • Witzigmann D.
      • Oka R.
      • Lin P.J.C.
      • Qi W.
      • et al.
      In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA.
      ,
      • Villiger L.
      • Grisch-Chan H.M.
      • Lindsay H.
      • Ringnalda F.
      • Pogliano C.B.
      • Allegri G.
      • et al.
      Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.
      and prime editing.
      • Boeck D.
      • Rothgangl T.
      • Villiger L.
      • Schmidheini L.
      • Mathis N.
      • Ioannidi E.
      • et al.
      Treatment of a metabolic liver disease by in vivo prime editing in mice.
      Prime editing has also been applied to correct the E342K mutation (G to A) in SERPINA1 (PiZ allele) in a mouse model of alpha-1 antitrypsin deficiency.
      • Liu P.
      • Liang S.-Q.
      • Zheng C.
      • Mintzer E.
      • Zhao Y.G.
      • Ponnienselvan K.
      • et al.
      Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice.
      These proof of principle experiments have demonstrated editing efficiency as high as 70%, and the rescue of phenotypes such as weight loss or normalisation of protein levels.
      According to proof-of-concept data, liver-directed mRNA therapy and gene editing are entering early phase clinical trials.
      In conclusion, genome/epigenome editing holds great promise for correcting liver diseases, particularly in children, but remains mostly in preclinical research stages. Stop codon readthrough is another emerging option that enables cells to ignore the premature stop signal caused by nonsense mutations and produce a full-length protein. The first approved drug using this approach was ataluren for the treatment of Duchenne muscular dystrophy. However, its utility for liver-related disorders remains to be determined.
      • Vauthier V.
      • Housset C.
      • Falguières T.
      Targeted pharmacotherapies for defective ABC transporters.
      Premature stop codons have been reported in several liver disorders, including autosomal dominant polycystic liver disease or lysosomal-storage disorders.
      • Drenth J.P.H.
      • te Morsche R.H.M.
      • Smink R.
      • Bonifacino J.S.
      • Jansen J.B.M.J.
      Germline mutations in PRKCSH are associated with autosomal dominant polycystic liver disease.
      ,
      • Brooks D.A.
      • Muller V.J.
      • Hopwood J.J.
      Stop-codon read-through for patients affected by a lysosomal storage disorder.

      RNAi and ASO therapeutic approaches

      GalNac-conjugated siRNA enables specific targeting to hepatocytes and has given rise to several recently approved therapeutics.
      Modification of mRNA processing and translation with short complementary RNA fragments is an increasingly used therapeutic option.
      • Bennett C.F.
      Therapeutic antisense oligonucleotides are coming of age.
      Two major modalities using this mechanism are siRNAs and antisense oligonucleotides (ASOs).
      • Bennett C.F.
      Therapeutic antisense oligonucleotides are coming of age.
      ,
      • Hu B.
      • Zhong L.
      • Weng Y.
      • Peng L.
      • Huang Y.
      • Zhao Y.
      • et al.
      Therapeutic siRNA: state of the art.
      The efficacy of antisense mechanisms was first demonstrated in chick embryos, in which synthetic oligonucleotides blocked the translation of Rous sarcoma viral RNA and thereby inhibited viral replication.
      • Zamecnik P.C.
      • Stephenson M.L.
      Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide.
      However, the road to human applications has been challenging, requiring modifications in oligonucleotide chemistry and formulations, and posing safety issues. Because of these hurdles, 20 years elapsed before the first ASO was approved in 1998: fomivirsen was administered via intraocular injection and was used to treat cytomegalovirus retinitis in patients with complications of late-stage AIDS.
      • Bajan S.
      • Hutvagner G.
      RNA-based therapeutics: from antisense oligonucleotides to miRNAs.
      Another 15 years passed before the first systemically administered ASO was approved by the FDA. Mipomersen administered subcutaneously in individuals with homozygous familial hypercholesterolaemia has been found to inhibit the production of apolipoprotein B100 (Table 4).
      • Quemener A.M.
      • Bachelot L.
      • Forestier A.
      • Donnou-Fournet E.
      • Gilot D.
      • Galibert M.
      The powerful world of antisense oligonucleotides: from bench to bedside.
      Administration over several weeks markedly decreases LDL-cholesterol levels.
      • Akdim F.
      • Tribble D.L.
      • Flaim J.D.
      • Yu R.
      • Su J.
      • Geary R.S.
      • et al.
      Efficacy of apolipoprotein B synthesis inhibition in subjects with mild-to-moderate hyperlipidaemia.
      ,
      • Ricotta D.N.
      • Frishman W.
      Mipomersen: a safe and effective antisense therapy adjunct to statins in patients with hypercholesterolemia.
      Table 4Overview of liver-targeted RNA-interference drugs with current market approval (FDA and/or EMA).
      DrugPlatformMarketing authorisation holderTargetIndicationDelivery routeApproval status
      Patisiran (Onpattro)siRNAAlnylam PharmaceuticalsTTR mRNAhATTRi.v.FDA (08/2018)

      EMA (08/2018)
      Givosiran (Givlaari)siRNAAlnylam PharmaceuticalsALAS1 mRNAAHPs.c.FDA (11/2019)

      EMA (03/2020)
      Lumasiran (Oxlumo)siRNAAlnylam PharmaceuticalsHAO1 mRNAPH Type 1s.c.FDA (11/2020)

      EMA (11/2020)
      Inclisiran (Leqvio)siRNANovartisPCSK9 mRNAprimary hypercholesterolaemia (heterozygous familial and non-familial), mixed dyslipidaemias.c.EMA (12/2020)
      Inotersen (Tegsedi)ASOIonis PharmaceuticalsTTR mRNAhATTRs.c.FDA (10/2018)

      EMA (07/2018)
      Volanesorsen (Waylivra)ASOIonis PharmaceuticalsAPOC3 mRNAFCSs.c.EMA (05/2019)
      Mipomersen (Kynamro)ASOGenzymeApoB100 mRNAhoFHs.c.FDA (01/2013),
      AHP, acute hepatic porphyria; ALAS1, delta-aminolevulinate synthase 1; ApoB100, apolipoprotein B100; APOC3, apolipoprotein C3; ASO, antisense oligonucleotide; FCS, familial chylomicronaemia syndrome; HAO1, hydroxyacid oxidase (glycolate oxidase) 1; hATTR, hereditary transthyretin-amyloidosis; hoFH, homozygous familial hypercholesterolaemia; i.v., intravenous; PH, primary hyperoxaluria; PCSK9, proprotein convertase subtilisin/kexin type 9; s.c., subcutaneous; siRNA, small-interfering RNA; TTR, transthyretin.
      ASOs consist of single-stranded oligonucleotides with backbone, sugar-, and base-specific modifications (12–30 nucleotides). Their structure enables high affinity interaction with plasma and cell surface proteins, thereby facilitating broad distribution to different tissues without formulation or excipients.
      • Bennett C.F.
      Therapeutic antisense oligonucleotides are coming of age.
      ASOs bind complementary (pre-) mRNA and sterically hinder protein translation. Additionally, ASOs designed as gapmers, containing a central core of largely unmodified DNA, can induce RNA cleavage and degradation via recruitment of RNase H1. Although hopes were high after the introduction of fomivirsen, the drug was withdrawn from the market because of the superior success of antiviral agents in treating AIDS.
      • Quemener A.M.
      • Bachelot L.
      • Forestier A.
      • Donnou-Fournet E.
      • Gilot D.
      • Galibert M.
      The powerful world of antisense oligonucleotides: from bench to bedside.
      ,
      • Nuciforo S.
      • Heim M.H.
      Organoids to model liver disease.
      Moreover, the clinical use of mipomersen was limited by adverse effects including flu-like symptoms and hepatotoxicity.
      • Parham J.S.
      • Goldberg A.C.
      Mipomersen and its use in familial hypercholesterolemia.
      Because of these adverse effects, which were partly due to target-related increases in hepatic steatosis, mipomersen did not receive market authorisation by the EMA. Notably, hepatotoxicity was also observed in locked-nucleic acid-modified ASOs.
      • Burel S.A.
      • Hart C.E.
      • Cauntay P.
      • Hsiao J.
      • Machemer T.
      • Katz M.
      • et al.
      Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.
      ,
      • Swayze E.E.
      • Siwkowski A.M.
      • Wancewicz E.v.
      • Migawa M.T.
      • Wyrzykiewicz T.K.
      • Hung G.
      • et al.
      Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.
      Other major drawbacks included cases of thrombocytopenia and glomerulonephritis. For example, in a phase III clinical trial of inotersen, a second-generation ASO used for the treatment of hereditary TTR amyloidosis (hATTR), grade 4 thrombocytopenia occurred in 3 of the 112 treated participants and led to intracranial haemorrhage and death in 1 patient.
      • Benson M.D.
      • Waddington-Cruz M.
      • Berk J.L.
      • Polydefkis M.
      • Dyck P.J.
      • Wang A.K.
      • et al.
      Inotersen treatment for patients with hereditary transthyretin amyloidosis.
      Although the currently approved ASOs primarily target neuromuscular disorders such as Duchenne muscular dystrophy (eteplirsen [Exondys 51], casimersen [Amondys 45], and golodirsen [Vyondys 53]) and spinal muscular atrophy (nusinersen [Spinraza]), volanesorsen (Waylivra) blocks the production of apolipoprotein C3 and has been approved for the treatment of familial chylomicronaemia syndrome (FCS; Table 4). In the corresponding phase III trial, which recruited 66 patients with FCS, treatment with volanesorsen resulted in an 84% decrease in serum apolipoprotein C3 levels and a 77% decrease in mean triglyceride levels, whereas an 18% increase was observed in the placebo group.
      • Witztum J.L.
      • Gaudet D.
      • Freedman S.D.
      • Alexander V.J.
      • Digenio A.
      • Williams K.R.
      • et al.
      Volanesorsen and triglyceride levels in familial chylomicronemia syndrome.
      Similar results have been reported in individuals with severe hypertriglyceridaemia. Importantly, the administration of volanesorsen resulted in fewer cases of pancreatitis, a typical complication of hypertriglyceridaemia.
      • Paik J.
      • Duggan S.
      Volanesorsen: first global approval.
      Injection site reactions were observed in 20 of 33 treated participants with FCS, and 15 developed platelet counts below 100,000/μl.
      • Witztum J.L.
      • Gaudet D.
      • Freedman S.D.
      • Alexander V.J.
      • Digenio A.
      • Williams K.R.
      • et al.
      Volanesorsen and triglyceride levels in familial chylomicronemia syndrome.
      These major adverse effects may limit the therapeutic utility of this interesting compound; therefore, efforts should focus on improving the cell type-specific delivery of ASOs. In this respect, the introduction of N-acetylgalactosamine (GalNAc)-conjugated ASOs appears particularly promising, because it directs the ASO to hepatocytes via high-affinity interactions with the asialoglycoprotein receptor and results in a 20- to 30-fold increase in potency.
      • Huang Y.
      Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics.
      This approach allows for lower and less frequent dosing. For example, typical GalNAc ASO regimens require 50–80 mg/month, in contrast to prior non-GalNAc regimens such as volanesorsen and inotersen, which require 300 mg/week. Accordingly, all current ASO regimens in the liver use GalNAc conjugation. For example, pelacarsen inhibits hepatic expression of the atherogenic and thrombogenic lipoprotein(a) (LPA). Phase II trials have reported a dose-dependent decrease in LPA levels and an encouraging safety profile.
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      An overview of ASOs in mid-to-late-phase clinical trials is presented in Table 5.
      Table 5Overview of liver-targeted RNA-interference-based drugs in late-phase clinical trials.
      DrugPlatformSponsorIndicationTargetDelivery routeStudy phaseNCT, EudraCT etc.
      VutrisiransiRNAAlnylam PharmaceuticalshATTR and WT-ATTR with associated cardiomyopathyTTR mRNAs.c.III (NDA and MAA signed)NCT03759379

      NCT04153149
      FitusiransiRNAGenzymeHaemophilia A or BAT mRNAs.c.II/IIINCT03974113

      NCT03549871

      NCT03754790

      NCT03417245

      NCT03417102
      NedosiransiRNADicerna PharmaceuticalsPH (Types 1, 2, 3)LDH mRNAs.c.II/IIINCT04042402

      NCT03847909

      NCT04555486

      NCT05001269
      ARO-AATsiRNAArrowhead Pharmaceuticals

      Takeda
      AATDAAT mRNAs.c.IIbNCT03362242

      NCT03946449

      NCT03945292
      ARO-APOC3siRNAArrowhead PharmaceuticalsSevere hypertriglyceridaemia, mixed dyslipidaemia,

      FCS
      APOC3 mRNAs.c.IIbNCT04720534

      NCT04998201
      ARO-ANG3siRNAArrowhead PharmaceuticalsMixed dyslipidaemiaANGPTL3 mRNAs.c.IIbNCT04832971
      ARO-HSDsiRNAArrowhead PharmaceuticalsLiver diseaseHSD17B13 mRNAs.c.IIaNCT04202354
      JNJ-3989siRNAJanssen PharmaceuticalsChronic hepatitis BFull HBV transcriptome (cccDNA and integrated DNA)s.c.IIa/bNCT03365947

      NCT03982186

      NCT04129554

      NCT04535544
      VIR-2218siRNAVir Biotechnology

      Alnylam Pharmaceuticals
      Chronic hepatitis BFull HBV transcriptome (cccDNA and integrated DNA)s.c.IINCT04507269

      NCT04856085

      NCT03672188

      NCT04412863

      NCT04749368
      EplontersenASOIonis PharmaceuticalshATTR and WT-ATTR with associated polyneuropathy and/or cardiomyopathyTTR mRNAs.c.IIINCT04136171

      NCT04136184
      PelacarsenASOIonis Pharmaceuticals

      Novartis
      Patients with hyperlipoproteinaemia at high risk for/with established ASCVDLPA mRNAs.c.IIa/bNCT03070782

      NCT04993664
      VupanorsenASOIonis Pharmaceuticals

      Pfizer
      Patients with hyperlipoproteinaemia at high risk for ASCVDANGPTL3 mRNAs.c.IINCT04516291

      NCT03360747

      NCT03371355

      2020-002796-35
      IONIS-APOCIII-LRxASOIonis PharmaceuticalsPatients with FCSAPOC3 mRNAs.c.IIINCT03385239

      NCT04568434

      NCT04568434
      IONIS-AGT-LRxASOIonis PharmaceuticalsPatients with uncontrolled hypertensionAGT mRNAs.c.IINCT04083222

      NCT03714776

      NCT04714320

      NCT04836182
      FesomersenASOIonis Pharmaceuticals

      Bayer
      Patients at high risk of inappropriate thrombosisFXI mRNAs.c.IINCT03582462
      AZD8233ASOIonis Pharmaceuticals

      AstraZeneca
      Patients with hypercholesterolaemia at high risk for ASCVDPCSK9 mRNAs.c.IINCT04155645

      NCT04641299

      NCT04823611

      NCT04964557
      IONIS-FB-LRxASOIonis Pharmaceuticals

      Roche
      Patients with diseases caused by inappropriate complement activationCFB mRNAs.c.IINCT04014335

      NCT03815825
      ION224ASOIonis PharmaceuticalsPatients with non-alcoholic steatohepatitis (or NASH)DGAT2 mRNAs.c.IINCT04932512
      MiravirsenASOSantaris PharmaceuticalsChronic hepatitis CmiR-122s.c.IINCT01200420

      NCT02452814

      NCT02508090

      NCT01727934

      NCT01872936
      RG-101ASORegulus TherapeuticsChronic hepatitis CmiR-122s.c.II2015-001535-21

      2015-004702-42

      2016-002069-77
      DonidalorsenASOIonis PharmaceuticalsHereditary angioedemaPrekallikrein mRNAs.c.IINCT04030598
      (Table includes only phase II/III trial numbers).
      AAT, alpha-1 antitrypsin; AATD, alpha-1 antitrypsin deficiency; AGT, angiotensinogen; ANGPTL3, angiopoietin like protein 3; APOC3, apolipoprotein C3; ASCVD, atherosclerotic cardiovascular disease; AT, antithrombin; cccDNA, covalently closed circular DNA; CFB, complement factor B; DGAT2, diacylglycerol O-acyltransferase 2; FCS, familial chylomicronaemia syndrome; FXI, coagulation factor XI; hATTR, hereditary transthyretin-amyloidosis; HSD17B13, 17-β hydroxysteroid dehydrogenase 13; LDH, lactate dehydrogenase; LPA, lipoprotein(a); MAA, market authorization application; miR, micro-RNA; NDA, new drug application; PCSK9, proprotein convertase subtilisin/kexin type 9; PH, primary hyperoxaluria; s.c., subcutaneous; TTR, transthyretin; WT-TTR, wild-type transthyretin-amyloidosis.
      In contrast to ASOs, double-stranded siRNAs (19–22 base pairs) have less pronounced interactions with cell surface or plasma proteins, and therefore require an active delivery mechanism. siRNAs inactivate their target mRNAs via the RNA-induced silencing complex (RISC): the duplex is loaded into the RISC via chaperones, and after removal of the sense strand, the active RISC is formed. The remaining strand serves as a guide for binding of the targeted mRNA, which is then degraded by endonucleases included in the RISC.
      • Hu B.
      • Zhong L.
      • Weng Y.
      • Peng L.
      • Huang Y.
      • Zhao Y.
      • et al.
      Therapeutic siRNA: state of the art.
      The first successful in vitro silencing, reported 20 years ago,
      • Elbashir S.M.
      • Harborth J.
      • Lendeckel W.
      • Yalcin A.
      • Weber K.
      • Tuschl T.
      Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
      ,
      • Caplen N.J.
      • Parrish S.
      • Imani F.
      • Fire A.
      • Morgan R.A.
      Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
      increased interest in siRNA-based drugs. Nevertheless, several obstacles needed to be overcome, such as the poor stability and therapeutic efficacy of unmodified or only slightly modified siRNA molecules.
      • Hu B.
      • Zhong L.
      • Weng Y.
      • Peng L.
      • Huang Y.
      • Zhao Y.
      • et al.
      Therapeutic siRNA: state of the art.
      Adjustments such as chemical modifications, selection of an appropriate sequence, and/or delivery techniques were necessary before patisiran (Onpattro) was approved as the first siRNA-based drug in 2018. The RNAi trigger is composed of physiological nucleotides and targets the hepatic expression of TTR in the treatment of polyneuropathy in patients with hATTR (Table 4). Patisiran uses LNP-based encapsulation of the RNAi to increase its bioavailability.
      • Adams D.
      • Gonzalez-Duarte A.
      • O’Riordan W.D.
      • Yang C.-C.
      • Ueda M.
      • Kristen A.v.
      • et al.
      Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis.
      Because of the formulation’s potential to cause administration-associated inflammatory reactions or unspecific lipid interactions with cell membranes, pre-medication with glucocorticoids and antihistamines is necessary. Several other investigational drugs have also used LNP formulations, but most of these studies were discontinued because other platforms were demonstrated to be superior in potency and safety, particularly regarding liver-targeted delivery. The currently approved siRNA-based drugs rely primarily on conjugation of the RNAi with GalNAc. The compound is then internalised through receptor-mediated endocytosis and released into the cytoplasm.
      • Nair J.K.
      • Willoughby J.L.S.
      • Chan A.
      • Charisse K.
      • Alam MdR.
      • Wang Q.
      • et al.
      Multivalent N-Acetylgalactosamine-Conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.
      The strong tissue specificity of these conjugates minimises the risk of adverse events. Simultaneously, GalNAc-conjugates achieve higher hepatic siRNA exposure after subcutaneous rather than intravenous administration, because the limited numbers of asialoglycoprotein receptors on hepatocytes’ surfaces are incapable of internalising the excess siRNA flooding the liver after an intravenous bolus injection. In such situations, the excess siRNA is rapidly excreted by the kidneys. The current developments in siRNAs have replaced natural RNA with metabolically stable modifications, such as 2’O-methyl- or 2’O-fluoro-substituents, thereby enabling long-term silencing and correspondingly long dosing intervals.
      • Huang Y.
      • Cheng Q.
      • Ji J.-L.
      • Zheng S.
      • Du L.
      • Meng L.
      • et al.
      Pharmacokinetic behaviors of intravenously administered siRNA in glandular tissues.
      With the exception of patisiran, all other currently approved siRNA drugs, givosiran (Givlaari), lumasiran (Oxlumo), and inclisiran (Leqvio) use the hepatocyte-targeting GalNAc platform, thereby establishing hepatocytes as a focus of siRNA research (Table 4). Givosiran was the second RNAi drug to receive market approval and is indicated for the treatment of acute hepatic porphyria.
      • Scott L.J.
      Givosiran: first approval.
      In acute hepatic porphyria, the induction of hepatic delta-aminolevulinate synthase 1 (ALAS1) in genetically predisposed individuals results in the accumulation of neurotoxic heme intermediates.
      • Bissell D.M.
      • Anderson K.E.
      • Bonkovsky H.L.
      Monthly subcutaneous givosiran administration, compared with placebo, has been found to decrease hepatic ALAS1 levels by more than 80% and to be accompanied by a 74% lower mean annualised attack rate.
      • Balwani M.
      • Sardh E.
      • Ventura P.
      • Peiró P.A.
      • Rees D.C.
      • Stölzel U.
      • et al.
      Phase 3 trial of RNAi therapeutic givosiran for acute intermittent porphyria.
      The drug was generally well tolerated. A key safety concern was the liver enzyme elevation.
      • Balwani M.
      • Sardh E.
      • Ventura P.
      • Peiró P.A.
      • Rees D.C.
      • Stölzel U.
      • et al.
      Phase 3 trial of RNAi therapeutic givosiran for acute intermittent porphyria.
      Although the reason for this elevation remains unclear, silencing of hepatic ALAS1 has been hypothesised to affect heme-dependent enzymes, such as the cytochrome P450 family.
      • Vassiliou D.
      • Sardh E.
      • Harper P.
      • Simon A.R.
      • Clausen V.A.
      • Najafian N.
      • et al.
      A drug-drug interaction study evaluating the effect of givosiran, a small interfering ribonucleic acid, on cytochrome P450 activity in the liver.
      Injection site reactions, rash, nausea, fatigue, and renal adverse events were more common in the treated group than the placebo group.
      • Pallet N.
      • Mami I.
      • Schmitt C.
      • Karim Z.
      • François A.
      • Rabant M.
      • et al.
      High prevalence of and potential mechanisms for chronic kidney disease in patients with acute intermittent porphyria.
      In 2020, lumasiran and inclisiran were approved. Lumasiran targets primary hyperoxaluria type 1 (PH1) which is caused by a mutation in the hydroxyacid oxidase 1 gene. In PH1, this enzymatic defect leads to the conversion of glyoxylate to oxalate. Oxalate forms insoluble calcium crystals, which accumulate primarily in the kidneys. Patients with PH1 develop recurrent nephrolithiasis and calcinosis, end-stage renal disease and subsequently manifestations in additional organs (eyes, heart, bones, and skin), with devastating consequences.
      • Hopp K.
      • Cogal A.G.
      • Bergstralh E.J.
      • Seide B.M.
      • Olson J.B.
      • Meek A.M.
      • et al.
      Phenotype-genotype correlations and estimated carrier frequencies of primary hyperoxaluria.
      ,
      • Sas D.J.
      • Harris P.C.
      • Milliner D.S.
      Recent advances in the identification and management of inherited hyperoxalurias.
      Lumasiran inhibits hydroxyacid oxidase 1, thereby normalising urinary oxalate levels and decreasing the formation of oxalate stones/deposits.
      • Garrelfs S.F.
      • Frishberg Y.
      • Hulton S.A.
      • Koren M.J.
      • O’Riordan W.D.
      • Cochat P.
      • et al.
      Lumasiran, an RNAi therapeutic for primary hyperoxaluria type 1.
      ,
      • Salido E.
      • Pey A.L.
      • Rodriguez R.
      • Lorenzo V.
      Primary hyperoxalurias: disorders of glyoxylate detoxification.
      Inclisiran inhibits hepatic expression of PCSK9, a protein that induces the degradation of LDL receptors.
      • Ray K.K.
      • Wright R.S.
      • Kallend D.
      • Koenig W.
      • Leiter L.A.
      • Raal F.J.
      • et al.
      Two phase 3 trials of inclisiran in patients with elevated LDL cholesterol.
      PCSK9 silencing increases the clearance of LDL from the systemic circulation and markedly decreases LDL plasma levels.
      • Raal F.J.
      • Kallend D.
      • Ray K.K.
      • Turner T.
      • Koenig W.
      • Wright R.S.
      • et al.
      Inclisiran for the treatment of heterozygous familial hypercholesterolemia.
      Apart from injection site reactions, no major adverse events have been reported in the lumasiran/inclisiran trials, thus suggesting that the effects of givosiran might be due to ALAS1 silencing or off-target events rather than a class effect. An overview of RNAi-based drugs that have received market authorisation is presented in Table 4.
      Spurred by recent approvals, several clinical trials have evaluated the safety and efficacy of siRNA therapeutics in liver diseases. For example, a phase II open-label trial has reported efficient silencing of alpha-1 antitrypsin expression in individuals with severe alpha-1 antitrypsin deficiency due to homozygous expression of the mutant and hepatotoxic PI∗Z allele. The inhibition not only decreased hepatic accumulation of toxic alpha-1 antitrypsin variant, but also improved liver enzyme levels and liver fibrosis markers.
      • Strnad P.
      • Mandorfer M.
      • Choudhury G.
      • Griffiths W.
      • Trautwein C.
      • Loomba R.
      • et al.
      ARO-AAT, an investigational RNAi therapeutic, demonstrates improvement in liver fibrosis with reduction in intrahepatic Z-AAT burden.
      The RNAi approach is also being used as a tool to treat chronic hepatitis B. The GalNAc-conjugated VIR-2218 (Vir Biotechnology) silences all major transcripts of HBV across different genotypes. A recent open-label phase II trial has shown substantial decreases in HBV surface antigen levels.
      • Gane E.
      • Lim Y.-S.
      • Tangkijvanich P.
      • O’Beirne J.
      • Lim T.H.
      • Bakardjiev A.
      • et al.
      Preliminary safety and antiviral activity of VIR-2218, an X-targeting HBV RNAi therapeutic, in chronic hepatitis B patients.
      ,
      • Gane E.
      • Lim Y.
      • Cloutier D.
      • Shen L.
      • Cathcart A.
      • Ding X.
      • et al.
      Safety and antiviral activity of VIR-2218, an X-targeting RNAi therapeutic, in participants with chronic hepatitis B Infection: week 48 follow-up results.
      Even stronger, more sustained repression of HBV surface antigen levels has been demonstrated in combination with the immunomodulating PEG-interferon 2 alpha.
      • Gane E.
      • Lim Y.-S.
      • Tangkijvanich P.
      • O’Beirne J.
      • Lim T.H.
      • Bakardjiev A.
      • et al.
      Preliminary safety and antiviral activity of VIR-2218, an X-targeting HBV RNAi therapeutic, in chronic hepatitis B patients.
      Similarly encouraging results have been achieved by JNJ-3989 (Janssen Pharmaceuticals), which also broadly silences the HBV transcriptome.
      • Yuen M.-F.
      • Locarnini S.
      • Lim T.H.
      • Strasser S.
      • Sievert W.
      • Cheng W.
      • et al.
      Short term RNA interference therapy in chronic hepatitis B using JNJ-3989 brings majority of patients to HBsAg < 100 IU/ml threshold.
      An overview of further investigations of siRNA-based drugs is presented in Table 5.

      Conclusion

      Genetic treatment strategies have the potential to transform modern medicine because they can act on targets not easily accessed by small molecules or therapeutic antibodies. Gene addition approaches using rAAV vectors have shown promising results in phase III trials in haemophilia and are currently being evaluated in a broader range of liver diseases. In contrast, hepatocytes have been the main focus of gene silencing efforts because of the ease of their specific targeting and their crucial roles in multiple disease-related pathways. The advantage of these silencing drugs is their long duration of biological efficacy, which can be further extended with biochemical modifications. For example, whereas PCSK9 antibodies are typically administered every 2–4 weeks, the PCSK9 siRNA inclisiran lasts 6 months. Additionally, the different modes of action enable sequential therapy. Thus, inclisiran may become useful in individuals who develop anti-drug antibodies. Because silencing approaches preferentially target the liver, new drug-specific or class-related adverse effects may arise. Despite these caveats, the essential role of the liver as the central hub for protein and lipid metabolism has been demonstrated. Therefore, the liver is a target for the treatment of both liver-related and systemic diseases. The advances described herein not only herald novel treatments but also improve our understanding of homeostatic and pathological processes in the liver.

      Abbreviations

      AAV, adeno-associated virus; ALAS1, delta-aminolevulinate synthase 1; ASO(s), antisense oligonucleotide(s); DSB, double-strand DNA break; ERT, enzyme replacement/substitution therapy; Fah, fumarylacetoacetate hydrolase; FCS, familial chylomicronaemia syndrome; FIX, coagulation factor IX; FVIII, coagulation factor VIII; GalNAc; N-acetylgalactosamine; hATTR, hereditary transthyretin-amyloidosis; HCC, hepatocellular carcinoma; HDR, homology-directed repair; LNP, lipid nanoparticle; LPA, lipoprotein(a); MTM, myotubular myopathy; NAb, neutralising antibody; NHEJ, non-homologous end joining; PCSK9, proprotein convertase subtilisin kexin type 9; PEG, polyethylene glycol; PFIC, progressive familial intrahepatic cholestasis; PH1, primary hyperoxaluria type 1; rAAV, recombinant AAV; RISC, RNA-induced silencing complex; siRNA, small-interfering RNA; TTR, transthyretin; WD, Wilson disease; WT, wild-type.

      Financial support

      PS is supported by the German Research Foundation (DFG) consortium SFB 1382 “Gut-liver axis” and DFG grant STR 1095/6-1 (Heisenberg professorship). ERA is supported by a Karolinska Institutet Career Ladder grant 2-2110/2019-7 . AG received funding from Institut National de la Santé et de la Recherche Médicale (Inserm).

      Authors’ contributions

      Conceptualization: ERA; EG; AG; KR; PS. Writing - original draft: ERA; EG; AG; KR; PS. Writing - review and editing: ERA; EG; AG; KR; PS. Visualization: ERA; EG; AG; KR; PS. All authors had full access to all the data and approved the final version of this manuscript.

      Conflicts of interest

      ERA holds a research grant from modeRNA (no personal remuneration). PS reports receiving grant support and lecture fees from Grifols and CSL Behring, grant support and advisory board fees from Arrowhead Pharmaceuticals, grant support from Vertex Pharmaceuticals, advisory board fees from Dicerna Pharmaceuticals and Ono Pharmaceuticals, and lecture fees from Alnylam Pharmaceuticals. EG has received consultancy fees from Mirum Pharmaceuticals, Albireo, and Laboratoires CTRS.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      The expert suggestions from Adam Mullick (Ionis Pharmaceuticals), Klaus Charisse, Björn Ambrosius (Alnylam Pharmaceuticals), Luise Raut (Sanofi-Aventis), Bernard Benichou (Vivet Therapeutics) and Giuseppe Ronzitti (Genethon) are gratefully acknowledged.

      Supplementary data

      The following are the supplementary data to this article:

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