The liver is a major organ to maintain homeostasis by promo-ting detoxification and lipid and protein metabolism. Liver dysfunction results in toxin accumulation, organ damage and the development of pathological conditions. Various liver diseases are caused by gene mutations. Liver transplantation was considered as the only therapeutic option for liver diseases. However, liver transplantation is associated with several limitations, including shortage of liver donors and immune rejection (1). The advent of gene editing methods has enabled the repair of pathological mutations. Thus, gene editing technology will replace liver transplantation as a therapeutic strategy for liver diseases.
The first successful gene therapy in humans was reported approximately 30 years ago. The peripheral blood T lymphocytes transduced with a retrovirus encoding
The first gene therapy for liver disease in humans was not successful. Jesse Gelsinger (aged 18 years) with partial ornithine transcarbamylase (OTC) deficiency, a rare liver disorder caused by a genetic mutation, participated in a clinical trial in which the wild-type OTC-encoding gene was delivered using an adenoviral vector. The patient developed pathological immune response after treatment, which led to multiple organ failure and death (Fig. 1) (4). Several efforts have since been undertaken to overcome the pathological immune response associated with gene therapy.
In the last two decades, various gene editing tools including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have been developed and these tools promote double-strand breaks (DSBs) in the target gene. Subsequently, DSBs activate DNA repair responses including non-homologous end joining (NHEJ), leads to the random insertions and deletions (indels) at the site of cleavage and the repair of pathological mutations. Homology-directed repair (HDR) can change the DNA at the cleavage location in the presence of a homologous DNA template. However, the efficiency of HDR is lower than that of NHEJ owing to the frequency of unwanted indels, especially in non-dividing cells. Therefore, alternative strategies are needed to repair point mutations without introducing DSBs.
A new method known as “prime editing” has been developed to fix single nucleotide polymorphism (SNP) without DSBs. Prime editors (PEs) require an engineered reverse transcriptase fused to nCas9 and a prime editing guide RNA (pegRNA) that can induce both insertions and deletions in all single nucleotide variants (SNVs) of human cells (7). Prime editors exhibit enormous editing capacities, but the limitations include off-target editing, poor immune response and few delivery options (Table 1) (8).
In this review, we summarize gene mutations related to liver diseases in the descending order of their prevalence and discuss previous approaches to repair these mutations using gene editing tools (Table 2). Successful recent applications of
AATD is one of the most common liver disorder and an autosomal recessive metabolic disease with a high prevalence in Scandinavia and North America. The disease results from c.1096G>A mutation in
Some strategies involve the insertion of wild-type
Hemophilia caused by mutations in
Initial attempts to repair mutations via genome editing targeted these inversions using HA-derived or HB-derived iPSCs. The two inversions in hiPSCs were repaired using CRISPR/Cas9 or TALENS (19, 20). The efficiency of reversion was up to 6.7% and the wild-type F8 was expressed in the engineered cells. Furthermore, HA mice transplanted with engineered endothelial cells was significantly increased in the F8 enzymatic acti-vity compared to non-transplanted HA mice (19).
Another approach for the repair of inversion was used TALENs to insert the exon 23-26 fragment of wild-type F8 cDNA correctly at the intersection of exon 22 and intron 22 in HA-hiPSCs via HDR. The mRNA and protein levels of F8 were rescued in the engineered hiPSC-derived endothelial cells (21). Several studies reported general strategies to repair all genetic variants in patients with HA by nuclease-mediated insertion of wild-type F8 cDNA fragment into the F8 locus, Alb locus or H11 safe harbor (22). Interestingly, a B-domain deleted form of
A similar approach was used in HB, which is an ideal disease for liver-directed genome editing strategy due to marked upregulation of hF9 activity. The cDNA of
In 2018, the first patient treated with SB-FIX (Clinical Trial: NCT02695160), which is ZEN-regulated genome editing transformed by rAAV inserted the wild-type of Factor IX cDNA into the Alb locus. Barzel
Alternative general knock-in strategies adopted for HB mice models entailed transgene insertion into
PKU is caused by mutations in
Patients with PKU have mutations involving both alleles of
WD is a rare genetic disorder caused by loss-of-function mutations in the copper-transporting P-type ATPase-encoding gene (
OTC deficiency is a rare X-linked genetic disorder caused by absence of the OTC enzyme, in which plays the role in elimination of nitrogen, which results in the form of ammonia (hyperammonemia) via the urea cycle. The accumulation of ammonia caused by the lack of the OTC enzyme moves to central nervous system through the blood and leads to the symptoms with OTC deficiency. The treatment of the disease involves the treatment with ammonia scavengers, such as sodium benzoate and dietary protein restriction. The transplantation of liver is considered in cases of hyperammonemia (39).
One study repaired
HT1 is a rare genetic disorder of tyrosine catabolism caused by a loss-of-function mutation in a gene encoding fumarylacetoacetate hydrolase (FAH). FAH deficiency promotes the accumulation of fumarylacetoacetate, maleylacetoacetate and their derivatives, and consequently causes by the damage of liver and renal tubular. Nitisinone rescues the pathological phenotype and acute liver injury by inhibiting hydroxyphenylpyruvate dioxygenase (HPD) in tyrosine catabolism upstream of FAH (42).
HT1 is amenable to gene repair therapy as the repaired hepatocytes undergo optimal proliferation in the liver (43). The two main genome editing strategies used for HT1 management include repair of
In 2014, Yin
Arginase-1 deficiency is a genetic disorder associated with urea metabolism in which the hydrolysis of arginine to urea and ornithine is impaired due to mutations in
Recently, we demonstrated the efficacy of an
Our finding indicated that genetic liver disease can be treated using
A recent study reported that a single administration of NLTA-2001, an
Gene editing components can be easily delivered to the liver intravenously since liver metabolizes all foreign particles. Recent studies have demonstrated that the direct delivery of LNPs conjugated with apolipoprotein E to the hepatocytes was markedly safer than the AAV-mediated delivery. Hence, LNPs enable
This work was supported by the research fund of Hanyang University (HY-201900000003369).
The authors have no conflicting interests.
Information about base and prime editors
|CBEs||C·G to T·A||Cytosine deaminase||Cas9-mediated off target||(5, 6)|
|d/nCas9 (dead (d) or nickase (n)||Unwanted ssDNA deamination|
|ABEs||A·T to G·C||Adenine deaminase||Cas9-mediated off target||(5, 6)|
|d/nCas9 (dead (d) or nickase (n)||Random deamination|
|PEs||Insertion & deletion to any SNV||Reverse transcriptase||Off-target editing||(7, 8)|
|nCas9 (nickase (n)||Immune response|
CBEs: cytosine base editors, ABEs: adenine base editors, PEs: prime editors, C: cytosine, G: guanine, T: thymine, A: adenine, UGI: uracil glycosylase inhibitor, sgRNA: single-guide RNA, SNV: single nucleotide variants, pegRNA: prime-editing guide RNA.
Gene editing methods to treat liver diseases
|Liver disease||Prevalence||Model||Target gene||Mutation||Method||Ref.|
|α-1 antitrypsin deficiency (AATD)||1:1,500-7,000||iPSCs from patients||ZFN-piggyBac||(9-7)|
|Hemophilia A (HA)||1:5,000||hiPSCs from patients||Deletions, insertions, inversions, and point mutations||CRISPR/Cas9||(18-29)|
|Engineered hiPSCs||TALENs; ZFNs|
|HA/CD4 null mice||Promoterless rAAV|
|Phenylketonuria (PKU)||1:10,000-15,000||COS-7 cells||FokI-dCas9 system||(30-35)|
|Pahenu adult mouse||CRISPR-Cas|
|Wilson’s disease (WD)||1:30,000||WD mouse||AAV containing WT||(36-38)|
|Ornithine transcarbamylase (OTC) deficiency||1:70,000||spfash mouse||CRISPR/Cas9||(39-41)|
|Tyrosinemia type 1 (HT1)||1:100,000||fah-/- mice||Promoterless rAAV||(42-53)|
|Fahneo/PM mice||saCas9; Nme-Cas9|
|fah-/- primary hepatocytes||Promoterless rAAV
|Arginase-1 deficiency||1:1,000,000||Induced mouse model||Deletions in exon 7 and 8||TALEN||(54-56)|