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Therapeutic editing of hepatocyte genome in vivo

  • Marina Ruiz de Galarreta
    Affiliations
    Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA

    Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA
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  • Amaia Lujambio
    Correspondence
    Corresponding author. Address: Department of Oncological Sciences, Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue Hess 6-111, New York, NY 10029, USA. Tel.: +1 212 824 9338.
    Affiliations
    Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA

    Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA

    Graduate School of Biomedical Sciences at Icahn School of Medicine at Mount Sinai, New York, USA
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      Summary

      The recent development of gene editing platforms enables making precise changes in the genome of eukaryotic cells. Programmable nucleases, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleases have revolutionized the way research is conducted as they facilitate the rapid production of mutant or knockout cellular and animal models. These same genetic tools can potentially be applied to cure or alleviate a variety of diseases, including genetic diseases that lack an efficient therapy. Thus, gene editing platforms could be used for correcting mutations that cause a disease, restoration of the expression of genes that are missing, or be used for the removal of deleterious genes or viral genomes. In the context of liver diseases, genome editing could be developed to treat not only hereditary monogenic liver diseases but also hepatitis B infection and diseases that have both genetic and non-genetic components. While the prospect of translating these therapeutic strategies to a clinical setting is highly appealing, there are numerous challenges that need to be addressed first. Safety, efficiency, specificity, and delivery are some of the obstacles that will need to be addressed before each specific gene treatment is safely used in patients. Here, we discuss the most used gene editing platforms, their mechanisms of action, their potential for liver disease treatment, the most pressing challenges, and future prospects.

      Keywords

      Introduction

      Hepatocytes are the most numerous type of cells in the liver and carry out a wide variety of functions including protein and lipid metabolism, carbohydrate use and storage, formation and secretion of bile, and detoxification of body metabolites such as ammonia and bilirubin.
      • Gissen P.
      • Arias I.M.
      Structural and functional hepatocyte polarity and liver disease.
      Considering the vast array of activities performed by hepatocytes it is not surprising that alterations affecting these cells can give rise to many different diseases. For example, disruption of key metabolic pathways in the hepatocytes can lead to the accumulation of proteins or toxins, which can in turn have deleterious effects.
      • Alam S.
      • Sood V.
      Metabolic liver disease: when to suspect and how to diagnose?.
      Many of the liver-related diseases are directly caused by genetic mutations and it has been reported that there are hundreds of monogenic disorders linked to the liver.
      • Fagiuoli S.
      • Daina E.
      • D'Antiga L.
      • Colledan M.
      • Remuzzi G.
      Monogenic diseases that can be cured by liver transplantation.
      While the understanding of the genetic basis of many of these liver disorders have significantly improved over the last years, the translation of this knowledge into successful small molecule therapies has been limited, emphasizing the need for alternative therapeutic approaches.
      • Thoene J.G.
      Orphan drugs and orphan tests in the USA.
      Recent advances in the development of technologies that are able to accurately modify DNA could potentially enable the treatment of certain monogenic liver diseases, such as anti-trypsin deficiency, hemochromatosis, and tyrosinemia. Furthermore, similar approaches could also be implemented to tackle non-genetic liver diseases.
      Until recently, the main strategies to manipulate gene expression were limited to gene therapy and RNA interference (RNAi) technologies.
      • Fellmann C.
      • Lowe S.W.
      Stable RNA interference rules for silencing.
      Gene therapy can restore gene expression through the reintroduction of a wild-type copy of the malfunctioning gene.
      • Naldini L.
      In vivo gene delivery by lentiviral vectors.
      This strategy has been used to treat hemophilia by integrating Factor IX gene into the genome of hepatocytes (NCT02484092). Unfortunately, the clinical implementation of gene therapy has been hampered by the risks and limitations associated with this technology. On the one hand, random integration of genetic elements into the DNA can have adverse consequences, either disrupting or promoting the expression of adjacent genes and potentially causing cancer. On the other hand, gene therapy is restricted to diseases caused by loss of function alterations in genes that in addition need to have a small size to allow proper packaging and delivery.
      • Baum C.
      • von Kalle C.
      • Staal F.J.
      • Li Z.
      • Fehse B.
      • Schmidt M.
      • et al.
      Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.
      RNAi can be used to repress the expression of aberrant genes by impacting the messenger RNA (mRNA) of the target gene. While RNAi is being used to treat several malignancies, including hypercholesterolemia caused by mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) and subsequent overactivation (NCT01437059),
      • Vaishnaw A.K.
      • Gollob J.
      • Gamba-Vitalo C.
      • Hutabarat R.
      • Sah D.
      • Meyers R.
      • et al.
      A status report on RNAi therapeutics.
      • Kanasty R.
      • Dorkin J.R.
      • Vegas A.
      • Anderson D.
      Delivery materials for siRNA therapeutics.
      it also presents several limitations, including incomplete target inhibition, suboptimal specificity, and potential off-target effects.
      • Castanotto D.
      • Rossi J.J.
      The promises and pitfalls of RNA-interference-based therapeutics.
      • Jackson A.L.
      • Linsley P.S.
      Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application.
      More recently, the development of genome editing technologies based on meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleases has dramatically facilitated targeted genome manipulation, opening new avenues for both research and therapy.
      In this review, we will describe the main genome editing platforms and their mechanisms of action, and we will discuss the potential for their use to treat liver diseases and the major obstacles that need to be addressed in order for their clinical translation. We will mainly focus on monogenic liver diseases with known mutations but we will also cover other clinical applications, such as treatment of viral infections.

      Gene editing exploits DNA repair mechanisms

      Gene editing, also known as genetic manipulation or gene targeting, has been a key research tool that has enabled the study of gene function in the context of live organisms.
      • Porteus M.H.
      Towards a new era in medicine: therapeutic genome editing.
      • Thomas K.R.
      • Capecchi M.R.
      Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.
      • Smithies O.
      • Gregg R.G.
      • Boggs S.S.
      • Koralewski M.A.
      • Kucherlapati R.S.
      Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination.
      After the first successful gene editing efforts, it was soon speculated that direct genetic manipulation could potentially be applied to cure genetic diseases; however, its low efficiency has always been a limiting factor for its clinical implementation. Mechanistically, it is now well established that double-strand breaks (DSBs) can stimulate the cellular repair machinery and therefore enhance gene editing.
      • Porteus M.H.
      Towards a new era in medicine: therapeutic genome editing.
      • Rouet P.
      • Smih F.
      • Jasin M.
      Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.
      • Smih F.
      • Rouet P.
      • Romanienko P.J.
      • Jasin M.
      Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.
      On the one hand, DSBs in the target gene enhance homologous recombination up to five orders of magnitude. On the other hand, DSBs can increase the generation of mutations at the site of the DSB up to nine orders of magnitude.
      • Porteus M.H.
      Towards a new era in medicine: therapeutic genome editing.
      • Rouet P.
      • Smih F.
      • Jasin M.
      Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.
      • Smih F.
      • Rouet P.
      • Romanienko P.J.
      • Jasin M.
      Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.
      DSBs recruit endogenous repair machinery for either non-homologous end-joining (NHEJ) or homology directed repair (HDR) to the DSB site to mediate genome editing depending on the cell state and the presence of a repair template (Fig. 1).
      • West S.C.
      • Chappell C.
      • Hanakahi L.A.
      • Masson J.Y.
      • McIlwraith M.J.
      • Van Dyck E.
      Double-strand break repair in human cells.
      Figure thumbnail gr1
      Fig. 1Double strand breaks (DSBs) and DNA repair mechanisms. (A) DSBs (red scissors) can be repaired by non-homologous end-joining (NHEJ) repair mechanisms, giving rise to small insertions (violet) and deletions (light blue) that can disrupt gene expression. (B) Two DSBs can also be corrected by NHEJ and can generate inversions, inverting the DNA located between the two DSBs (in green, reverted), or deletions, by removing the DNA located between the two DSBs (in green). (C) DSBs can be repaired by homology directed repair (HDR) mechanisms in the presence of a donor template, allowing the incorporation of insertions (violet) or mutations (yellow star) that can disrupt gene expression. Black vertical bars indicate homology.
      NHEJ is the most “basic” repair mechanism and it does not require the presence of a DNA template. NHEJ repairs the lesion by joining the two ends of the DSB, often introducing small insertions or deletions in the breaking site.
      • West S.C.
      • Chappell C.
      • Hanakahi L.A.
      • Masson J.Y.
      • McIlwraith M.J.
      • Van Dyck E.
      Double-strand break repair in human cells.
      In general, these indels affect a few nucleotides and therefore can cause frameshift mutations that can lead to the production of aberrant truncated proteins or mRNA degradation by nonsense-mediated decay.
      • Hentze M.W.
      • Kulozik A.E.
      A perfect message: RNA surveillance and nonsense-mediated decay.
      When there are two DSBs instead of one, NHEJ can create deletions, inversion, or translocations. Either way, NHEJ can be triggered to repress the expression of aberrant proteins. HDR is a more “sophisticated” repair mechanism, but as such, it is also more inefficient. HDR requires the presence of a template DNA, which directs the accurate repair of the DSB.
      • Krejci L.
      • Altmannova V.
      • Spirek M.
      • Zhao X.
      Homologous recombination and its regulation.
      Depending on the DNA template used and the location of the homology arms, HDR can introduce changes in a single nucleotide or affect larger regions. Thus, this repair mechanism can be used to correct or replace a dysfunctional gene, restoring gene function, or to mutate or delete an aberrantly expressing gene. It is therefore possible to harness the endogenous DNA repair machineries to engineer or reverse a wide variety of genomic alterations.
      Gene editing is based on the targeted induction of double strand breaks and subsequent DNA repair.

      Gene editing platforms

      There are several requirements for successful gene editing: a nuclease that introduces a DSB, intact DNA repair machineries, and in the case of HDR-directed DNA repair, a DNA template. In addition, it is critical that the nuclease performs DSBs in the sequence of interest. So far, four different platforms have been co-opted to introduce site-specific DSBs: meganucleases, ZFNs, TALENs, and CRISPR-associated nucleases (Fig. 2). They differ in the DNA-recognition mechanisms and type of DSB induced, and as a consequence, present different advantages and disadvantages.
      Figure thumbnail gr2
      Fig. 2The four main gene editing platforms. The different nucleases (pale red) bind to DNA through protein-DNA interactions (meganucleases, ZFNs, TALENs) or protein-RNA-DNA interactions (CRISPR-Cas nucleases). ZFNs and TALENs contain protein domains that can bind 3 or 1 nucleotide(s), respectively, in a sequence specific manner. The nucleases induce different types of DSBs, represented by the red lines. DSB, double-strand break; nt, nucleotide; PAM, protospacer-adjacent motif; sgRNA, single guide RNA. The vertical black bars indicate homology.
      Meganuclease technology is based on the ability of homing endonucleases to bind and cut DNA in a sequence specific manner.
      • Maeder M.L.
      • Gersbach C.A.
      Genome-editing technologies for gene and cell therapy.
      Some examples of naturally occurring homing endonucleases are I-CreI and I-SceI enzymes.
      • Chevalier B.S.
      • Stoddard B.L.
      Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
      These homing endonucleases can be engineered to extend the repertoire of DNA binding sequences.
      • Smith J.
      • Grizot S.
      • Arnould S.
      • Duclert A.
      • Epinat J.C.
      • Chames P.
      • et al.
      A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.
      • Rosen L.E.
      • Morrison H.A.
      • Masri S.
      • Brown M.J.
      • Springstubb B.
      • Sussman D.
      • et al.
      Homing endonuclease I-CreI derivatives with novel DNA target specificities.
      • Sussman D.
      • Chadsey M.
      • Fauce S.
      • Engel A.
      • Bruett A.
      • Monnat Jr, R.
      • et al.
      Isolation and characterization of new homing endonuclease specificities at individual target site positions.
      • Seligman L.M.
      • Chisholm K.M.
      • Chevalier B.S.
      • Chadsey M.S.
      • Edwards S.T.
      • Savage J.H.
      • et al.
      Mutations altering the cleavage specificity of a homing endonuclease.
      • Redondo P.
      • Prieto J.
      • Munoz I.G.
      • Alibes A.
      • Stricher F.
      • Serrano L.
      • et al.
      Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.
      Of note, meganucleases generate DSBs with a 3′ overhang. Additional advantages of this platform are that the recognition site for meganucleases is around 14 base pairs long, which increases specificity, and the size of the meganucleases is relatively small, facilitating potential in vivo delivery.
      • Maeder M.L.
      • Gersbach C.A.
      Genome-editing technologies for gene and cell therapy.
      • Smith J.
      • Grizot S.
      • Arnould S.
      • Duclert A.
      • Epinat J.C.
      • Chames P.
      • et al.
      A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.
      Nevertheless, the re-engineering of meganucleases needed to increase the range of DNA-target sequences is complex and laborious, vastly limiting the applicability of this platform.
      ZFNs are artificial proteins in which a zinc finger DNA binding domain is fused to the non-specific nuclease domain derived from FokI.
      • Kim Y.G.
      • Cha J.
      • Chandrasegaran S.
      Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
      Zinc finger DNA binding domains can also be engineered to target different DNA sequences using different approaches, such as rational design or using combinatorial libraries.
      • Desjarlais J.R.
      • Berg J.M.
      Toward rules relating zinc finger protein sequences and DNA binding site preferences.
      • Blancafort P.
      • Magnenat L.
      • Barbas 3rd., C.F.
      Scanning the human genome with combinatorial transcription factor libraries.
      • Joung J.K.
      • Ramm E.I.
      • Pabo C.O.
      A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions.
      • Choo Y.
      • Klug A.
      Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage.
      • Rebar E.J.
      • Pabo C.O.
      Zinc finger phage: affinity selection of fingers with new DNA-binding specificities.
      ZFNs were the first platform to successfully be used for genome editing of human somatic cells
      • Porteus M.H.
      • Baltimore D.
      Chimeric nucleases stimulate gene targeting in human cells.
      • Urnov F.D.
      • Miller J.C.
      • Lee Y.L.
      • Beausejour C.M.
      • Rock J.M.
      • Augustus S.
      • et al.
      Highly efficient endogenous human gene correction using designed zinc-finger nucleases.
      and were also harnessed to seamlessly create mutant animals, such as the rat.
      • Tesson L.
      • Usal C.
      • Menoret S.
      • Leung E.
      • Niles B.J.
      • Remy S.
      • et al.
      Knockout rats generated by embryo microinjection of TALENs.
      Structurally, tandem fingers wrap around the DNA, each finger covering 3 base pairs, so in order to increase specificity, several multi-finger arrays need to be assembled.
      • Bae K.H.
      • Kwon Y.D.
      • Shin H.C.
      • Hwang M.S.
      • Ryu E.H.
      • Park K.S.
      • et al.
      Human zinc fingers as building blocks in the construction of artificial transcription factors.
      One of the advantages of ZFNs is that FokI nuclease functions as a dimer so two ZFNs that bind at either side of the DSB are needed, which also increases specificity. However, it also escalates the size of the complex hindering delivery, the strategy is rather arduous, and it creates 5′ overhangs.
      TALE proteins were first identified in the plant pathogen Xanthomonas and they are able to specifically bind DNA.
      • Boch J.
      • Scholze H.
      • Schornack S.
      • Landgraf A.
      • Hahn S.
      • Kay S.
      • et al.
      Breaking the code of DNA binding specificity of TAL-type III effectors.
      • Moscou M.J.
      • Bogdanove A.J.
      A simple cipher governs DNA recognition by TAL effectors.
      Thus, 33 to 35 amino acid repeats are able to selectively bind a single base pair of DNA,
      • Deng D.
      • Yan C.
      • Pan X.
      • Mahfouz M.
      • Wang J.
      • Zhu J.K.
      • et al.
      Structural basis for sequence-specific recognition of DNA by TAL effectors.
      • Mak A.N.
      • Bradley P.
      • Cernadas R.A.
      • Bogdanove A.J.
      • Stoddard B.L.
      The crystal structure of TAL effector PthXo1 bound to its DNA target.
      and these TALE repeats can be assembled to recognize longer sequences of DNA. Similar to ZFNs, TALEs are fused to the catalytic domain of the FokI endonuclease to give rise to TALENs.
      • Miller J.C.
      • Tan S.
      • Qiao G.
      • Barlow K.A.
      • Wang J.
      • Xia D.F.
      • et al.
      A TALE nuclease architecture for efficient genome editing.
      • Li T.
      • Huang S.
      • Jiang W.Z.
      • Wright D.
      • Spalding M.H.
      • Weeks D.P.
      • et al.
      TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.
      The main advantage of TALENs is that they can virtually target any DNA sequence, since the only restriction is the presence of a T at the 5′ end.
      • Cermak T.
      • Doyle E.L.
      • Christian M.
      • Wang L.
      • Zhang Y.
      • Schmidt C.
      • et al.
      Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.
      Moreover, TALENs are very specific and not too troublesome to be engineered. However, TALENs present a large size as around 34 amino acids are required to bind a single nucleotide,
      • Cermak T.
      • Doyle E.L.
      • Christian M.
      • Wang L.
      • Zhang Y.
      • Schmidt C.
      • et al.
      Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.
      hampering the potential in vivo delivery of the constructs.
      CRISPR-Cas RNA-guide nucleases were discovered in bacteria more than two decades ago.
      • Mojica F.J.
      • Ferrer C.
      • Juez G.
      • Rodriguez-Valera F.
      Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning.
      The role of CRISPR-Cas RNA-guide nucleases is to protect from invading viruses, working as a primitive adaptive immune system.
      • Bolotin A.
      • Quinquis B.
      • Sorokin A.
      • Ehrlich S.D.
      Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.
      • Barrangou R.
      • Fremaux C.
      • Deveau H.
      • Richards M.
      • Boyaval P.
      • Moineau S.
      • et al.
      CRISPR provides acquired resistance against viruses in prokaryotes.
      CRISPR-Cas works slightly different than meganucleases, ZFNs, and TALENs, as the recognition of the DNA is mediated by an RNA molecule and not only by a protein. In particular, CRISPRs are transcribed and processed into CRISPR RNAs (cRNAs) that associate with a trans-activating crRNA (tracrRNAs), which in turn binds to a CRISPR-associated (Cas) nuclease, which then cleaves the DNA.
      • Wiedenheft B.
      • Sternberg S.H.
      • Doudna J.A.
      RNA-guided genetic silencing systems in bacteria and archaea.
      Later work demonstrated that the system was composed of three elements, crRNA, tracrRNA and the Cas9 protein,
      • Deltcheva E.
      • Chylinski K.
      • Sharma C.M.
      • Gonzales K.
      • Chao Y.
      • Pirzada Z.A.
      • et al.
      CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
      • Garneau J.E.
      • Dupuis M.E.
      • Villion M.
      • Romero D.A.
      • Barrangou R.
      • Boyaval P.
      • et al.
      The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
      and could be simplified to just two components by fusing the crRNA and the tracrRNA into a single guide RNA (sgRNA).
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
      The main advantage of the CRISPR/Cas system is that the gRNAs can be engineered to target different sequences in the DNA, including mammalian cells.
      • Mali P.
      • Yang L.
      • Esvelt K.M.
      • Aach J.
      • Guell M.
      • DiCarlo J.E.
      • et al.
      RNA-guided human genome engineering via Cas9.
      • Jinek M.
      • East A.
      • Cheng A.
      • Lin S.
      • Ma E.
      • Doudna J.
      RNA-programmed genome editing in human cells.
      • Cho S.W.
      • Kim S.
      • Kim J.M.
      • Kim J.S.
      Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • et al.
      Multiplex genome engineering using CRISPR/Cas systems.
      The only sequence restriction for the CRISPR/Cas system is that there needs to be a 3 nucleotide-long sequence, the protospacer-adjacent motif (PAM), located at the 3′ of the target site. The PAM sequence can vary between different nucleases from different species, which could broaden the repertoire of targetable sequences.
      • Chylinski K.
      • Makarova K.S.
      • Charpentier E.
      • Koonin E.V.
      Classification and evolution of type II CRISPR-Cas systems.
      The four different platforms offer different advantages and disadvantages, concerning their potential for in vivo delivery, selectivity, sequence specificity, and flexibility (Table 1). While meganucleases and ZFNs require complex protein engineering to be able to target novel sequences and the process is highly time-consuming (1 year or 3 months, respectively), TALENs are more straight-forward to design, taking around 2 weeks to engineer, and CRISPR/Cas system can be retargeted to new DNA sequences by simply changing the sequence of the sgRNA. Moreover, the CRISPR/Cas system can easily induce multiple DSBs (multiplexing) by expressing different sgRNAs, which allows more sophisticated gene editing.
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • et al.
      Multiplex genome engineering using CRISPR/Cas systems.
      Also, the four platforms differ in their cutting: meganucleases produce 3′ overhangs, ZFNs and TALENs create 5′ overhangs, and CRISPR/Cas induces blunt breaks. The type of genetic alteration and cell type of interest will dictate the most convenient platform for successful gene editing.
      There are four main gene editing platforms: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleases.
      Table 1Advantages and disadvantages of the four gene editing platforms.
      Gene editing platformFeaturesAdvantagesDisadvantages
      Meganucleases- DNA-protein recognition

      - 3′ overhang

      - 14-base recognition
      - High specificity

      - Relatively easy to deliver in vivo
      - Complex to engineer

      - Difficult to multiplex
      ZFN- DNA-protein recognition

      - 5′ overhang

      - 3-bases, 1 domain
      - High specificity

      - Relatively easy in vivo delivery
      - Complex to engineer

      - Difficult to multiplex
      TALENs- DNA-protein recognition

      - 5′ overhang

      - 1-base, 1 domain
      - High specificity

      - Relatively easy to engineer
      - Limited in vivo delivery

      - Difficult to multiplex
      CRISPR/Cas9- DNA-RNA-protein recognition

      - blunt break

      - 20 + 3 base recognition
      - Easy to engineer

      - Easy to multiplex
      - Lower specificity

      - Limited in vivo delivery
      ZFNs, zinc finger nucleases; TALENS, transcription activator-like effector nucleases; CRISPR, clustered regularly interspaced short palindromic repeat-associated nucleases.

      Gene editing for the treatment of liver diseases

      Gene editing can be used for many different applications, such as introduction of deleterious mutations, gene deletion, correction of mutations, restoration of gene expression, or disruption of viral DNA. Some diseases can be caused by aberrant gene activity, due to excessive gene expression and/or the presence of activating mutations. In these cases, NHEJ-mediated deleterious mutations or deletions could be used to inactivate the aberrant gene copy while HDR-mediated gene editing could be harnessed to correct the mutated copy of the gene (Fig. 3). However, most hereditary liver diseases are caused by loss of function mutations or deficient gene expression, which could be reverted by HR-mediated gene correction or targeted addition of therapeutic transgenes (Table 2). Furthermore, targeted mutations and deletions could also be created to protect from diseases such as viral hepatitis infection or to eliminate viral DNA.
      Figure thumbnail gr3
      Fig. 3Different liver diseases caused by hereditary mutations or viral infection can potentially be treated with gene editing approaches. Yellow stars represent non-synonymous mutations; the red “scissors” mark indicates the location of the DSB; the vertical black bars indicate homology; WT, wild-type; HDR, homology directed repair; NHEJ, non-homologous end-joining.
      Table 2An overview of the liver diseases targeted by gene editing treatments.
      DiseaseType of genetic alterationGene editing treatmentGene editing platform employed (preclinical)Delivery method (preclinical)Efficiency (preclinical)Therapeutic threshold (preclinical)[Refs.]
      HaemophiliaLoss of function mutations in Factor IXCorrection or integration of Factor IX through HDRZFNsAAVs7%Yes (>1%)
      • Li H.
      • Haurigot V.
      • Doyon Y.
      • Li T.
      • Wong S.Y.
      • Bhagwat A.S.
      • et al.
      In vivo genome editing restores haemostasis in a mouse model of haemophilia.
      • Anguela X.M.
      • Sharma R.
      • Doyon Y.
      • Miller J.C.
      • Li H.
      • Haurigot V.
      • et al.
      Robust ZFN-mediated genome editing in adult hemophilic mice.
      TyrosinemiaLoss of function mutations in FAHCorrection or integration of through HDRCRISPR/Cas9Hydrodynamic injections; or viral and lipid particles0.4–6%Yes
      • Yin H.
      • Xue W.
      • Chen S.
      • Bogorad R.L.
      • Benedetti E.
      • Grompe M.
      • et al.
      Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
      • 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.
      Alpha 1 antitrypsin deficiencyLoss of function mutations in SERPINA1Correction or integration of SERPINA1 through HDR
      HemochromatosisLoss of function mutations in HFECorrection or integration of HFE through HDR
      Wilson diseaseLoss of function mutations in ATP7BCorrection or integration of ATP7B through HDR
      HypercholesterolemiaGain of function mutations in PCSK9Correction or deletion of ATP7B through HDR and NHEJ, respectively.CRISPR/Cas9AAVs50%Yes
      • Ding Q.
      • Strong A.
      • Patel K.M.
      • Ng S.L.
      • Gosis B.S.
      • Regan S.N.
      • et al.
      Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing.
      Hepatitis B infectionExogenous DNADeletion or mutation of viral DNA through NHEJCRISPR/Cas9, TALENs, ZFNsHydrodynamic injections; AAVs27%-70%Yes
      • Lin S.R.
      • Yang H.C.
      • Kuo Y.T.
      • Liu C.J.
      • Yang T.Y.
      • Sung K.C.
      • et al.
      The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo.
      • Bloom K.
      • Ely A.
      • Mussolino C.
      • Cathomen T.
      • Arbuthnot P.
      Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.
      • Weber N.D.
      • Stone D.
      • Sedlak R.H.
      • De Silva Feelixge H.S.
      • Roychoudhury P.
      • Schiffer J.T.
      • et al.
      AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
      HDR, homology directed repair; ZFNs, zinc finger nucleases; AAVs, adeno-associated virus; CRISPR, clustered regularly interspaced short palindromic repeat; FAH, fumarylacetoacetate hydrolase; SERPINA1, serpin family A member 1; ATP7B, ATPase copper transporting beta; PCSK9, proprotein convertase subtilisin/kexin type 9; TALENs, transcription activator-like effector nucleases; NHEJ, non-homologous end-joining.
      Hemochromatosis is a good example of a hereditary liver disease caused by loss of function mutations that give rise to liver damage. It is mainly caused by mutations in HFE (hemochromatosis gene) but can also be triggered by mutations in HAMP (hepcidin antimicrobial peptide), HFE2 (hemochromatosis type 2), SLC40A1 (solute carrier family 40 member 1), and TFR2 (transferrin receptor 2).
      • Pietrangelo A.
      Hereditary hemochromatosis–a new look at an old disease.
      These mutations lead to excessive accumulation of iron in the body in general but particularly in the liver, which can induce severe liver cell death. This liver damage can advance into cirrhosis and eventually lead to liver cancer, further emphasizing the severity of the disease.
      Wilson disease, which is caused by copper accumulation, is mainly due to a loss of function mutation in the ATP7B (ATPase copper transporting beta) gene, which impairs the transport and elimination of excessive copper from the liver, resulting in liver damage and subsequent development of liver disease.
      • Das S.K.
      • Ray K.
      Wilson's disease: an update.
      Another example of liver hereditary disease due to a loss of function mutation is alpha 1-antitrypsin deficiency, caused by mutations in SERPINA1 (serpin family A member 1) gene. This can lead to the accumulation of elastase, which can in turn damage the lungs and the liver.
      • Fairbanks K.D.
      • Tavill A.S.
      Liver disease in alpha 1-antitrypsin deficiency: a review.
      All these diseases could potentially be cured by restoring the proper activity of the genes in every key cell, either by HDR-mediated gene correction to revert the loss of function mutation to the wild-type sequence, or by targeting the introduction of a functional copy of the gene. To achieve HDR-mediated gene repair, the presence of a donor template is needed, with the correct version of the gene fragment. The donor template can be incorporated as single-stranded oligonucleotides (ssODNs), with an homology of up to 80 base pairs, although shorter homology sequences can also be successfully used.
      • Chen F.
      • Pruett-Miller S.M.
      • Huang Y.
      • Gjoka M.
      • Duda K.
      • Taunton J.
      • et al.
      High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.
      • Renaud J.B.
      • Boix C.
      • Charpentier M.
      • De Cian A.
      • Cochennec J.
      • Duvernois-Berthet E.
      • et al.
      Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases.
      However, for cells that are difficult to transfect in vivo, viral vectors can be used as a source of donor DNA.
      • Miller D.G.
      • Wang P.R.
      • Petek L.M.
      • Hirata R.K.
      • Sands M.S.
      • Russell D.W.
      Gene targeting in vivo by adeno-associated virus vectors.
      • Handel E.M.
      • Gellhaus K.
      • Khan K.
      • Bednarski C.
      • Cornu T.I.
      • Muller-Lerch F.
      • et al.
      Versatile and efficient genome editing in human cells by combining zinc-finger nucleases with adeno-associated viral vectors.
      • Hirsch M.L.
      • Green L.
      • Porteus M.H.
      • Samulski R.J.
      Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair.
      In the past, gene therapy was used with the aim of introducing a copy of the functional gene, using viral vectors to restore the expression of the given gene. However, the random integration of those viruses has been problematic,
      • Hacein-Bey-Abina S.
      • Von Kalle C.
      • Schmidt M.
      • McCormack M.P.
      • Wulffraat N.
      • Leboulch P.
      • et al.
      LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.
      hampering the clinical development of gene therapies. A safer alternative is to induce HDR-mediated targeted gene integration, in which the gene to restore is flanked by homology arms matching the insertion site sequence.
      • Moehle E.A.
      • Rock J.M.
      • Lee Y.L.
      • Jouvenot Y.
      • DeKelver R.C.
      • Gregory P.D.
      • et al.
      Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.
      One option is to incorporate the functional gene into a “safe harbor” that allows good expression of the gene.
      • Lombardo A.
      • Cesana D.
      • Genovese P.
      • Di Stefano B.
      • Provasi E.
      • Colombo D.F.
      • et al.
      Site-specific integration and tailoring of cassette design for sustainable gene transfer.
      Another option is to integrate the wild-type copy of the gene into the endogenous locus, allowing for endogenous gene regulation, which is expected to be more physiological.
      • Li H.
      • Haurigot V.
      • Doyon Y.
      • Li T.
      • Wong S.Y.
      • Bhagwat A.S.
      • et al.
      In vivo genome editing restores haemostasis in a mouse model of haemophilia.
      While the majority of liver diseases are caused by loss of function genetic mutations, there are other liver diseases that are initiated by aberrant or excessive activity of a gene. For example, familial hypercholesterolemia can be caused by nonsense mutations in PCSK9 gene.
      • Cohen J.
      • Pertsemlidis A.
      • Kotowski I.K.
      • Graham R.
      • Garcia C.K.
      • Hobbs H.H.
      Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
      PCSK9 overexpression or aberrant activation induces a reduction in the number of low-density lipoprotein receptors, which in turn leads to the accumulation of low-density lipoprotein in circulation and eventually to accumulation in the liver. One genetic strategy to revert the effects of PCSK9 overactivation is to exploit the error-prone NHEJ to introduce small indels at the target site. When the DSB site is placed in the coding region of a gene, the resulting indels will often cause frameshifts that can lead to truncating forms and loss of function (Fig. 3). Several DSBs could also be introduced to promote the deletion of the aberrant gene,
      • Lee H.J.
      • Kim E.
      • Kim J.S.
      Targeted chromosomal deletions in human cells using zinc finger nucleases.
      and in the case of activating mutations, gene correction could be achieved by HDR-mediated DNA repair, as explained above. Gene disruption or deletion could also be used to impair gene infection by viruses or to directly eliminate viral DNA. This could be particularly useful for hepatitis B infections,
      • Lin S.R.
      • Yang H.C.
      • Kuo Y.T.
      • Liu C.J.
      • Yang T.Y.
      • Sung K.C.
      • et al.
      The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo.
      • Bloom K.
      • Ely A.
      • Mussolino C.
      • Cathomen T.
      • Arbuthnot P.
      Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.
      • Weber N.D.
      • Stone D.
      • Sedlak R.H.
      • De Silva Feelixge H.S.
      • Roychoudhury P.
      • Schiffer J.T.
      • et al.
      AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
      as a successful therapy is lacking and its viral DNA gets integrated into the hepatocyte genome.
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      Taken together, gene editing technologies could be exploited to treat a range of liver diseases, with a genetic or non-genetic component.
      Gene editing could be harnessed to cure liver diseases caused by loss of function mutations, such as hemochromatosis, or aberrant gene activation, such as hypercholesterolemia.

      Preclinical studies testing hepatocyte genome editing strategies

      The ability to manipulate the hepatocyte genome offers multiple avenues for the treatment of a myriad of liver diseases. Numerous preclinical studies demonstrating the feasibility and therapeutic potential of gene editing have been performed, supporting further investigation and translation into the clinics. Great advances have been made in the potential treatment of hemophilia by genetic manipulation of hepatocytes. While strictly not a “liver” disease, Factor IX is produced by hepatocytes, which can be subjected to genome editing easier than other cell types in vivo. Moreover, restoration of Factor IX activity to only 1% of the physiological levels can make the disease much milder, reducing the risk of severe bleedings. In a mouse model of hemophilia, 7% correction of mutated Factor IX was achieved by using ZFNs, a wild-type DNA copy, and delivery by adeno-associated viruses.
      • Li H.
      • Haurigot V.
      • Doyon Y.
      • Li T.
      • Wong S.Y.
      • Bhagwat A.S.
      • et al.
      In vivo genome editing restores haemostasis in a mouse model of haemophilia.
      The study was performed in young mice, in which the hepatocytes are still proliferating and HDR-mediated mechanisms are still active. Importantly, the strategy has also worked in adult hepatocytes that have exited the cell cycle, through a combination of HDR- and NHEJ-mediated repair mechanisms,
      • Anguela X.M.
      • Sharma R.
      • Doyon Y.
      • Miller J.C.
      • Li H.
      • Haurigot V.
      • et al.
      Robust ZFN-mediated genome editing in adult hemophilic mice.
      extending the applicability of the approach. By following similar strategies, CRISPR-Cas9 was successfully used to treat a mouse model of hereditary tyrosinemia caused by fumarylacetoacetate hydrolase (FAH)-deficiency.
      • Yin H.
      • Xue W.
      • Chen S.
      • Bogorad R.L.
      • Benedetti E.
      • Grompe M.
      • et al.
      Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
      Low levels of FAH can lead to the insufficient processing of tyrosine and subsequent fumarylacetoacetate accumulation, which is toxic for the liver cells. The delivery of CRISPR-Cas9 complexes was achieved by either hydrodynamic injection
      • Yin H.
      • Xue W.
      • Chen S.
      • Bogorad R.L.
      • Benedetti E.
      • Grompe M.
      • et al.
      Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
      or combination of viral and lipid particles,
      • 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.
      and allowed the correction of 0.4% or 6% of hepatocytes, respectively, which eventually repopulated the liver, correcting the disease phenotype.
      There are also some studies supporting the potential therapeutic application of NHEJ-mediated gene editing to address diseases that require gene inactivation. For example, CRISPR-Cas9-mediated loss of function mutations in PCSK9 were generated in mice, using adenoviruses directed at hepatocytes, which led to the mutation of 50% of the hepatocytes and a 40% decrease in the levels of cholesterol in the blood.
      • Ding Q.
      • Strong A.
      • Patel K.M.
      • Ng S.L.
      • Gosis B.S.
      • Regan S.N.
      • et al.
      Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing.
      Gene editing can also be used to eliminate or target viral genomes. In the context of the liver, the elimination of hepatitis B virus can be achieved in preclinical models by using CRISPR-Cas9, TALENs, and ZFNs nuclease platforms.
      • Lin S.R.
      • Yang H.C.
      • Kuo Y.T.
      • Liu C.J.
      • Yang T.Y.
      • Sung K.C.
      • et al.
      The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo.
      • Bloom K.
      • Ely A.
      • Mussolino C.
      • Cathomen T.
      • Arbuthnot P.
      Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.
      • Weber N.D.
      • Stone D.
      • Sedlak R.H.
      • De Silva Feelixge H.S.
      • Roychoudhury P.
      • Schiffer J.T.
      • et al.
      AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
      While the strategies work remarkably well, it also emphasizes the limitations of the approach, as the genetic manipulation of every infected cell is needed to completely eradicate the disease.
      • Porteus M.H.
      Towards a new era in medicine: therapeutic genome editing.
      Moreover, viruses could mutate to escape the gene editing, suggesting that multiplexed strategies aimed at deleting viral DNA and/or mutate multiple sites will be needed.
      • Maeder M.L.
      • Gersbach C.A.
      Genome-editing technologies for gene and cell therapy.
      Although there are multiple limitations associated to these therapeutic approaches, these preclinical studies strongly suggest that gene editing platforms could be used to cure or alleviate a range of liver diseases caused by genetic mutations or viral infection, supporting the translation into a clinical setting alone or in combination with other treatments.
      Preclinical studies in animal and cellular systems predict that reaching the therapeutic threshold with gene editing strategies is possible, although there are pressing challenges to overcome.

      Challenges to clinically implement gene editing of hepatocytes

      Targeted gene editing could potentially serve to cure or alleviate a variety of liver diseases. However, translating the gene editing based strategies into a clinical setting faces several challenges related to safety, efficacy, specificity, or cost, which will need to be addressed for a successful translation. First of all, each disease and potentially each patient may require a unique strategy, particularly if the mutation is very rare and needs to be corrected by HDR. Therefore, the cost of clinical development and regulatory approval to develop each of these therapeutic strategies may be expensive.
      • Maeder M.L.
      • Gersbach C.A.
      Genome-editing technologies for gene and cell therapy.
      Second, it is important to establish, for each type of disease, the therapeutic threshold, which refers to the level of genetic or cell modification that is required to confer a positive therapeutic impact in the disease course.
      • Cox D.B.
      • Platt R.J.
      • Zhang F.
      Therapeutic genome editing: prospects and challenges.
      The ability to reach the therapeutic threshold will be influenced by the efficacy of the gene targeting, the efficiency of delivery, and the fitness of the cells.
      The efficiency of gene targeting and DNA repair mechanism highly depend on the cell type and the cellular state. In general, NHEJ is more active than HDR, which limits the potential for treating diseases that require gene correction or precise gene integration compared to those diseases that only require gene inactivation by NHEJ.
      • Moehle E.A.
      • Rock J.M.
      • Lee Y.L.
      • Jouvenot Y.
      • DeKelver R.C.
      • Gregory P.D.
      • et al.
      Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.
      • Cox D.B.
      • Platt R.J.
      • Zhang F.
      Therapeutic genome editing: prospects and challenges.
      • Ciccia A.
      • Elledge S.J.
      The DNA damage response: making it safe to play with knives.
      NHEJ is active throughout all the phases of the cell cycle and works well in hepatocytes.
      • Xue W.
      • Chen S.
      • Yin H.
      • Tammela T.
      • Papagiannakopoulos T.
      • Joshi N.S.
      • et al.
      CRISPR-mediated direct mutation of cancer genes in the mouse liver.
      • Rothkamm K.
      • Kruger I.
      • Thompson L.H.
      • Lobrich M.
      Pathways of DNA double-strand break repair during the mammalian cell cycle.
      While HDR works preferentially in dividing cells, during the S/G2 phase, it has also been shown that it can act in adult hepatocytes,
      • Anguela X.M.
      • Sharma R.
      • Doyon Y.
      • Miller J.C.
      • Li H.
      • Haurigot V.
      • et al.
      Robust ZFN-mediated genome editing in adult hemophilic mice.
      • 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.
      which are mainly arrested in the G1 phase. HDR efficiency is influenced by the type of gene editing to be performed: thus, small deletion or mutations are much easier to achieve than large gene insertions.
      • Moehle E.A.
      • Rock J.M.
      • Lee Y.L.
      • Jouvenot Y.
      • DeKelver R.C.
      • Gregory P.D.
      • et al.
      Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.
      Furthermore, HDR efficiency is also affected by the length of the homology arms and the type of DNA template, as the single-stranded and viral DNA more efficient than double-stranded DNA.
      • Chen F.
      • Pruett-Miller S.M.
      • Huang Y.
      • Gjoka M.
      • Duda K.
      • Taunton J.
      • et al.
      High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.
      • Miller D.G.
      • Wang P.R.
      • Petek L.M.
      • Hirata R.K.
      • Sands M.S.
      • Russell D.W.
      Gene targeting in vivo by adeno-associated virus vectors.
      One important consideration is that while the gene editing machinery is expressed in the cell, NHEJ will be active and could mutate the corrected versions, implying that introducing synonymous mutations resistant to DSBs could be a good alternative option. Despite the challenges associated with HDR, proof of concept preclinical HDR treatments have been described for mouse models of hemophilia B and hereditary tyrosinemia.
      • Li H.
      • Haurigot V.
      • Doyon Y.
      • Li T.
      • Wong S.Y.
      • Bhagwat A.S.
      • et al.
      In vivo genome editing restores haemostasis in a mouse model of haemophilia.
      • Yin H.
      • Xue W.
      • Chen S.
      • Bogorad R.L.
      • Benedetti E.
      • Grompe M.
      • et al.
      Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
      Nevertheless, strategies to improve the efficiency of gene targeting in general and of HDR-mediated DNA repair in particular will be needed to reach an optimal therapeutic threshold. One alternative strategy to increase the expression of restored genes is to integrate them in the albumin locus downstream of the endogenous albumin promoter.
      • Barzel A.
      • Paulk N.K.
      • Shi Y.
      • Huang Y.
      • Chu K.
      • Zhang F.
      • et al.
      Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.
      • Sharma R.
      • Anguela X.M.
      • Doyon Y.
      • Wechsler T.
      • DeKelver R.C.
      • Sproul S.
      • et al.
      In vivo genome editing of the albumin locus as a platform for protein replacement therapy.
      Albumin is highly expressed in hepatocytes and the integration of the wild-type gene into this locus would ensure high expression. As an example, by targeting the albumin promoter the authors could achieve 7–20% of Factor IX levels, despite an HDR rate of 0.5%.
      • Barzel A.
      • Paulk N.K.
      • Shi Y.
      • Huang Y.
      • Chu K.
      • Zhang F.
      • et al.
      Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.
      However, albumin gene is frequently mutated in human hepatocellular carcinomas (HCCs)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      and its disruption could potentially be tumorigenic, indicating that functional studies will need to be performed before therapeutically exploiting the locus.
      Another important challenge hindering the clinical translation of gene targeting strategies is the poor delivery of big complexes into the target cells.
      • Cox D.B.
      • Platt R.J.
      • Zhang F.
      Therapeutic genome editing: prospects and challenges.
      Hepatocytes are relatively easier to target than most cell types in the human body but still significant improvements are needed. The delivery method can influence not only the extent of cells that are reached but also the type of expression in the target cell. Thus, continued expression of nucleases may induce genomic instability or predispose a person to cancer.
      • Frock R.L.
      • Hu J.
      • Meyers R.M.
      • Ho Y.J.
      • Kii E.
      • Alt F.W.
      Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.
      So far, the most promising delivery method to the liver is based on adeno-associated viral (AAV) vectors, which are already approved in the clinical setting.
      • Samulski R.J.
      • Muzyczka N.
      AAV-mediated gene therapy for research and therapeutic purposes.
      However, its small packaging size, around 4.8 kilobases, is a critical limitation. For example, a dimeric ZFN pair can fit into a single AAV while two AAVs are needed for TALEN packaging. Smaller versions of Cas9 can be packaged into a single AAV as well.
      • Ran F.A.
      • Cong L.
      • Yan W.X.
      • Scott D.A.
      • Gootenberg J.S.
      • Kriz A.J.
      • et al.
      In vivo genome editing using Staphylococcus aureus Cas9.
      • Friedland A.E.
      • Baral R.
      • Singhal P.
      • Loveluck K.
      • Shen S.
      • Sanchez M.
      • et al.
      Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications.
      However, AAV-mediated delivery of nucleases presents several disadvantages.
      • Cox D.B.
      • Platt R.J.
      • Zhang F.
      Therapeutic genome editing: prospects and challenges.
      • 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.
      First, AAV-mediated nuclease expression is permanent and could lead to DNA damage, improper gene editing, and deleterious cellular effects. Second, many patients may have developed immunity against AAVs and they could be eliminated by the immune system. Third, AAV2, a specific type of AAVs, has been shown to induce HCC in some patients.
      • Nault J.C.
      • Datta S.
      • Imbeaud S.
      • Franconi A.
      • Mallet M.
      • Couchy G.
      • et al.
      Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.
      • Nault J.C.
      • Datta S.
      • Imbeaud S.
      • Franconi A.
      • Zucman-Rossi J.
      Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma.
      While the AAV-based vectors used in gene therapy are different from AAV2, these results highlight the need to exercise caution when using AAV-based approaches. Nanoparticle and lipid-based delivery of mRNA or proteins could also be an interesting option.
      • Kormann M.S.
      • Hasenpusch G.
      • Aneja M.K.
      • Nica G.
      • Flemmer A.W.
      • Herber-Jonat S.
      • et al.
      Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.
      • Zuris J.A.
      • Thompson D.B.
      • Shu Y.
      • Guilinger J.P.
      • Bessen J.L.
      • Hu J.H.
      • et al.
      Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.
      This strategy should in theory allow for the dose to be modulated and is transient, eliminating the problems associated with the constitutive expression of nucleases. Finally, another alternative would be to manipulate human hepatocytes ex vivo and then transplant them into the patient’s liver. The delivery in cell culture is much easier and the corrected cells can be selected prior to transplantation into the liver. However, this latter strategy also presents further limitations. For example, the ex vivo culture of primary hepatocytes is challenging, the hepatocytes may not survive the genetic manipulation, and the engraftment of manipulated hepatocytes back in in vivo is also not optimal.
      • Gao S.
      • Seker E.
      • Casali M.
      • Wang F.
      • Bale S.S.
      • Price G.M.
      • et al.
      Ex vivo gene delivery to hepatocytes: techniques, challenges, and underlying mechanisms.
      Another aspect to consider is the potential toxicity associated with the gene targeting. First of all, the different nuclease platforms may be seen by the immune system as foreign, as shown in vivo in mice.
      • Wang D.
      • Mou H.
      • Li S.
      • Li Y.
      • Hough S.
      • Tran K.
      • et al.
      Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses.
      In addition, the DSBs created by the nucleases could potentially lead to aberrant chromosomal rearrangements,
      • Maddalo D.
      • Manchado E.
      • Concepcion C.P.
      • Bonetti C.
      • Vidigal J.A.
      • Han Y.C.
      • et al.
      In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.
      with unanticipated adverse effects. Moreover, off-target effects – the induction of mutations in different locations in the genome other than the target sequence – could potentially have unexpected and deleterious effects. Bioinformatics analysis and deep sequencing techniques can be used to predict and detect off-target effects, respectively, which should allow for the selection of highly specific constructs.
      • Cradick T.J.
      • Qiu P.
      • Lee C.M.
      • Fine E.J.
      • Bao G.
      COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites.
      • Hendel A.
      • Fine E.J.
      • Bao G.
      • Porteus M.H.
      Quantifying on- and off-target genome editing.
      • Tsai S.Q.
      • Zheng Z.
      • Nguyen N.T.
      • Liebers M.
      • Topkar V.V.
      • Thapar V.
      • et al.
      GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
      • Kim D.
      • Bae S.
      • Park J.
      • Kim E.
      • Kim S.
      • Yu H.R.
      • et al.
      Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.
      However, current sequencing methodologies present limitations indicating that they may not be able to identify all the present off-targets. Therefore, strategies that completely eliminate off-target effects will be needed before safely translating these platforms into the clinics. For example, a mutant Cas9 that only introduces single-strand breaks can be used in combination with two sgRNAs in order to increase specificity
      • Ran F.A.
      • Hsu P.D.
      • Lin C.Y.
      • Gootenberg J.S.
      • Konermann S.
      • Trevino A.E.
      • et al.
      Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
      or high-fidelity Cas9 variants.
      • Tsai S.Q.
      • Wyvekens N.
      • Khayter C.
      • Foden J.A.
      • Thapar V.
      • Reyon D.
      • et al.
      Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.
      • Guilinger J.P.
      • Thompson D.B.
      • Liu D.R.
      Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
      • Fu Y.
      • Sander J.D.
      • Reyon D.
      • Cascio V.M.
      • Joung J.K.
      Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
      • Kleinstiver B.P.
      • Pattanayak V.
      • Prew M.S.
      • Tsai S.Q.
      • Nguyen N.T.
      • Zheng Z.
      • et al.
      High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
      • Slaymaker I.M.
      • Gao L.
      • Zetsche B.
      • Scott D.A.
      • Yan W.X.
      • Zhang F.
      Rationally engineered Cas9 nucleases with improved specificity.
      Further research into different Cas nucleases from different species may lead to safer and more efficient strategies.
      Finally, the fitness of the target cells can play an important role in determining the best gene editing strategy.
      • Cox D.B.
      • Platt R.J.
      • Zhang F.
      Therapeutic genome editing: prospects and challenges.
      For example, if the cell expressing the mutation presents reduced fitness compared to the corrected cell, the edited cells will be positively selected reducing the need for high efficiency. This is the case of tyrosinemia caused by FAH deficiency. It has been proven in mice that just a 1–5% of successful editing is enough to achieve the therapeutic threshold.
      • Yin H.
      • Xue W.
      • Chen S.
      • Bogorad R.L.
      • Benedetti E.
      • Grompe M.
      • et al.
      Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
      • 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, if the edited cell presents defective fitness, then, the therapeutic benefit will be low and gene editing strategies would not be recommended. This indicates that diseases that require the complete correction of the cells, such as liver cancer or hepatitis B-infected hepatocytes, may not be the best indication for gene targeting therapies. Nevertheless, genome editing technologies can be used to identify novel drug target genes in cancer or hepatitis, which can then be targeted with more traditional therapeutic strategies, such as small molecule inhibitors. For example, high-throughput loss of function experiments using CRISPR-Cas9 are successful at identifying genes whose inhibition affects cancer cell viability, something that could be translated therapeutically.
      • Shalem O.
      • Sanjana N.E.
      • Hartenian E.
      • Shi X.
      • Scott D.A.
      • Mikkelsen T.S.
      • et al.
      Genome-scale CRISPR-Cas9 knockout screening in human cells.
      • Wang T.
      • Wei J.J.
      • Sabatini D.M.
      • Lander E.S.
      Genetic screens in human cells using the CRISPR-Cas9 system.
      Similar strategies could be used to increase the therapeutic options for HCC or hepatitis B, broadening the applications of gene editing technologies for the treatment or study of liver diseases.
      Main challenges limiting the clinical translation of gene editing therapies are: safety, specificity, delivery, and efficiency, which can ultimately affect the therapeutic threshold.

      Final remarks

      Gene editing technologies have revolutionized the way research is conducted and have the potential of dramatically expanding the repertoire of therapeutic options in a large variety of diseases. In the context of the liver, targeted genome editing of hepatocytes could help cure or alleviate genetic hereditary diseases or hepatitis B infection. However, several obstacles need to be addressed to clinically translate these early successes. Further studies addressing efficiency, therapeutic threshold, toxicity, and safety will be essential to fulfill the promises of gene editing based therapies. These issues are mainly being addressed with the development of strategies that increase specificity
      • Tsai S.Q.
      • Wyvekens N.
      • Khayter C.
      • Foden J.A.
      • Thapar V.
      • Reyon D.
      • et al.
      Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.
      • Guilinger J.P.
      • Thompson D.B.
      • Liu D.R.
      Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
      • Fu Y.
      • Sander J.D.
      • Reyon D.
      • Cascio V.M.
      • Joung J.K.
      Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
      • Kleinstiver B.P.
      • Pattanayak V.
      • Prew M.S.
      • Tsai S.Q.
      • Nguyen N.T.
      • Zheng Z.
      • et al.
      High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
      • Slaymaker I.M.
      • Gao L.
      • Zetsche B.
      • Scott D.A.
      • Yan W.X.
      • Zhang F.
      Rationally engineered Cas9 nucleases with improved specificity.
      and decrease off-target effects. Moreover, efforts to improve delivery are being made and could integrate strategies that decrease toxicity. Finally, a better understanding of the genetic basis of every liver disease will inform the best gene editing targets. Thus, different gene editing platforms could then target specific diseases. Taken together, the optimization of gene editing technologies will allow its clinical translation and the cure of several hereditary liver diseases.

      Financial support

      The authors gratefully thank the following funding agencies: MRG is funded by Fundación Alfonso Martín Escudero and Department of Defense ( DoD ) Translational Team Science Award ( CA150272P2 ). AL is funded by Department of Defense (DoD) Translational Team Science Award ( CA150272P2 ), DoD Career Development Award ( CA150178 ), American Association for the Study of Liver Diseases ( AASLD ) Pinnacle Award, and Icahn School of Medicine at Mount Sinai Seed Funding.

      Conflict of interest

      The authors declared that they have no conflict of interest or financial support in relation to this manuscript.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contribution

      MRG and AL equally contributed to the writing of this manuscript.

      Supplementary data

      References

        • Gissen P.
        • Arias I.M.
        Structural and functional hepatocyte polarity and liver disease.
        J Hepatol. 2015; 63: 1023-1037
        • Alam S.
        • Sood V.
        Metabolic liver disease: when to suspect and how to diagnose?.
        Indian J Pediatr. 2016; 83: 1321-1333
        • Fagiuoli S.
        • Daina E.
        • D'Antiga L.
        • Colledan M.
        • Remuzzi G.
        Monogenic diseases that can be cured by liver transplantation.
        J Hepatol. 2013; 59: 595-612
        • Thoene J.G.
        Orphan drugs and orphan tests in the USA.
        Community Genet. 2004; 7: 169-172
        • Fellmann C.
        • Lowe S.W.
        Stable RNA interference rules for silencing.
        Nat Cell Biol. 2014; 16: 10-18
        • Naldini L.
        In vivo gene delivery by lentiviral vectors.
        Thromb Haemost. 1999; 82: 552-554
        • Baum C.
        • von Kalle C.
        • Staal F.J.
        • Li Z.
        • Fehse B.
        • Schmidt M.
        • et al.
        Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.
        Mol Ther. 2004; 9: 5-13
        • Vaishnaw A.K.
        • Gollob J.
        • Gamba-Vitalo C.
        • Hutabarat R.
        • Sah D.
        • Meyers R.
        • et al.
        A status report on RNAi therapeutics.
        Silence. 2010; 1: 14
        • Kanasty R.
        • Dorkin J.R.
        • Vegas A.
        • Anderson D.
        Delivery materials for siRNA therapeutics.
        Nat Mater. 2013; 12: 967-977
        • Castanotto D.
        • Rossi J.J.
        The promises and pitfalls of RNA-interference-based therapeutics.
        Nature. 2009; 457: 426-433
        • Jackson A.L.
        • Linsley P.S.
        Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application.
        Nat Rev Drug Discov. 2010; 9: 57-67
        • Porteus M.H.
        Towards a new era in medicine: therapeutic genome editing.
        Genome Biol. 2015; 16: 286
        • Thomas K.R.
        • Capecchi M.R.
        Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.
        Cell. 1987; 51: 503-512
        • Smithies O.
        • Gregg R.G.
        • Boggs S.S.
        • Koralewski M.A.
        • Kucherlapati R.S.
        Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination.
        Nature. 1985; 317: 230-234
        • Rouet P.
        • Smih F.
        • Jasin M.
        Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.
        Mol Cell Biol. 1994; 14: 8096-8106
        • Smih F.
        • Rouet P.
        • Romanienko P.J.
        • Jasin M.
        Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.
        Nucleic Acids Res. 1995; 23: 5012-5019
        • West S.C.
        • Chappell C.
        • Hanakahi L.A.
        • Masson J.Y.
        • McIlwraith M.J.
        • Van Dyck E.
        Double-strand break repair in human cells.
        Cold Spring Harb Symp Quant Biol. 2000; 65: 315-321
        • Hentze M.W.
        • Kulozik A.E.
        A perfect message: RNA surveillance and nonsense-mediated decay.
        Cell. 1999; 96: 307-310
        • Krejci L.
        • Altmannova V.
        • Spirek M.
        • Zhao X.
        Homologous recombination and its regulation.
        Nucleic Acids Res. 2012; 40: 5795-5818
        • Maeder M.L.
        • Gersbach C.A.
        Genome-editing technologies for gene and cell therapy.
        Mol Ther. 2016; 24: 430-446
        • Chevalier B.S.
        • Stoddard B.L.
        Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
        Nucleic Acids Res. 2001; 29: 3757-3774
        • Smith J.
        • Grizot S.
        • Arnould S.
        • Duclert A.
        • Epinat J.C.
        • Chames P.
        • et al.
        A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.
        Nucleic Acids Res. 2006; 34: e149
        • Rosen L.E.
        • Morrison H.A.
        • Masri S.
        • Brown M.J.
        • Springstubb B.
        • Sussman D.
        • et al.
        Homing endonuclease I-CreI derivatives with novel DNA target specificities.
        Nucleic Acids Res. 2006; 34: 4791-4800
        • Sussman D.
        • Chadsey M.
        • Fauce S.
        • Engel A.
        • Bruett A.
        • Monnat Jr, R.
        • et al.
        Isolation and characterization of new homing endonuclease specificities at individual target site positions.
        J Mol Biol. 2004; 342: 31-41
        • Seligman L.M.
        • Chisholm K.M.
        • Chevalier B.S.
        • Chadsey M.S.
        • Edwards S.T.
        • Savage J.H.
        • et al.
        Mutations altering the cleavage specificity of a homing endonuclease.
        Nucleic Acids Res. 2002; 30: 3870-3879
        • Redondo P.
        • Prieto J.
        • Munoz I.G.
        • Alibes A.
        • Stricher F.
        • Serrano L.
        • et al.
        Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.
        Nature. 2008; 456: 107-111
        • Kim Y.G.
        • Cha J.
        • Chandrasegaran S.
        Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
        Proc Natl Acad Sci U S A. 1996; 93: 1156-1160
        • Desjarlais J.R.
        • Berg J.M.
        Toward rules relating zinc finger protein sequences and DNA binding site preferences.
        Proc Natl Acad Sci U S A. 1992; 89: 7345-7349
        • Blancafort P.
        • Magnenat L.
        • Barbas 3rd., C.F.
        Scanning the human genome with combinatorial transcription factor libraries.
        Nat Biotechnol. 2003; 21: 269-274
        • Joung J.K.
        • Ramm E.I.
        • Pabo C.O.
        A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions.
        Proc Natl Acad Sci U S A. 2000; 97: 7382-7387
        • Choo Y.
        • Klug A.
        Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage.
        Proc Natl Acad Sci U S A. 1994; 91: 11163-11167
        • Rebar E.J.
        • Pabo C.O.
        Zinc finger phage: affinity selection of fingers with new DNA-binding specificities.
        Science. 1994; 263: 671-673
        • Porteus M.H.
        • Baltimore D.
        Chimeric nucleases stimulate gene targeting in human cells.
        Science. 2003; 300: 763
        • Urnov F.D.
        • Miller J.C.
        • Lee Y.L.
        • Beausejour C.M.
        • Rock J.M.
        • Augustus S.
        • et al.
        Highly efficient endogenous human gene correction using designed zinc-finger nucleases.
        Nature. 2005; 435: 646-651
        • Tesson L.
        • Usal C.
        • Menoret S.
        • Leung E.
        • Niles B.J.
        • Remy S.
        • et al.
        Knockout rats generated by embryo microinjection of TALENs.
        Nat Biotechnol. 2011; 29: 695-696
        • Bae K.H.
        • Kwon Y.D.
        • Shin H.C.
        • Hwang M.S.
        • Ryu E.H.
        • Park K.S.
        • et al.
        Human zinc fingers as building blocks in the construction of artificial transcription factors.
        Nat Biotechnol. 2003; 21: 275-280
        • Boch J.
        • Scholze H.
        • Schornack S.
        • Landgraf A.
        • Hahn S.
        • Kay S.
        • et al.
        Breaking the code of DNA binding specificity of TAL-type III effectors.
        Science. 2009; 326: 1509-1512
        • Moscou M.J.
        • Bogdanove A.J.
        A simple cipher governs DNA recognition by TAL effectors.
        Science. 2009; 326: 1501
        • Deng D.
        • Yan C.
        • Pan X.
        • Mahfouz M.
        • Wang J.
        • Zhu J.K.
        • et al.
        Structural basis for sequence-specific recognition of DNA by TAL effectors.
        Science. 2012; 335: 720-723
        • Mak A.N.
        • Bradley P.
        • Cernadas R.A.
        • Bogdanove A.J.
        • Stoddard B.L.
        The crystal structure of TAL effector PthXo1 bound to its DNA target.
        Science. 2012; 335: 716-719
        • Miller J.C.
        • Tan S.
        • Qiao G.
        • Barlow K.A.
        • Wang J.
        • Xia D.F.
        • et al.
        A TALE nuclease architecture for efficient genome editing.
        Nat Biotechnol. 2011; 29: 143-148
        • Li T.
        • Huang S.
        • Jiang W.Z.
        • Wright D.
        • Spalding M.H.
        • Weeks D.P.
        • et al.
        TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.
        Nucleic Acids Res. 2011; 39: 359-372
        • Cermak T.
        • Doyle E.L.
        • Christian M.
        • Wang L.
        • Zhang Y.
        • Schmidt C.
        • et al.
        Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.
        Nucleic Acids Res. 2011; 39: e82
        • Mojica F.J.
        • Ferrer C.
        • Juez G.
        • Rodriguez-Valera F.
        Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning.
        Mol Microbiol. 1995; 17: 85-93
        • Bolotin A.
        • Quinquis B.
        • Sorokin A.
        • Ehrlich S.D.
        Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.
        Microbiology. 2005; 151: 2551-2561
        • Barrangou R.
        • Fremaux C.
        • Deveau H.
        • Richards M.
        • Boyaval P.
        • Moineau S.
        • et al.
        CRISPR provides acquired resistance against viruses in prokaryotes.
        Science. 2007; 315: 1709-1712
        • Wiedenheft B.
        • Sternberg S.H.
        • Doudna J.A.
        RNA-guided genetic silencing systems in bacteria and archaea.
        Nature. 2012; 482: 331-338
        • Deltcheva E.
        • Chylinski K.
        • Sharma C.M.
        • Gonzales K.
        • Chao Y.
        • Pirzada Z.A.
        • et al.
        CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
        Nature. 2011; 471: 602-607
        • Garneau J.E.
        • Dupuis M.E.
        • Villion M.
        • Romero D.A.
        • Barrangou R.
        • Boyaval P.
        • et al.
        The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
        Nature. 2010; 468: 67-71
        • Jinek M.
        • Chylinski K.
        • Fonfara I.
        • Hauer M.
        • Doudna J.A.
        • Charpentier E.
        A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
        Science. 2012; 337: 816-821
        • Mali P.
        • Yang L.
        • Esvelt K.M.
        • Aach J.
        • Guell M.
        • DiCarlo J.E.
        • et al.
        RNA-guided human genome engineering via Cas9.
        Science. 2013; 339: 823-826
        • Jinek M.
        • East A.
        • Cheng A.
        • Lin S.
        • Ma E.
        • Doudna J.
        RNA-programmed genome editing in human cells.
        Elife. 2013; 2: e00471
        • Cho S.W.
        • Kim S.
        • Kim J.M.
        • Kim J.S.
        Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.
        Nat Biotechnol. 2013; 31: 230-232
        • Cong L.
        • Ran F.A.
        • Cox D.
        • Lin S.
        • Barretto R.
        • Habib N.
        • et al.
        Multiplex genome engineering using CRISPR/Cas systems.
        Science. 2013; 339: 819-823
        • Chylinski K.
        • Makarova K.S.
        • Charpentier E.
        • Koonin E.V.
        Classification and evolution of type II CRISPR-Cas systems.
        Nucleic Acids Res. 2014; 42: 6091-6105
        • Pietrangelo A.
        Hereditary hemochromatosis–a new look at an old disease.
        N Engl J Med. 2004; 350: 2383-2397
        • Das S.K.
        • Ray K.
        Wilson's disease: an update.
        Nat Clin Pract Neurol. 2006; 2: 482-493
        • Fairbanks K.D.
        • Tavill A.S.
        Liver disease in alpha 1-antitrypsin deficiency: a review.
        Am J Gastroenterol. 2008; 103 ([Quiz 2142]): 2136-2141
        • Chen F.
        • Pruett-Miller S.M.
        • Huang Y.
        • Gjoka M.
        • Duda K.
        • Taunton J.
        • et al.
        High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.
        Nat Methods. 2011; 8: 753-755
        • Renaud J.B.
        • Boix C.
        • Charpentier M.
        • De Cian A.
        • Cochennec J.
        • Duvernois-Berthet E.
        • et al.
        Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases.
        Cell Rep. 2016; 14: 2263-2272
        • Miller D.G.
        • Wang P.R.
        • Petek L.M.
        • Hirata R.K.
        • Sands M.S.
        • Russell D.W.
        Gene targeting in vivo by adeno-associated virus vectors.
        Nat Biotechnol. 2006; 24: 1022-1026
        • Handel E.M.
        • Gellhaus K.
        • Khan K.
        • Bednarski C.
        • Cornu T.I.
        • Muller-Lerch F.
        • et al.
        Versatile and efficient genome editing in human cells by combining zinc-finger nucleases with adeno-associated viral vectors.
        Hum Gene Ther. 2012; 23: 321-329
        • Hirsch M.L.
        • Green L.
        • Porteus M.H.
        • Samulski R.J.
        Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair.
        Gene Ther. 2010; 17: 1175-1180
        • Hacein-Bey-Abina S.
        • Von Kalle C.
        • Schmidt M.
        • McCormack M.P.
        • Wulffraat N.
        • Leboulch P.
        • et al.
        LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.
        Science. 2003; 302: 415-419
        • Moehle E.A.
        • Rock J.M.
        • Lee Y.L.
        • Jouvenot Y.
        • DeKelver R.C.
        • Gregory P.D.
        • et al.
        Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.
        Proc Natl Acad Sci U S A. 2007; 104: 3055-3060
        • Lombardo A.
        • Cesana D.
        • Genovese P.
        • Di Stefano B.
        • Provasi E.
        • Colombo D.F.
        • et al.
        Site-specific integration and tailoring of cassette design for sustainable gene transfer.
        Nat Methods. 2011; 8: 861-869
        • Li H.
        • Haurigot V.
        • Doyon Y.
        • Li T.
        • Wong S.Y.
        • Bhagwat A.S.
        • et al.
        In vivo genome editing restores haemostasis in a mouse model of haemophilia.
        Nature. 2011; 475: 217-221
        • Cohen J.
        • Pertsemlidis A.
        • Kotowski I.K.
        • Graham R.
        • Garcia C.K.
        • Hobbs H.H.
        Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
        Nat Genet. 2005; 37: 161-165
        • Lee H.J.
        • Kim E.
        • Kim J.S.
        Targeted chromosomal deletions in human cells using zinc finger nucleases.
        Genome Res. 2010; 20: 81-89
        • Lin S.R.
        • Yang H.C.
        • Kuo Y.T.
        • Liu C.J.
        • Yang T.Y.
        • Sung K.C.
        • et al.
        The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo.
        Molecular therapy Nucleic acids. 2014; 3: e186
        • Bloom K.
        • Ely A.
        • Mussolino C.
        • Cathomen T.
        • Arbuthnot P.
        Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.
        Mol Ther. 2013; 21: 1889-1897
        • Weber N.D.
        • Stone D.
        • Sedlak R.H.
        • De Silva Feelixge H.S.
        • Roychoudhury P.
        • Schiffer J.T.
        • et al.
        AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
        PLoS One. 2014; 9: e97579
        • Schulze K.
        • Imbeaud S.
        • Letouze E.
        • Alexandrov L.B.
        • Calderaro J.
        • Rebouissou S.
        • et al.
        Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
        Nat Genet. 2015; 47: 505-511
        • Cox D.B.
        • Platt R.J.
        • Zhang F.
        Therapeutic genome editing: prospects and challenges.
        Nat Med. 2015; 21: 121-131
        • Anguela X.M.
        • Sharma R.
        • Doyon Y.
        • Miller J.C.
        • Li H.
        • Haurigot V.
        • et al.
        Robust ZFN-mediated genome editing in adult hemophilic mice.
        Blood. 2013; 122: 3283-3287
        • Yin H.
        • Xue W.
        • Chen S.
        • Bogorad R.L.
        • Benedetti E.
        • Grompe M.
        • et al.
        Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
        Nat Biotechnol. 2014; 32: 551-553
        • 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.
        Nat Biotechnol. 2016; 34: 328-333
        • Ding Q.
        • Strong A.
        • Patel K.M.
        • Ng S.L.
        • Gosis B.S.
        • Regan S.N.
        • et al.
        Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing.
        Circ Res. 2014; 115: 488-492
        • Ciccia A.
        • Elledge S.J.
        The DNA damage response: making it safe to play with knives.
        Mol Cell. 2010; 40: 179-204
        • Xue W.
        • Chen S.
        • Yin H.
        • Tammela T.
        • Papagiannakopoulos T.
        • Joshi N.S.
        • et al.
        CRISPR-mediated direct mutation of cancer genes in the mouse liver.
        Nature. 2014; 514: 380-384
        • Rothkamm K.
        • Kruger I.
        • Thompson L.H.
        • Lobrich M.
        Pathways of DNA double-strand break repair during the mammalian cell cycle.
        Mol Cell Biol. 2003; 23: 5706-5715
        • Barzel A.
        • Paulk N.K.
        • Shi Y.
        • Huang Y.
        • Chu K.
        • Zhang F.
        • et al.
        Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.
        Nature. 2015; 517: 360-364
        • Sharma R.
        • Anguela X.M.
        • Doyon Y.
        • Wechsler T.
        • DeKelver R.C.
        • Sproul S.
        • et al.
        In vivo genome editing of the albumin locus as a platform for protein replacement therapy.
        Blood. 2015; 126: 1777-1784
        • Frock R.L.
        • Hu J.
        • Meyers R.M.
        • Ho Y.J.
        • Kii E.
        • Alt F.W.
        Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.
        Nat Biotechnol. 2015; 33: 179-186
        • Samulski R.J.
        • Muzyczka N.
        AAV-mediated gene therapy for research and therapeutic purposes.
        Annu Rev Virol. 2014; 1: 427-451
        • Ran F.A.
        • Cong L.
        • Yan W.X.
        • Scott D.A.
        • Gootenberg J.S.
        • Kriz A.J.
        • et al.
        In vivo genome editing using Staphylococcus aureus Cas9.
        Nature. 2015; 520: 186-191
        • Friedland A.E.
        • Baral R.
        • Singhal P.
        • Loveluck K.
        • Shen S.
        • Sanchez M.
        • et al.
        Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications.
        Genome Biol. 2015; 16: 257
        • 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.
        Hum Gene Ther. 2016; 27: 947-961
        • Nault J.C.
        • Datta S.
        • Imbeaud S.
        • Franconi A.
        • Mallet M.
        • Couchy G.
        • et al.
        Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.
        Nat Genet. 2015; 47: 1187-1193
        • Nault J.C.
        • Datta S.
        • Imbeaud S.
        • Franconi A.
        • Zucman-Rossi J.
        Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma.
        Mol Cell Oncol. 2016; 3: e1095271
        • Kormann M.S.
        • Hasenpusch G.
        • Aneja M.K.
        • Nica G.
        • Flemmer A.W.
        • Herber-Jonat S.
        • et al.
        Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.
        Nat Biotechnol. 2011; 29: 154-157
        • Zuris J.A.
        • Thompson D.B.
        • Shu Y.
        • Guilinger J.P.
        • Bessen J.L.
        • Hu J.H.
        • et al.
        Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.
        Nat Biotechnol. 2015; 33: 73-80
        • Gao S.
        • Seker E.
        • Casali M.
        • Wang F.
        • Bale S.S.
        • Price G.M.
        • et al.
        Ex vivo gene delivery to hepatocytes: techniques, challenges, and underlying mechanisms.
        Ann Biomed Eng. 2012; 40: 1851-1861
        • Wang D.
        • Mou H.
        • Li S.
        • Li Y.
        • Hough S.
        • Tran K.
        • et al.
        Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses.
        Hum Gene Ther. 2015; 26: 432-442
        • Maddalo D.
        • Manchado E.
        • Concepcion C.P.
        • Bonetti C.
        • Vidigal J.A.
        • Han Y.C.
        • et al.
        In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.
        Nature. 2014; 516: 423-427
        • Cradick T.J.
        • Qiu P.
        • Lee C.M.
        • Fine E.J.
        • Bao G.
        COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites.
        Mol Ther Nucleic Acids. 2014; 3: e214
        • Hendel A.
        • Fine E.J.
        • Bao G.
        • Porteus M.H.
        Quantifying on- and off-target genome editing.
        Trends Biotechnol. 2015; 33: 132-140
        • Tsai S.Q.
        • Zheng Z.
        • Nguyen N.T.
        • Liebers M.
        • Topkar V.V.
        • Thapar V.
        • et al.
        GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
        Nat Biotechnol. 2015; 33: 187-197
        • Kim D.
        • Bae S.
        • Park J.
        • Kim E.
        • Kim S.
        • Yu H.R.
        • et al.
        Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.
        Nat Methods. 2015; 12 ([231 p following 243]): 237-243
        • Ran F.A.
        • Hsu P.D.
        • Lin C.Y.
        • Gootenberg J.S.
        • Konermann S.
        • Trevino A.E.
        • et al.
        Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
        Cell. 2013; 154: 1380-1389
        • Tsai S.Q.
        • Wyvekens N.
        • Khayter C.
        • Foden J.A.
        • Thapar V.
        • Reyon D.
        • et al.
        Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.
        Nat Biotechnol. 2014; 32: 569-576
        • Guilinger J.P.
        • Thompson D.B.
        • Liu D.R.
        Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
        Nat Biotechnol. 2014; 32: 577-582
        • Fu Y.
        • Sander J.D.
        • Reyon D.
        • Cascio V.M.
        • Joung J.K.
        Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
        Nat Biotechnol. 2014; 32: 279-284
        • Kleinstiver B.P.
        • Pattanayak V.
        • Prew M.S.
        • Tsai S.Q.
        • Nguyen N.T.
        • Zheng Z.
        • et al.
        High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
        Nature. 2016; 529: 490-495
        • Slaymaker I.M.
        • Gao L.
        • Zetsche B.
        • Scott D.A.
        • Yan W.X.
        • Zhang F.
        Rationally engineered Cas9 nucleases with improved specificity.
        Science. 2016; 351: 84-88
        • Shalem O.
        • Sanjana N.E.
        • Hartenian E.
        • Shi X.
        • Scott D.A.
        • Mikkelsen T.S.
        • et al.
        Genome-scale CRISPR-Cas9 knockout screening in human cells.
        Science. 2014; 343: 84-87
        • Wang T.
        • Wei J.J.
        • Sabatini D.M.
        • Lander E.S.
        Genetic screens in human cells using the CRISPR-Cas9 system.
        Science. 2014; 343: 80-84

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