
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01866-2
10.1016/j.isci.2024.110641
110641
Review
Recent progress in gene therapy for familial hypercholesterolemia treatment
Luo Yaxin 12
Hou Yaofeng 12
Zhao Wenwen 12
Yang Bei yangbei@shanghaitech.edu.cn
1234∗
1 Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China
2 Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
3 Shanghai Clinical Research and Trial Center, Shanghai 201210, China
4 Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Shanghai 200031, China
∗ Corresponding author yangbei@shanghaitech.edu.cn
10 8 2024
20 9 2024
10 8 2024
27 9 110641© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Familial hypercholesterolemia (FH) is a genetic disorder that affects 1 in 300 people, leading to high cholesterol levels and significantly increased cardiovascular risk. The limitations of existing FH treatments underscore the need for innovative therapeutics, and gene therapy offers a promising alternative to address FH more effectively. In this review, we survey approved gene therapy drugs first and then delve into the landscape of gene addition, gene inactivation, and gene editing therapies for hypercholesterolemia, highlighting both approved interventions and those in various stages of development. We also discussed recent advancements in gene editing tools that are essential for their application in gene therapy. Safety considerations inherent to gene therapy are also discussed, emphasizing the importance of mitigating potential risks associated with such treatments. Overall, this review highlights the progress and prospects of gene therapies for FH treatments, underscoring their potential to revolutionize the management of this prevalent and challenging condition.

Graphical abstract

Clinical genetics; Endocrinology; Human metabolism

Subject areas

Clinical genetics
Endocrinology
Human metabolism
==== Body
pmcIntroduction

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder,1 with prevalence rates as high as ∼ 1:300 for heterozygous FH (HeFH) and approximately ∼1:300,000 for homozygous FH (HoFH).2 FH may be caused by mutations in several genes that control low-density lipoprotein cholesterol (LDL-C) removal in the liver, including the low-density lipoprotein cholesterol receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), and LDL receptor adaptor protein 1 (LDLRAP1). The vast majority of FH cases (80–90%) involve mutations in LDLR, which binds and endocytoses low-density lipoproteins (LDLs) in the blood.3 Individuals suffering from FH exhibit substantially elevated LDL-C blood levels from a young age, predisposing them to chronic hypercholesterolemia.1 As elevated cholesterol levels are directly associated with an increased risk of atherosclerosis and atherosclerotic cardiovascular diseases (ASCVDs),4 individuals with FH generally experience an elevated incidence and mortality rates of ASCVDs compared to the general population.3

Diagnosis of FH mainly relies on serum LDL-C levels, family medical history, clinical manifestations such as skin/tendon xanthomas, corneal arcus, and so forth, and genetic testing.5 Among these methods, genetic testing represents the most accurate diagnostic approach, and can also offer insights into treatment strategies and prognosis for patients.6 Nevertheless, the accessibility of genetic testing is low due to its high-cost.7 The primary objective of FH treatment is to reduce the LDL-C blood levels, thereby mitigating the risk or delaying the onset and progression of ASCVDs.8 Complemented by non-pharmacological interventions such as lifestyle adjustments9,10,11 and lipoprotein apheresis,12,13 pharmacological interventions stand as the current cornerstone of FH treatment.14,15

Among pharmacological interventions, statins, PCSK9 inhibitors, and ezetimibe are widely employed as first-line treatments. Statins, renowned for their extensive use worldwide, exert their effects by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis (Figure 1).16 However, although statins can typically reduce blood LDL-C by 20–50%, concerns persist regarding their adverse effects, which happen at a frequency ranging from 0.1% to 10%.17,18 Among the predominant musculoskeletal issues caused by Statins, myalgia is the most common, myositis is relatively rare, and rhabdomyolysis, though very rare, is a serious concern as it can lead to kidney damage and other complications.19 Furthermore, certain statins carry a potential risk of inducing diabetes, limiting the tolerability of high-dose therapy.20 In clinical practice, stains are often administered concurrently with Ezetimibe or PCSK9 inhibitors.21 Ezetimibe impedes intestinal cholesterol absorption through its inhibition of Niemann-Pick C1-Like 1 (NPC1L1), thereby reducing the cholesterol levels (Figure 1).22 Nevertheless, it also poses several adverse effects, including upper respiratory tract infections (URTI), arthralgia, and extremity pain.23 On the other hand, PCSK9 reduces the number of LDLRs on the surface of hepatocytes by enhancing their degradation.24 Therefore, PCSK9 inhibitors can promote the recycling of LDLR and bolster the LDLR-mediated clearance of LDL-C from the bloodstream (Figure 1).25 However, though only on the market for a relatively short period, PCSK9 inhibitors have also been associated with allergic reactions,26 neurocognitive effects,27,28 and liver enzyme abnormalities.29Figure 1 The working mechanism of first-line drug statins, PCSK9 inhibitors, and ezetimibe

Statins inhibit HMG-CoA reductase, thereby reducing cholesterol biosynthesis and lowering plasma cholesterol levels. Ezetimibe inhibits the absorption of dietary cholesterol by NPC1L1 in the intestine. PCSK9 inhibitors improve the stability of LDLR, thereby enhancing the LDLR-mediated clearance of cholesterol from blood.

Besides the above mainstay FH medications, a few other treatments have been approved in recent years to help patients with severe FH restore normal LDL-C levels, including antisense oligonucleotide (ASO) therapy. An ASO inhibitor targeting APOB has been shown to reduce LDL-C levels by an additional 24.7% when combined with high-dose statin therapy.30 Lomitapide, an inhibitor of microsomal triglyceride transport protein (MTP), indirectly inhibits the assembly and secretion of apolipoprotein B (apoB) with lipoproteins in the liver and intestine, resulting in an additional 50% reduction in LDL-C levels when used alongside statins. However, it can also cause significant gastrointestinal side effects.31 Lipoprotein Apheresis can reduce LDL levels by up to 65%. However, it is a costly procedure and financially unfeasible for most patients.13,32 Currently, liver transplantation is the only method capable of rapidly normalizing cholesterol levels in patients with HoFH. Nevertheless, it remains an uncommon treatment due to immune rejection risks and the severe shortage of organ donors.33

In summary, the current treatment of FH is challenged by various factors, including low diagnosis rates, high drug costs, severe side effects, intricate drug interactions, and considerable variability in individual drug response. To this end, the burgeoning field of gene therapy offers a promising therapeutic alternative for patients with FH which may revolutionize the current FH treatment landscape. In this review, we provide an overview of the present landscape of gene therapies for FH, with a specific emphasis on several gene editing-based therapies currently under development.

The landscape of gene therapy

With the ongoing development of biotechnology, several innovative treatment options have emerged in the past decades. Among these, gene therapy stands out as one of the most promising approaches for addressing human hereditary diseases. In general, gene therapy treats genetic disorders by introducing genetic engineering materials into human cells, with the primary goal of modulating therapeutic gene expression at levels sufficient to alleviate or even cure the symptoms of the disorders.34 From a narrow perspective, two strategies are mainly employed in gene therapy, gene addition, and gene editing. While gene addition introduces a functional copy of a gene into cells to compensate for a faulty or missing gene, gene editing instills precise the modification of the cellular genome to correct or modify genetic defects associated with diseases and holds the potential of a “one-time editing, benefit forever” prospect.35,36

Approved gene addition and gene editing therapies

The past decade has witnessed an explosion in gene therapy research and application.37 Gene therapy can be given in two ways, in vivo and ex vivo. In vivo gene therapies deliver therapeutic genes or gene-modifying components directly to the target organ or tissue via delivery vectors such as adeno-associated virus vectors (AAVs) or lipid nanoparticles (LNPs).38,39,40,41 In contrast, ex vivo gene therapy involves the isolation of recipient cells from the patient, followed by the introduction of therapeutic genes or gene-modifying components into these cells. Subsequently, the modified cells are infused back into the patient.42,43,44,45 Currently, over 500 gene therapy clinical studies have been initiated globally, spanning cancers, hereditary genetic disorders, cardiovascular diseases, and other conditions. Among all the conditions, cancer and single-gene rare diseases stand out as the most extensively researched areas.46

As of 2023, dozens of gene therapy drugs have been approved for marketing worldwide, including 4 ex vivo gene therapies and 11 in vivo gene therapies (Table 1). The world’s first gene therapy to receive regulatory approval was Gendicine. Approved by the Chinese State Food and Drug Administration (SFDA) in 2003, Gendicine treats head and neck squamous cell carcinoma (HNSCC) by intratumorally delivering a functional copy of the tumor suppressor gene p53 on a recombinant adenovirus vector (rAd-p53).47 However, Gendicine has not been approved for use in the United States or other countries. The first gene therapy approved by the European Medicines Agency (EMA) is Glybera, which received conditional marketing approval in 2012. Glybera leverages an AAV vector to deliver a functional copy of the lipoprotein lipase (LPL) gene for the treatment of familial lipoprotein lipase deficiency through intravenous infusion.48 Another significant approval was granted to Strimvelis by the EMA in 2016, which treats rare genetic disorder called severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID).49,50 Using a gammaretrovirus vector to transfer a functional copy of the adenosine deaminase (ADA) gene into hematopoietic stem cells (HSCs) isolated from the patients, Strimvelis represents a notable example of ex vivo gene therapy.50 The first gene therapy approval in the United States was given to Luxturna by the Food and Drug Administration (FDA) in 2017, which delivers a functional RPE65 gene vectored on AAV2 through subretinal injection to treat Leber Congenital Amaurosis (LCA).51 Following these approvals, 10 other gene therapies that use either AAV, adenovirus (ADV), or retroviral vectors to deliver functional copies of therapeutic genes for the treatment of a range of genetic disorders were approved in 2019–2023 (Table 1). Most of these approvals are one-time in vivo gene therapies administered intravenously/intrathecally or ex vivo gene therapies given to autologous HSCs. However, VYJUVEK for the treatment of Dystrophic epidermolysis bullosa (DEB) is an exception. Administered topically, VYJUVEK represents the first redosable topical gene therapy in the world.52,53Table 1 Gene therapies approved by the SFDA, FDA or EMA

Product	Indications	Approval	Delivery route	Delivery vector	Mechanism	References	
Gendicine	head and neck cancer	2003	intratumoral injection	recombinant Adenovirus vector	deliver functional copies of the p53 gene	Peng et al.47	
Glybera	familial lipoprotein lipase deficiency	2012	intravenous infusion	AAV vector	deliver functional copies of the LPL gene	Gaudet et al.48	
Strimvelis	ADA-SCIDa	2016	ex vivo/HSCe	gamma-retroviral vector	deliver functional copies of the ADA gene	Cicalese et al.49	
Luxturna	LCAb or retinitis pigmentosa	2017	subretinal injection	AAV2	deliver functional copies of the RPE65 gene	Russell et al.51	
Zolgensma	spinal muscular atrophy (SMA)	2019	intravenous infusion	AAV9	deliver functional copies of the SMN1 gene	Mendell et al.54	
Zynteglo	transfusion-dependent β-thalassemia	2019	ex vivo/HSCe	gamma-retroviral vector	deliver functional copies of the β-globin gene	Locatelli et al.55	
ROCTAVIAN	hemophilia A	2019	intravenous infusion	AAV5	deliver functional copies of FVIII gene	Ozelo et al.56	
Libmeldy	metachromatic leukodystrophy (MLD)	2020	intrathecal injection	lentiviral vector	deliver functional copies of the arylsulfatase A gene	Sessa et al.57	
ADSTILADRIN	high-grade, BCG-unresponsive NMIBCc	2022	intravesical	adenovirus vector	deliver functional copies of the IFNα2b gene	Boorjian et al.58	
HEMGENIX	hemophilia B	2022	intravenous infusion	AAV5	deliver functional copies of the factor IX gene	Pipe et al.59	
SKYSONA	boys with early, active CALDd	2022	ex vivo/HSCe	lentiviral vector	deliver functional copies of the ABCD1 gene	Eichler et al.60	
ELEVIDYS	duchenne muscular dystrophy (DMD)	2023	intravenous infusion	AAV vector	deliver functional copies of an engineered dystrophin gene	Mendell et al.61	
VYJUVEK	dystrophic epidermolysis bullosa	2023	tropical	non-replicating Herpes simplex virus	deliver functional copies of the COL7A1 gene	Guide et al.52	
CASGEVY	sickle cell diseases	2024	ex vivo/HSCe	electroporation	deliver CRISPR/Cas9-sgRNA RNP to disrupt the BCL11A enhancer & restore γ-globin expression	Frangoul et al.62	
LYFGENIA	sickle cell diseases	2024	ex vivo/HSCe	lentiviral vector	deliver functional copies of the β-globin gene	Kanter et al.63	
a ADA-SCID: severe combined immunodeficiency due to adenosine deaminase deficiency.

b LCA: Leber Congenital Amaurosis.

c BCG-unresponsive NMIBC: Bacillus Calmette-Guerin (BCG)-unresponsive non-muscle invasive bladder cancer (NMIBC).

d CALD: cerebral adrenoleukodystrophy.

e HSC: hematopoietic stem cell.

In 2023, another milestone of gene therapy was reached by the groundbreaking approval of CASGEVY, which is the world’s first gene editing-based gene therapy.64 Through delivering the gene editing tool CRISPR/Cas9 to disrupt the enhancer region of BCL11A gene in autologous HSCs, CASGEVY restores the expression of γ-globin to replace the mutated β-globin for the treatment of sickle cell disease (SCD) in patients aged 12 years and older.62,64 Notably, CASGEVY does not use viral vectors for gene delivery. Instead, it is administered to patients-isolated HSCs through electroporation.62

Approved gene silencing therapies

From a broader perspective, gene silencing (at the RNA level) drugs can also be considered as gene therapies since they also introduce genetic engineering materials, such as small interfering RNA (siRNA) or ASOs, into human cells for the treatment of genetic disorders.65 siRNA is a 20–25 nt long double-stranded RNA, capable of eliciting gene silencing through RNA interference (RNAi). Different from siRNAs, ASOs and Splice switching oligonucleotides (SSOs) are short, single-stranded nucleic acid sequences, typically 16–53 nt in length. ASOs and SSOs specifically bind to target mRNAs or pre-mRNAs and control gene expression through translation blockage, splicing modulation, RNase H-mediated RNA degradation, and so forth.66 In general, these synthetic small nucleic acid molecules all modulate the expression of a disease-causing gene in some way to treat corresponding diseases. To date, more than 10 siRNA, ASO, and SSO drugs have been approved by EMA or FDA. Two recent reviews have made excellent summaries of these approved oligonucleotide drugs.65,67 Thus, they will not be discussed in detail here.

Gene therapy for hypercholesterolemia

Since the beginning of the 21st century, gene therapy has emerged as an important research area in modern biomedical engineering and clinical practice. In the realm of FH treatment, researchers also explored many new gene therapy possibilities. These endeavors can be grouped into three categories.

Gene addition therapy for hypercholesterolemia

This one-time therapy involves viral vectors- or exosomes-mediated in vivo delivery of functional gene copies to compensate for the body’s inability to produce normal proteins for cholesterol metabolism. The earliest clinical trial of gene addition therapies for FH can be traced back to the early 1990s, wherein retroviral vector containing LDLR gene was transduced into autologous hepatocytes obtained through partial hepatectomy before they were infused back into patients (NCT00004809) (Table 2). However, researchers only observed low levels of LDLR expression plus a variable and moderate decrease in plasma LDL-C levels in studied patients, both of which prevented the further progression of this trial.68 In 2015, Wang et al. employed AAV8 to deliver human LDLR gain-of-function mutants into an LDLR-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout). Remarkably, they observed a 98% reduction in cholesterol levels and no significant side effects in the mice, suggesting promise for this strategy in FH treatment.69 Although a phase I clinical trial using AAV for functional LDLR delivery was registered in 2016, interim efficacy analysis was not released due to the early termination of this trial (Table 2). The use of AAV for therapeutic LDLR gene delivery remains controversial due to the potential of AAV to trigger an immune response. Furthermore, the transduction efficiency of AAV in human hepatocytes is lower than that in mouse hepatocytes, suggesting that higher doses of AAV may be required for human applications and posing the risk of suboptimal cholesterol reduction in humans.70,71,72Table 2 Functional gene addition therapy for FH in clinical trials

Target	Delivery route	Delivery vector	Clinical Status	Indications	Ref. ID	
LDL-R	ex vivo	retrovirus vector	phase 1	HoFH	NCT00004809	
intravenous infusion	AAV8	phase 1/2	HoFH	NCT02651675	
intravenous infusion	Exosome	phase 1	HoFH	NCT05043181	

Meanwhile, exosomes are disk-shaped vesicles that originate from the endosomes of the cell nucleus. As “natural nanoparticles,” they can carry various types of nucleic acid cargoes, such as DNA and RNA, and transmit them across cellular membranes. By altering the targeting proteins, exosomes can also be directed to target specific organs or tissues.73 In 2021, Li et al. delivered the mRNA encoding functional LDLR via exosomes into LDLR knockout mice to alleviate atherosclerosis induced by a high-fat diet.74 This study observed an increase in LDLR levels and a concurrent decrease in serum LDL-C levels and mice liver lipid deposition, without the detection of exosome-associated toxicity.74 Encouraged by the positive preclinical results, a phase-I clinical trial employing the same strategy was registered under the ID NCT05043181 in the same year, although no interim data was released yet (Table 2). Currently, exosome-based therapies are still in the early stages of development. Enhancement in their purity and targeting specificity, alongside validations of their ability to carry lengthy mRNAs are expected steps before the approval and clinical applications of exosome-based therapies.75,76

Gene inactivation therapy for hypercholesterolemia

To date, two gene inactivation therapies, namely Mipomersen and Inclisiran, have been approved for treating HoFH and HeFH respectively, although the marketing of Mipomersen has been discontinued due to concerns about its adverse effects.77,78,79 As an ASO, Mipomerson targets the mRNAs of apolipoprotein B-100 (apoB-100) to prevent its translation, thereby reducing the production of LDL particles for the treatment of HoFH (Table 3).30 Meanwhile, Inclisiran is a siRNA that inhibits the expression of PCSK9, thereby increasing LDLR levels in the liver and improving the LDLR-mediated clearance of LDL-C from the blood.80 The long-lasting durability and safety of Inclisiran were well demonstrated in the ORION-3 clinical study.78 Over a period of four years, patients who received Inclisiran twice annually experienced an up to 78% reduction in PCSK9 expression levels and an average decrease in LDL-C levels of 42.2% (Table 3), albeit nearly all participants reported drug-related adverse effects, albeit mild, which primarily present as nasopharyngitis and injection site reactions (ISRs).78 Of note, both Inclisiran and Mipomersen need to be used as an adjunct to other lipid-lowering medications such as maximally tolerated statin therapy, in order to achieve greater reduction in LDL-C and better control of cardiovascular risks.Table 3 Lipid-lowering gene inactivation therapies approved and in clinical trials

Type	Target	Name	Clinical Status	Indications	Lipid-Lowering Effect (-%)	References	
siRNA	PCSK9	Inclisiran	approved	HeFH or ASCVD	44% (LDL-C)	Ray et al.78	
Angptl3	ARO-ANG3	phase 2b	HoFH/Mixed Dyslipidemia	up to 32% (non-HDL-C)	Watts et al.81	
APOC3	ARO-APOC3	Phase 2b	Mixed Dyslipidemia/Hypertriglyceridemia	up to 27% (non-HDL-C)	Vasas et al.82	
Lp(a)	Olpasiran	Phase 2	Elevated Lp(a)	up to 25% (LDL-C)	O'Donoghue et al.83	
Lp(a)	SLN360	phase 1	Elevated Lp(a)	up to 26% (LDL-C)	Nissen et al.84	
ASO	apoB-100	Mipomerson	Discontinued	HoFH	25% (LDL-C)	Raal et al.30	
PCSK9	AZD8233	phase 2b	Hyperlipidemia/Dyslipidemia	up to 79% (LDL-C)	Hofherr et al.85	
Angptl3	Vupanorsen	phase 2b	Dyslipidemia/FCSa	up to 28% (non-HDL-C)	Bergmark et al.86	
APOC3	Volanesorsen	phase 3	Hypertriglyceridemia/FCSa	46% (non-HDL-C)	Witztum et al.87	
APOC3	Olezarsen	Phase 2	Hypertriglyceridemia	up to 24% (non-HDL-C)	Tardif et al.88	
Lp(a)	Pelacarsen	phase 2	Elevated Lp(a) and CVDb	/	Tsimikas et al.89	
a FCS: Familial Chylomicronemia Syndrome.

b CVD: cardiovascular disease.

Besides PCSK9, ANGPTL3 is another intervention target for FH as its inhibition would promote the activity of LPL and endothelial lipase (EL), which hydrolyze triglycerides (TGs) and phospholipids respectively within very low-density lipoproteins (VLDLs) to prevent the conversion of VLDLs into LDLs.90,91 Xu et al. delivered ARO-ANG3, an siRNA targeting ANGPTL3, to five different mouse models and human hepatoma cells. During the experiment, an approximately 97% reduction in ANGPTL3 expression was observed on day 3, accompanied by a 73% reduction in LDL-C.92 Moreover, preliminary findings from a phase 1 clinical trial (NCT03747224) evaluating ARO-ANG3 demonstrated a ∼50% reduction in LDL-C levels among participants.93 Additionally, a phase 2b clinical trial (NCT04832971) further revealed that ARO-ANG3 led to up to 56% reduction in circulating TG levels and up to 32% reduction in non-high-density lipoprotein cholesterol (non-HDL-C) levels 24 weeks post-dosing (Table 3).81 It is important to note, however, that the drop in LDL-C level was not as prominent as that in TG levels.81

Besides siRNAs, ASO drugs targeting PCSK9 and ANGPTL3 have also advanced into phase 2 clinical trials. Of them, PCSK-targeting AZD8233 showed up to 79% reduction in LDL-C levels from baseline at 90 mg dosage (Table 3).85 Meanwhile, although the ANGPTL3-targeting Vupanorsen exhibited up to 28% and 57% reduction in non-HDL-C and TG levels respectively, the decrease in LDL-C levels only achieved statistical significance at high dose (Table 3).86 Accordingly, the indication of Vupanorsen has changed into dyslipidemia instead of HoFH. Notably, both ASOs exhibited signs of mild liver damage at high doses, suggesting more efforts in promoting their efficacy at low doses. However, as negatively charged and hydrophilic molecules, ASOs face challenges in efficiently crossing the cell membrane, which presents a significant hurdle in their application for gene therapy and may compromise their efficacy, especially at low doses. To boost their delivery efficiency, the LNP system has been employed to administer PCSK9-targeting ASO to mice, resulting in a 75% decrease in PCSK9 mRNA expression.94 Alternatively, ASOs may also be conjugated with target ligands to enhance their stability, cellular uptake, and organ specificity. Indeed, Vupanorsen is an N-acetyl galactosamine (GalNAc)-conjugated ASO targeting liver.95

Another target, APOC3, has also been the focus of lipid-lowering gene inactivation therapy development as well. APOC3 is a small apolipoprotein primarily found in triglyceride-rich lipoproteins (TRLs) and LDLs.96 It inhibits the activity of LPL and reduces the clearance of triglyceride-rich lipoproteins (TRLs) from the bloodstream.97,98 Though not considered as a direct target for FH, the inhibition of APOC3 can produce an LDL-lowering effect and reduce the risk of cardiovascular diseases. Volanesorsen, an APOC3-targeting ASO, has entered a phase 3 clinical trial and led to a ∼46% decrease in non-HDL-C levels in participants (Table 3).87 However, thrombocytopenia was observed in about half of the participants, therefore volanesorsen is approved with restrictions in Europe but not in the US.99 Olezarsen and ARO-APOC3, GalNAc-conjugated ASO and siRNA respectively, are designed to preserve the lipid-lowering effect of volanesorsen while minimizing its side effects.99 They have both finished phase 2 clinical trials and shown positive interim results (Table 3).82,88

Lipoprotein (a), or Lp(a) for short, is a unique lipoprotein particle composed of an LDL-like core plus an additional glycoprotein called apolipoprotein (a).100,101 Elevated levels of Lp(a) are recognized as an independent risk factor for ASCVD.102 As the inhibition of Lp(a) holds promise for reducing blood cholesterol levels and cardiovascular diseases risks, several gene-inactivation therapies targeting Lp(a) are also reviewed here, including two siRNAs and one ASO. Olpasiran and SLN360 are both GalNAc-conjugated siRNAs targeting Lp(a) mRNAs. In a phase 2 clinical trial conducted in patients with established ASCVD and elevated Lp(a) levels, Olpasiran effectively reduces the Lp(a) concentration by more than 95% and achieves up to 25% reduction in LDL-C level in the high-dose groups (Table 3).83 Meanwhile, SLN360, also known as Zerlasiran, reduces LDL-C levels by up to 26%, when administered at a single dose of 600 mg in a phase 1 clinical trial (Table 3).84 Pelacarsen, on the other hand, is an ASO against Lp(a). When administered at a weekly dose of 20 mg, Pelacarsen showed a reduction in Lp(a) levels of up to 79.5% and a modest decrease in LDL-C levels (Table 3).89 Notably, approximately 30% of the enrolled patients in the Pelacarsen clinical trial were diagnosed with FH, suggesting the potential of Pelacarsen in controlling LDL-C for FH treatment.89

Gene editing therapy for hypercholesterolemia

Following the emergence of CRISPR-Cas9 technology, the rapid development of the CRISPR/Cas9 system and the subsequent Base editing (BE) system have spurred widespread interest in gene editing therapy based on these systems. Recently, the first CRISPR/Cas9-based gene editing therapy, Casgevy, was even approved for the treatment of Sickle Cell Disease (SCD).103 The promise of gene editing therapy in treating genetic disorders also stimulated the exploration of gene editing-based FH treatment. In this section, we will review the rapid development of gene editing toolkit and gene editing-based FH therapies in development, targeting either PCSK9 or ANGPTL3. We will also briefly discuss the emerging new target ASGR1.

Development of gene editing toolkit

In recent years, a set of gene editing techniques capable of precisely modifying an organism’s genome and its transcripts at specific sites to knock out or modify disease-causing genes has been developed. Among the existing gene editing tools, the CRISPR-Cas9 system and its derivatives represent the latest advance in gene editing technology.104 Currently, different types of genome editors have derived from the CRISPR-Cas9 system, including nucleases, base editors (BEs), and prime editors (PEs), further expanding the toolkit for gene therapy.105

Of these tools, the CRISPR-Cas9 nucleases are more adept at gene inactivation and are unavoidably related to detrimental large DNA fragment deletions, cell-cycle arrest and p53-mediated apoptosis or even chromosomal translocations in edited cells given that they work by introducing DNA double-strand breakage (DSBs) into the genome (Figure 2).106,107,108 In contrast, BEs are capable of introducing precise and highly efficient single-point mutations in a target DNA sequence without causing DSBs or requiring a donor DNA template, thus offering significant advantages for gene therapy applications compared to traditional CRISPR-Cas9 nucleases (Figure 2).109 BEs generally comprise a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9) to act as the “locator,” and a fused single-stranded DNA deaminase such as APOBEC3A to act as the “effector.”104 Two main classes of BEs were developed first: cytosine base editors (CBEs), proficient at converting C-G base pairs into T-A,110 and adenine base editors (ABEs), adept at converting A-T into G-C.111 Together, CBEs and ABEs can efficiently mediate four types of transition mutations (C→T, A→G, T→C, G→A). These BEs offer remarkable efficiency and precision and have been widely applied across various contexts. More recently, the adenine base inversion editor (AYBE)112 and the glycosylase base editor (CGBE)113 are developed to catalyze A-to-T/C and C-to-G/A inversions respectively, further expanding the BEs repertoire.Figure 2 The pros and cons of the CRISPR/Cas9-derived nuclease, base editor, and prime editor systems

Though free of DSBs-related safety concerns, BEs do carry the risk of off-target editing due that its “locator” moiety may bind to off-target sites and its “effector” moiety may induce non-sgRNA-dependent off-target mutations in single-strand DNA (ssDNA) or single-strand RNA (ssRNA).104,109 To further enhance the precision and safety profile of BEs, Wang et al. developed a transformer base editor (tBE) system, which induces efficient on-target editing with only background levels of genome-wide and transcriptome-wide OT mutations (Figure 2).114 By fusing a cleavable deoxycytidine deaminase inhibitor (dCDI) element (i.e., mA3CDI) to the “effector” mA3CDA1, tBE remains inactive at OT sites, thereby eliminating unintended mutations. However, when located at the on-target sites, tBE transforms to cleave off the dCDI element and thus catalyzes intended deamination efficiently. The high precision and safety profile makes tBE a reliable and efficient tool for gene therapy.114

While BEs have shown remarkable capabilities, they do have limitations when it comes to executing certain types of gene editing, such as simultaneous base substitutions of mixed types or small insertions/deletions at precise locations.115 To address these limitations, the Prime Editor (PE) was developed. Different from BEs, PEs use a wild-type reverse transcriptase (RT) derived from the Loni murine leukemia virus (MMLV) to work as the “effector,” which can reverse-transcribe a template to replace the target sequence.116 In PE-meditated editing, prime editing guide RNA (pegRNA) first guides the nCas9-RT fusion protein to the target site, nCas9 then makes a nick in the non-target strand (NTS) near the target site, and RT uses the template provided in pegRNA for reverse transcription, thereby effectively inserting the desired modifications into the NTS and getting the modifications fixed during subsequent DNA repair.117 The design of PE enables it to introduce all 12 types of point mutations as well as small insertions and deletions without the generation of DSBs (Figure 2). However, it is important to note that PEs also have their limitations, particularly in terms of editing efficiency, and still require further optimization to maximize their potential in gene therapy applications (Figure 2).105

Gene editing therapy targeting proprotein convertase subtilisin/kexin type 9

PCSK9, originally identified as neuroapoptosis-regulated convertase 1 (NARC-1), is the ninth proprotein convertase discovered to date.118,119 It is primarily expressed in the liver and intestine, with lower expression observed in the kidneys, skin, and brain.120,121 PCSK9 binds to LDLR and promotes its degradation in the lysosome, leading to decreased LDLR density on the cell membrane and elevated LDL-C levels in the blood.24 Inhibiting PCSK9 can thus increase the number of LDLR on hepatocyte surfaces and enhance LDL-C clearance from the blood, thereby preventing or treating ASCVDs.122,123,124 Indeed, although gain-of-function mutations in PCSK9 can lead to FH,125 individuals with naturally occurring loss-of-function mutations in PCSK9 exhibit LDL-C levels approximately 40% lower than those who do not carry these mutations.124 They also demonstrate a lower risk of atherosclerosis126 and do not experience adverse health consequences.127

To further emphasize the significance of PCSK9 as a therapeutic target for FH, several medications targeting it have been approved and marketed in recent years. One such agent is Evolocumab, a monoclonal antibody against PCSK9, which has been shown to have a favorable long-term efficacy and safety profile.128 Likewise, Alirocumab is another monoclonal antibody targeting PCSK9129 and is used as a second-line drug in the treatment of hypercholesterolemia, often in conjunction with first-line drugs such as statins.130 Clinical studies have demonstrated that alirocumab is highly effective for the treatment of hypercholesterolemia, and can be used to treat patients with acute myocardial infarction and reduce the size of their atherosclerosis.131 Besides, semi-annual injections of Inclisiran, a siRNA that inhibits the translation of PCSK9, was able to produce a ∼42% decrease in LDL-C levels without significant adverse effects.78

The optimistic prospects of targeting PCSK9 for FH treatment motivated gene editing exploration toward it. To date, various gene editing tools have been employed to induce mutations at specific loci of PCSK9 for sustained gene regulation. The earliest attempt to edit PCSK9 was conducted in 2014 by Ding et al. in mice. Using an adenovirus-delivered CRISPR/Cas9 system, they achieved >50% PCSK9 editing in the mouse liver and decreased plasma cholesterol levels by 35–40% (Table 4).132 In 2021, Rothgangl et al. utilized LNPs to deliver mRNAs encoding ABEs to edit PCSK9 in mice. They achieved 61% editing efficiency and observed a 95% reduction in plasma PCSK9 levels and a 58% reduction in LDL-C levels on average (Table 4).133 The same study achieved a 26% editing efficiency and a 14% reduction in plasma LDL-C levels in macaques (Table 4).133 In the same year, another research team also utilized an LNP-delivered ABE system to modify PCSK9 in the livers of cynomolgus monkeys, achieving ∼60% blood LDL-C reduction (Table 4).134Table 4 Gene editing therapy for FH in development

Target	Delivery Strategy	Model	Editing tools	Editing efficiency	Lipid-Lowering Effect	References	
PCSK9	LNP	NHPsa	ABE8.8	∼79.4%	60%	Musunuru et al.134	
LNP	Mice	ABE	61%	58%	Rothgangl et al.133	
LNP	NHPsa	ABE	26%	14%	Rothgangl et al.133	
ADV	Mice	CRISPR-Cas9	>50%	35-40%	Ding et al.132	
ANGPTL3	ADV	LDLR−/− Mice	CBE3	∼35% (ANGPTL3)	51%	Chadwick et al.135	
LNP	Mice	CRISPR-Cas9	38.5%	56.8%	Qiu et al.136	
AAV9	Mice	ABE4max	63.30%	61%	Zuo et al.137	
GalNAc-LNP	Mice	ABE8.8	64%	/	Lee et al.138	
GalNAc-LNP	NHPs1	ABE8.8	∼61%	35%	Kasiewicz et al.139	
a NHPs: non-human primates.

The positive feedback from preclinical research also spurred the advancement of PCSK-9 editing clinical trials. Recently, Verve Therapeutics released interim data from its ongoing Phase 1b clinical trial of VERVE-101, which uses LNP-delivered ABE to switch off PCSK9 expression for the treatment of HeFH.140 The data showed a dose-dependent reduction in blood LDL-C levels among participants, with the highest dosage demonstrating a 55% decrease in blood LDL-C levels after a single treatment lasting up to 180 days or more.140 However, severe adverse events potentially related to the treatment were also observed in two patients from the high-dose group, raising safety concerns about VERVE-101. Notably, one patient with underlying ASCVD experienced a Grade 3 myocardial infarction, while the other exhibited transiently elevated liver enzymes and a drop in platelet levels.141 Besides, it is worth noting that the effectiveness of this PCSK9 targeting strategy for FH treatment is contingent upon the functionality of LDLR. For instance, in HoFH cases where LDLR is absent (LDLR−/−), targeting PCSK9 may not achieve the desired lipid-lowering effect.142,143 Thus, exploring other lipid-lowering targets for FH treatment is necessary.

Gene editing therapy targeting angiopoietin-like 3

ANGPTL3 acts as a natural inhibitor of LPL and EL, which hydrolyze TGs and phospholipids within chylomicrons and VLDLs into free fatty acids (FFA) and glycerol monoester.91,144 Reduction in ANGPTL3 expression would enhance LPL and EL activity, thereby accelerating the clearance of VLDLs and reducing the conversion of VLDLs into LDLs (Figure 3).91 In 2009, Romeo et al. discovered a significant association between mutations in ANGPTL3 and TG levels in humans.145 Subsequently, researchers from Harvard Medical School and other institutions jointly identified four patients with loss-of-function ANGPTL3 mutations. All four patients manifested broad-spectrum hypolipidemia (low LDL-C, HDL-C, and triacylglycerols), were in good health, and demonstrated normal fertility.146 In May 2017, a genetics study involving over 90,000 individuals was published in the New England Journal of Medicine, revealing that individuals with loss-of-function mutations in ANGPTL3 exhibit significantly lower LDL-C levels and a reduced risk of cardiovascular disease compared to the general population.147 Together, these discoveries reinforce that ANGPTL3 could represent a safe target for gene editing-based hypercholesterolemia treatment. Notably, ANGPTL3’s regulation of LDL-C is independent of the LDL receptor and other established mechanisms,148 thus suggesting a potential therapeutic role of ANGPTL3 in HoFH treatment.Figure 3 ANGPTL3 as gene therapy targets

ANGPTL3 inhibits the activities of LPL and EL, which hydrolyze TGs and phospholipids respectively within VLDLs. Reduction in ANGPTL3 expression enhances LPL and EL activity, thereby increasing the conversion of VLDL into VLDL remnants with accelerated clearance and reducing VLDL conversion into LDL. As elevated LDL levels in blood vessels contribute to atherosclerosis, targeting ANGPTL3 expression through gene editing (e.g., BE or CRISPR/Cas9) or gene inactivation tools (siRNA or ASO) can potentially reduce the risk of ASCVD.

Several studies have explored the use of base editors for ANGPTL3 editing. In 2018, Chadwick et al. utilized an adenovirus vector to deliver the CBE3 to hyperlipidemic LDLR−/− mice, achieving an average editing efficiency of ∼35% and 51% reduction in cholesterol levels after 14 days. Meanwhile, the researchers compared the therapeutic effects of editing ANGPTL3 with those of editing PCSK9, finding that targeting ANGPTL3 yielded superior lipid-lowering effects. They also edited both targets simultaneously but observed neither additivity nor synergism between the two.135 In 2021, instead of using the FDA-approved MC-3 LNP, Qiu et al. developed a 306-O12B LNP delivery platform and used it to deliver the CRISPR/Cas9 system for liver-targeted ANGPTL3 editing. They observed a median editing efficiency of 38. 5% in mouse liver and a significant reduction in blood ANGPTL3, LDL-C, and TG levels (65.2, 56.8, and 29.4%).136 In 2023, Zuo et al. delivered an optimized CBE, AncBE4max, via AAV to edit ANGPTL3 and observed an editing efficiency of 63.3 ± 2.3% at the target site. Four weeks after administration, the blood TG and total cholesterol levels decreased by 58% and 61%, respectively.137 To bypass the dependency of LNP-mediated hepatic delivery on the functionality of LDLR, Verve Therapeutics, in collaboration with the University of Pennsylvania, USA, designed a GalNAc-LNP system. Utilizing this delivery platform, they achieved an ANGPTL3 editing efficiency of 64% in mice liver and a 98% reduction in plasma ANGPTL3 protein on average.138 Moreover, they observed an average hepatic ANGPTL3 editing efficiency of 61% and a significant (89% and 35%) and durable (up to three months) reduction in plasma ANGPTL3 and LDL-C levels in cynomolgus monkeys.139

Overall, targeting ANGPTL3 represents an attractive target for treating patients with severe FH, particularly those with LDLR-deficient phenotypes. The significance of ANGPTL3 is supported by the up to 50% reduction in LDL-C levels observed even in patients with HoFH during a phase 3 clinical trial of Evinacumab, a monoclonal antibody against ANGPTL3.149 However, it is important to note that the safety and efficacy of ANGPTL3-targeted therapies still require long-term follow-up to determine their impact on ASCVD risks and any potential adverse effects or complications.

The emerging new target ASGR1

Lectins are cell-surface proteins that specifically recognize and bind to sugars on glycoproteins. They play a crucial role in coordinating intercellular functions across a variety of cell types.150 ASGR1 is a lectin involved in maintaining circulating glycoprotein homeostasis.151,152,153,154 It is the major subunit of the Asialoglycoprotein receptor (ASGPR), which is a heterotrimeric protein complex assembled from major and minor subunits. ASGPR maintains serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of serum glycoproteins bearing exposed terminal galactose or GalNAc residues (Figure 4).155,156,157 When asialoglycoproteins were injected into ASGPR-deficient mice, impaired clearance of these glycoproteins was observed.158 However, it is important to note that these glycoproteins do not accumulate in the serum, indicating that ASGPR is not the sole regulator of blood glycoprotein levels.158Figure 4 ASGR1 as gene therapy targets

ASGR1-mediated endocytosis and lysosomal degradation of serum glycoproteins lead to the mTORC1 pathway activation, AMPK pathway inhibition, as well as the translocation of transcription factor SREBPs from the ER to the nucleus, thereby leading to the upregulation of genes involved in cholesterol biosynthesis and PCSK9-mediated LDLR degradation. Importantly, ASGR1 also acts through the AMPK pathway to downregulate LXRα, which in turn downregulates ABC-family transporters ABCG5/G8 to inhibit cholesterol efflux into bile and intestine. In theory, ASGR1 knockout (ASGR1 KO) would inhibit intracellular cholesterol synthesis, promote ABCG5/G8-mediated cholesterol efflux, and promote LDLR recycling to enhance the LDLR-mediated clearance of blood cholesterol (dotted lines).

Specifically expressed in hepatocytes, ASGR1 serves as an ideal target for drug delivery to the liver.159 Indeed, the GalNAc-LNP took advantage of ASGR1 for its hepatic delivery.139 More importantly, ASGR1 seems to have a significant impact on lipid metabolism. A population genetics study conducted in 2016 among Icelanders revealed that individuals carrying a 12 bp deletion (Del12) in intron 4 of ASGR1 exhibit notably lower cholesterol levels (0.4 mmol per liter) and a 34% lower risk of coronary artery disease, as compared to the noncarriers.160 This Del12 mutation would activate a cryptic splice site, resulting in a frameshift and the synthesis of a truncated 89 aa protein (full-length ASGR1 is 291 aa long) that is susceptible to degradation, thereby representing an ASGR1 Loss-of-function phenotype. Besides, another loss-of-function ASGR1 variant, p.W158X, was also found to exhibit much lower LDL-C levels than non-carriers. p.W158X is caused by a 4 bp base insertion and would result in a premature stop codon at position 158. Hence, the loss-of-function variants of ASGR1 seem to be associated with lower cholesterol levels and reduced cardiovascular disease risk.160

In 2021, Xu et al. and Xie et al. employed CRISPR/Cas9 to disrupt the ASGR1 gene in mouse and pig embryos respectively.161,162 Using these genetically modified animals, the authors revealed that ASGR1 deficiency promotes the retention of sterol regulatory element binding proteins (SREBPs) in the endoplasmic reticulum (ER).161 SREBPs are transcription factors that are activated and translocated from the ER to the nucleus in response to low cellular cholesterol levels, thereby leading to the transcription of genes involved in lipogenesis biosynthesis and uptake.163,164 Therefore, ASGR1 deficiency inhibits cholesterol biosynthesis (Figure 4).161,162 Besides, as PCSK9 is also the downstream gene of SREBPs, the expression level of PCSK9 also decreased by 50% in ASGR1-deficient mice, hinting that ASGR1 deficiency might also enhance LDL-C clearance through PCSK9/LDLR axis (Figure 4).161 In 2022, the linkages between ASGR1 and SREBPs were further revealed to be mTORC1 and AMPK, which sense cellular nutrients and regulate various cellular metabolic pathways (Figure 4).165 ASGR1 deficiency impedes the glycoprotein endocytosis and decreases lysosomal amino acid levels, thereby inhibiting mTORC1 and activating AMPK pathways to interfere with the downstream lipogenesis pathways.165 More excitingly, the authors discovered that ASGR1 deficiency could also act through the AMPK pathway to upregulate LXRα, which in turn upregulates ABCA1 and ABCG5/G8 to facilitate cholesterol efflux (Figure 4).165 ABCG5/8 are ABC-family transporters responsible for cholesterol transport into bile, and their upregulation would increase biliary and fecal cholesterol excretion. Together, these findings suggest that ASGR1 may represent a highly promising new target for FH treatment, especially considering its unique role in regulating cholesterol efflux.

To date, studies exploring the potential of targeting ASGR1 for FH treatment are limited, only the work by Wang et al. has shown that an anti-ASGR1 neutralizing antibody could increase cholesterol excretion in mice and produce synergistic lipid-lowering effects when combined with statins or ezetimibe.165 More explorations into gene therapies targeting ASGR1 are expected in the future.

Overall, the rapid development of gene therapy holds promise for potentially curing patients with FH. As technologies develop, an imperative objective of research will be to provide cost-effective and enduring gene therapy options for all patients. However, existing gene therapy approaches are hampered by several limitations, including the complexity of delivery methods and safety concerns.

Safety considerations of gene therapy

Safety is the primary concern in the clinical application of any drug. Thus, ensuring the safety of gene therapy remains a critical consideration in both its basic research and clinical application. Unintended integration of therapeutic genes, potential antigenicity of viral vectors, and off-target editing are the primary safety concerns of gene therapy.166,167,168,169 Later in discussion, we discuss the key aspects of gene therapy safety concerns in detail.

Safety of vectors

Commonly used vectors for gene therapy delivery fall into two categories: viral and non-viral vectors. Viral vectors, such as AAVs, harness the infectivity of viruses to transfer exogenous genes, i.e., the payloads. After incorporating the payloads into the viral genome, the edited virus is then used to infect the patient’s cells and introduce target genes into these cells. Although viral vectors offer efficient gene delivery and long-term or even permanent gene expression, they may also lead to serious adverse consequences. For instance, certain gene therapies that use retroviral vectors may cause genomic instability and even trigger malignancies such as leukemia through unintended integration of exogenous genes.167 Meanwhile, AAV vectors, while lacking the ability to integrate, may still pose safety concerns such as hepatotoxicity, kidney damage, and neurological loss due to their ability to trigger immune responses and prolonged inflammation.170 Besides, viral vectors generally have limited cargo capacity, thus constraining the size of the payload. Therefore, careful consideration of the immunogenicity, payload size, and potential risk of insertional mutagenesis needs to be taken when using viral vectors for delivery.

Besides viral vectors, exogenous payloads can also be delivered through non-viral vectors such as liposomes, LNPs, nanoparticles, gene guns, and so forth.171 These vectors encapsulate or absorb the payloads before delivering them into cells. Non-viral vectors offer several advantages, including reduced immunogenicity, ease of production, and potential for carrying large cargo. They are also free of integration concerns associated with viral vectors.171 However, non-viral vectors generally exhibit low transfection efficiency and shorter duration of gene expression as compared to viral vectors. They also face challenges in achieving targeted delivery and pose risks of cytotoxicity and tissue damage.172 For instance, the adverse events observed in the high-dose group of the VERVE-101 trial are potentially attributable to the LNP delivery system, as similar liver toxicity was observed in animals dosed with empty LNPs or LNPs carrying inactive payloads.141 Consequently, Verve Therapeutics has temporarily paused the VERVE-101 trial and shifted its focus to the VERVE-102 trial, which delivers the same payload as VERVE-101 but utilizes a GalNAc-LNP delivery system instead.141

Safety of payloads

In gene therapy, the payloads could consist of proteins, mRNAs, ASOs, or siRNAs. Safety concerns associated with these payloads include immunogenicity, genotoxicity, off-target potentials, and so forth. For example, ASOs and siRNAs may bind to unintended mRNA sequences, leading to undesired gene silencing or modulation.173,174 Meanwhile, exogenous proteins, mRNAs, as well as ASOs and siRNAs can all provoke immune responses, potentially leading to the abnormal activation of the immune system and even the development of autoimmune disorders.174,175,176 Besides, excessive expression of the delivered gene may disrupt physiological homeostasis, causing toxicity or adverse effects. Therefore, careful consideration of the immunogenicity, stability, specificity, and controllability of payloads is crucial to maximize the efficacy of gene therapies while minimizing their adverse effects. It is also advisable to avoid selecting oncogenic genes or genes that may produce adverse effects as payloads. Moreover, attention should also be paid to whether the cells may undergo stress response or apoptosis due to the presence of foreign genes. Besides, the expression level, duration, and distribution of the gene in the body need to be continuously monitored to prevent the occurrence of other potential side effects.

Safety of gene editing tools

Recent research indicates potential safety concerns associated with the CRISPR/Cas9 system due to off-target effects.177 Moreover, the Cas9 cleavage-mediated DSBs can lead to deletions of large DNA segments, causing genomic instability and even chromosomal translocations.106,108 Furthermore, DSBs may also activate DNA damage response pathways to trigger cell-cycle arrest and p53-mediated apoptosis in edited cells.107,178,179 In contrast, BEs do not generate DSBs in general and thus are free of large DNA fragment deletions or chromosomal translocations. Nonetheless, they carry the risk of off-target editing at both the DNA and RNA levels.104,109 It is worth noting that mismatches in the sgRNA can lead to the incorrect binding of the base editor at off-target sites with sequence similarity to the target site, resulting in sgRNA-dependent off-target mutations. Meanwhile, given that the “effector” of BEs possesses inherent deamination activity toward ssDNA or ssRNA, the base editor may also induce non-sgRNA-dependent off-target mutations at a genome-wide and transcriptome-wide level.180,181 Notably, to further enhance the safety profile of BEs for clinical applications, a new BE system named tBE was recently developed, which could reduce the genome-wide and transcriptome-wide OT mutations to the background level, thus presenting an ideal tool for gene therapy.114

Conclusions

Patients with severe FH are at a high risk of developing cardiovascular disease and are often unresponsive to conventional therapies. Gene therapy targeting disease-causing factors such as LDLR, PCSK9, ANGPTL3, and APOB represents a promising new strategy for FH treatment. Several gene therapy drugs for FH treatment are currently undergoing clinical trials, with some showing encouraging interim results. With the continuous development of gene editing tools, mRNA, siRNA, and ASOs technology, and delivery systems, it is anticipated that patients with FH may benefit from these gene therapy drugs and achieve long-lasting relief or even cure from them in the future. Nevertheless, it is also worth noting that the development and manufacturing of gene therapies require substantial investments. Consequently, gene therapies often carry a high price tag and substantial implementation requirements, which may render them inaccessible to certain people or regions. In this context, any scientific or technical innovation that can reduce the cost of gene therapy is of great significance, as it will greatly improve the accessibility and practical application of gene therapy.

Limitations of the study

Due to space limitations, some important works may not be fully discussed and acknowledged.

Acknowledgments

This work is supported by grant 2019YFA0802804 to B.Y from the 10.13039/501100012166 National Key Research and Development Program of China ., grants 32371272 and 32070170 to B.Y. from the 10.13039/501100001809 National Natural Science Foundation of China (NSFC), and grant 23ZR1442500 to B.Y. from the 10.13039/501100003399 Shanghai Municipal Science and Technology Commission . This work was also supported in part by a Shanghai Municipal Education Commission (SMEC) grant to the Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University. The figures were first drafted with BioRender.com and then modified in Adobe Illustrator (Adobe).

Author contributions

B.Y. and Y.X.L. drafted the article, Y.X.L., B.Y., and Y.F.H. prepared the illustrations, all authors have reviewed the literature and edited the article. All authors have read the final article and approve its submission.

Declaration of interests

The authors declare no competing interests.
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References

1 Defesche J.C. Gidding S.S. Harada-Shiba M. Hegele R.A. Santos R.D. Wierzbicki A.S. Familial hypercholesterolaemia Nat. Rev. Dis. Prim. 3 2017 17093 10.1038/nrdp.2017.93
2 Santos R.D. Gidding S.S. Hegele R.A. Cuchel M.A. Barter P.J. Watts G.F. Baum S.J. Catapano A.L. Chapman M.J. Defesche J.C. Defining severe familial hypercholesterolaemia and the implications for clinical management: a consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel Lancet Diabetes Endocrinol. 4 2016 850 861 10.1016/s2213-8587(16)30041-9 27246162
3 Brandts J. Ray K.K. Familial Hypercholesterolemia: JACC Focus Seminar 4/4 J. Am. Coll. Cardiol. 78 2021 1831 1843 10.1016/j.jacc.2021.09.004 34711342
4 Gofman J.W. Lindgren F. Elliott H. Mantz W. Hewitt J. Strisower B. Herring V. Lyon T.P. The role of lipids and lipoproteins in atherosclerosis Science 111 1950 166 186 15403115
5 Watts G.F. Gidding S.S. Mata P. Pang J. Sullivan D.R. Yamashita S. Raal F.J. Santos R.D. Ray K.K. Familial hypercholesterolaemia: evolving knowledge for designing adaptive models of care Nat. Rev. Cardiol. 17 2020 360 377 10.1038/s41569-019-0325-8 31974482
6 Sturm A.C. Knowles J.W. Gidding S.S. Ahmad Z.S. Ahmed C.D. Ballantyne C.M. Baum S.J. Bourbon M. Carrié A. Cuchel M. Clinical Genetic Testing for Familial Hypercholesterolemia: JACC Scientific Expert Panel J. Am. Coll. Cardiol. 72 2018 662 680 10.1016/j.jacc.2018.05.044 30071997
7 Watts G.F. Gidding S.S. Hegele R.A. Raal F.J. Sturm A.C. Jones L.K. Sarkies M.N. Al-Rasadi K. Blom D.J. Daccord M. International Atherosclerosis Society guidance for implementing best practice in the care of familial hypercholesterolaemia Nat. Rev. Cardiol. 20 2023 845 869 10.1038/s41569-023-00892-0 37322181
8 Lui D.T.W. Lee A.C.H. Tan K.C.B. Management of Familial Hypercholesterolemia: Current Status and Future Perspectives J. Endocr. Soc. 5 2021 bvaa122 10.1210/jendso/bvaa122
9 Broekhuizen K. van Poppel M.N.M. Koppes L.L.J. Brug J. van Mechelen W. A tailored lifestyle intervention to reduce the cardiovascular disease risk of individuals with Familial Hypercholesterolemia (FH): design of the PRO-FIT randomised controlled trial BMC Publ. Health 10 2010 69 10.1186/1471-2458-10-69
10 Hegele R.A. Environmental modulation of atherosclerosis end points in familial hypercholesterolemia Atherosclerosis Suppl. 2 2002 5 7 10.1016/S1567-5688(01)00013-7
11 Austin M.A. Hutter C.M. Zimmern R.L. Humphries S.E. Familial Hypercholesterolemia and Coronary Heart Disease: A HuGE Association Review Am. J. Epidemiol. 160 2004 421 429 10.1093/aje/kwh237 15321838
12 Moriarty P.M. Lipoprotein apheresis: present and future uses Curr. Opin. Lipidol. 26 2015 544 552 10.1097/mol.0000000000000234 26780007
13 Safarova M.S. Moriarty P.M. Lipoprotein Apheresis: Current Recommendations for Treating Familial Hypercholesterolemia and Elevated Lipoprotein(a) Curr. Atherosclerosis Rep. 25 2023 391 404 10.1007/s11883-023-01113-2
14 Wierzbicki A.S. Humphries S.E. Minhas R. Guideline Development Group Familial hypercholesterolaemia: summary of NICE guidance Br. Med. J. 337 2008 a1095 10.1136/bmj.a1095
15 Kayikcioglu M. Tokgozoglu L. Current Treatment Options in Homozygous Familial Hypercholesterolemia Pharmaceuticals 16 2022 64 36678563
16 Taylor F. Huffman M.D. Macedo A.F. Moore T.H.M. Burke M. Davey Smith G. Ward K. Ebrahim S. Statins for the primary prevention of cardiovascular disease Cochrane Database Syst. Rev. 2013 2013 Cd004816 10.1002/14651858.CD004816.pub5
17 Ward N.C. Watts G.F. Eckel R.H. Statin Toxicity Circ. Res. 124 2019 328 350 10.1161/circresaha.118.312782 30653440
18 Howard J.P. Wood F.A. Finegold J.A. Nowbar A.N. Thompson D.M. Arnold A.D. Rajkumar C.A. Connolly S. Cegla J. Stride C. Side Effect Patterns in a Crossover Trial of Statin, Placebo, and No Treatment J. Am. Coll. Cardiol. 78 2021 1210 1222 10.1016/j.jacc.2021.07.022 34531021
19 Pedro-Botet J. Núñez-Cortés J.M. Flores J. Rius J. Muscle symptoms related with statin therapy in general practice Atherosclerosis 241 2015 e197
20 Chogtu B. Magazine R. Bairy K.L. Statin use and risk of diabetes mellitus World J. Diabetes 6 2015 352 357 10.4239/wjd.v6.i2.352 25789118
21 Ramkumar S. Raghunath A. Raghunath S. Statin Therapy: Review of Safety and Potential Side Effects Acta Cardiol. Sin. 32 2016 631 639 10.6515/acs20160611a 27899849
22 Phan B.A.P. Dayspring T.D. Toth P.P. Ezetimibe therapy: mechanism of action and clinical update Vasc. Health Risk Manag. 8 2012 415 427 10.2147/vhrm.S33664 22910633
23 Kim B.K. Hong S.J. Lee Y.J. Hong S.J. Yun K.H. Hong B.K. Heo J.H. Rha S.W. Cho Y.H. Lee S.J. Long-term efficacy and safety of moderate-intensity statin with ezetimibe combination therapy versus high-intensity statin monotherapy in patients with atherosclerotic cardiovascular disease (RACING): a randomised, open-label, non-inferiority trial Lancet 400 2022 380 390 10.1016/s0140-6736(22)00916-3 35863366
24 Lagace T.A. PCSK9 and LDLR degradation: regulatory mechanisms in circulation and in cells Curr. Opin. Lipidol. 25 2014 387 393 10.1097/mol.0000000000000114 25110901
25 Handelsman Y. Lepor N.E. PCSK9 Inhibitors in Lipid Management of Patients With Diabetes Mellitus and High Cardiovascular Risk: A Review J. Am. Heart Assoc. 7 2018 e008953 10.1161/JAHA.118.008953
26 Gürgöze M.T. Muller-Hansma A.H.G. Schreuder M.M. Galema-Boers A.M.H. Boersma E. Roeters van Lennep J.E. Adverse Events Associated With PCSK9 Inhibitors: A Real-World Experience Clin. Pharmacol. Ther. 105 2019 496 504 10.1002/cpt.1193 30053327
27 di Mauro G. Zinzi A. Scavone C. Mascolo A. Gaio M. Sportiello L. Ferrajolo C. Rafaniello C. Rossi F. Capuano A. PCSK9 Inhibitors and Neurocognitive Adverse Drug Reactions: Analysis of Individual Case Safety Reports from the Eudravigilance Database Drug Saf. 44 2021 337 349 10.1007/s40264-020-01021-3 33351170
28 Mefford M.T. Rosenson R.S. Cushman M. Farkouh M.E. McClure L.A. Wadley V.G. Irvin M.R. Bittner V. Safford M.M. Somaratne R. PCSK9 Variants, Low-Density Lipoprotein Cholesterol, and Neurocognitive Impairment Circulation 137 2018 1260 1269 10.1161/CIRCULATIONAHA.117.029785 29146683
29 Zafar Y. Sattar Y. Ullah W. Roomi S. Rashid M.U. Khan M.S. Schmidt L. Proprotein convertase subtilisin/Kexin type-9 (PCSK-9) inhibitors induced liver injury - a retrospective analysis J. Community Hosp. Intern. Med. Perspect. 10 2020 32 37 10.1080/20009666.2019.1710952 32128056
30 Raal F.J. Santos R.D. Blom D.J. Marais A.D. Charng M.J. Cromwell W.C. Lachmann R.H. Gaudet D. Tan J.L. Chasan-Taber S. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial Lancet 375 2010 998 1006 10.1016/s0140-6736(10)60284-x 20227758
31 Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein Biochim. Biophys. Acta 1345 1997 136 150 10.1016/s0005-2760(96)00168-3 9106493
32 Gidding S.S. Champagne M.A. de Ferranti S.D. Defesche J. Ito M.K. Knowles J.W. McCrindle B. Raal F. Rader D. Santos R.D. The Agenda for Familial Hypercholesterolemia: A Scientific Statement From the American Heart Association Circulation 132 2015 2167 2192 10.1161/cir.0000000000000297 26510694
33 Moyle M. Tate B. Homozygous familial hypercholesterolaemia presenting with cutaneous xanthomas: response to liver transplantation Australas. J. Dermatol. 45 2004 226 228 15527434
34 Scheller E.L. Krebsbach P.H. Gene therapy: design and prospects for craniofacial regeneration J. Dent. Res. 88 2009 585 596 10.1177/0022034509337480 19641145
35 Razi Soofiyani S. Baradaran B. Lotfipour F. Kazemi T. Mohammadnejad L. Gene therapy, early promises, subsequent problems, and recent breakthroughs Adv. Pharmaceut. Bull. 3 2013 249 255 10.5681/apb.2013.041
36 Pathak S. Gene therapy – Principles and Applications Glob. J. Transfus. Med. 7 2022 3 6 10.4103/gjtm.gjtm_87_21
37 Anguela X.M. High K.A. Entering the Modern Era of Gene Therapy Annu. Rev. Med. 70 2019 273 288 10.1146/annurev-med-012017-043332 30477394
38 Ling Q. Herstine J.A. Bradbury A. Gray S.J. AAV-based in vivo gene therapy for neurological disorders Nat. Rev. Drug Discov. 22 2023 789 806 10.1038/s41573-023-00766-7 37658167
39 Li C. Georgakopoulou A. Newby G.A. Chen P.J. Everette K.A. Paschoudi K. Vlachaki E. Gil S. Anderson A.K. Koob T. In vivo HSC prime editing rescues sickle cell disease in a mouse model Blood 141 2023 2085 2099 10.1182/blood.2022018252 36800642
40 Shi D. Toyonaga S. Anderson D.G. In Vivo RNA Delivery to Hematopoietic Stem and Progenitor Cells via Targeted Lipid Nanoparticles Nano Lett. 23 2023 2938 2944 10.1021/acs.nanolett.3c00304 36988645
41 Li X. La Salvia S. Liang Y. Adamiak M. Kohlbrenner E. Jeong D. Chepurko E. Ceholski D. Lopez-Gordo E. Yoon S. Extracellular Vesicle–Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart Circulation 148 2023 405 425 10.1161/CIRCULATIONAHA.122.063759 37409482
42 Wu X. He X. Liu F. Jiang X. Wang P. Zhang J. Jiang J. Development and clinical translation of ex vivo gene therapy Comput. Struct. Biotechnol. J. 20 2022 2986 3003 10.1016/j.csbj.2022.06.015 35782737
43 Vavassori V. Ferrari S. Beretta S. Asperti C. Albano L. Annoni A. Gaddoni C. Varesi A. Soldi M. Cuomo A. Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells Blood 142 2023 812 826 10.1182/blood.2022019333 37294917
44 Fischer A. Gene therapy for inborn errors of immunity: past, present and future Nat. Rev. Immunol. 23 2023 397 408 10.1038/s41577-022-00800-6 36434109
45 Everette K.A. Newby G.A. Levine R.M. Mayberry K. Jang Y. Mayuranathan T. Nimmagadda N. Dempsey E. Li Y. Bhoopalan S.V. Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice Nat. Biomed. Eng. 7 2023 616 628 10.1038/s41551-023-01026-0 37069266
46 Singh G. Resveratrol Delivery via Gene Therapy: Entering the Modern Era Turk. J. Pharm. Sci. 19 2022 104 109 10.4274/tjps.galenos.2020.89577 35227259
47 Peng Z. Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers Hum. Gene Ther. 16 2005 1016 1027 10.1089/hum.2005.16.1016 16149900
48 Gaudet D. Méthot J. Déry S. Brisson D. Essiembre C. Tremblay G. Tremblay K. de Wal J. Twisk J. van den Bulk N. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial Gene Ther. 20 2013 361 369 10.1038/gt.2012.43 22717743
49 Cicalese M.P. Ferrua F. Castagnaro L. Pajno R. Barzaghi F. Giannelli S. Dionisio F. Brigida I. Bonopane M. Casiraghi M. Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency Blood 128 2016 45 54 10.1182/blood-2016-01-688226 27129325
50 South E. Cox E. Meader N. Woolacott N. Griffin S. Strimvelis(®) for Treating Severe Combined Immunodeficiency Caused by Adenosine Deaminase Deficiency: An Evidence Review Group Perspective of a NICE Highly Specialised Technology Evaluation Pharmacoecon. Open 3 2019 151 161 10.1007/s41669-018-0102-3 30334168
51 Russell S. Bennett J. Wellman J.A. Chung D.C. Yu Z.F. Tillman A. Wittes J. Pappas J. Elci O. McCague S. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial Lancet 390 2017 849 860 10.1016/s0140-6736(17)31868-8 28712537
52 Guide S.V. Gonzalez M.E. Bağcı I.S. Agostini B. Chen H. Feeney G. Steimer M. Kapadia B. Sridhar K. Quesada Sanchez L. Trial of Beremagene Geperpavec (B-VEC) for Dystrophic Epidermolysis Bullosa N. Engl. J. Med. 387 2022 2211 2219 10.1056/NEJMoa2206663 36516090
53 Khan A. Riaz R. Ashraf S. Akilimali A. Revolutionary breakthrough: FDA approves Vyjuvek, the first topical gene therapy for dystrophic epidermolysis bullosa Ann. Med. Surg. 85 2023 6298 6301 10.1097/ms9.0000000000001422
54 Mendell J.R. Al-Zaidy S. Shell R. Arnold W.D. Rodino-Klapac L.R. Prior T.W. Lowes L. Alfano L. Berry K. Church K. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy N. Engl. J. Med. 377 2017 1713 1722 10.1056/NEJMoa1706198 29091557
55 Locatelli F. Thompson A.A. Kwiatkowski J.L. Porter J.B. Thrasher A.J. Hongeng S. Sauer M.G. Thuret I. Lal A. Algeri M. Betibeglogene Autotemcel Gene Therapy for Non-β(0)/β(0) Genotype β-Thalassemia N. Engl. J. Med. 386 2022 415 427 10.1056/NEJMoa2113206 34891223
56 Ozelo M.C. Mahlangu J. Pasi K.J. Giermasz A. Leavitt A.D. Laffan M. Symington E. Quon D.V. Wang J.D. Peerlinck K. Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A N. Engl. J. Med. 386 2022 1013 1025 10.1056/NEJMoa2113708 35294811
57 Sessa M. Lorioli L. Fumagalli F. Acquati S. Redaelli D. Baldoli C. Canale S. Lopez I.D. Morena F. Calabria A. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial Lancet 388 2016 476 487 10.1016/S0140-6736(16)30374-9 27289174
58 Boorjian S.A. Alemozaffar M. Konety B.R. Shore N.D. Gomella L.G. Kamat A.M. Bivalacqua T.J. Montgomery J.S. Lerner S.P. Busby J.E. Intravesical nadofaragene firadenovec gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: a single-arm, open-label, repeat-dose clinical trial Lancet Oncol. 22 2021 107 117 10.1016/s1470-2045(20)30540-4 33253641
59 Pipe S.W. Leebeek F.W.G. Recht M. Key N.S. Castaman G. Miesbach W. Lattimore S. Peerlinck K. Van der Valk P. Coppens M. Gene Therapy with Etranacogene Dezaparvovec for Hemophilia B N. Engl. J. Med. 388 2023 706 718 10.1056/NEJMoa2211644 36812434
60 Eichler F. Duncan C. Musolino P.L. Orchard P.J. De Oliveira S. Thrasher A.J. Armant M. Dansereau C. Lund T.C. Miller W.P. Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy N. Engl. J. Med. 377 2017 1630 1638 10.1056/NEJMoa1700554 28976817
61 Mendell J.R. Sahenk Z. Lehman K.J. Lowes L.P. Reash N.F. Iammarino M.A. Alfano L.N. Lewis S. Church K. Shell R. Long-term safety and functional outcomes of delandistrogene moxeparvovec gene therapy in patients with Duchenne muscular dystrophy: A phase 1/2a nonrandomized trial Muscle Nerve 69 2024 93 98 10.1002/mus.27955 37577753
62 Frangoul H. Altshuler D. Cappellini M.D. Chen Y.-S. Domm J. Eustace B.K. Foell J. de la Fuente J. Grupp S. Handgretinger R. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia N. Engl. J. Med. 384 2021 252 260 10.1056/NEJMoa2031054 33283989
63 Kanter J. Walters M.C. Krishnamurti L. Mapara M.Y. Kwiatkowski J.L. Rifkin-Zenenberg S. Aygun B. Kasow K.A. Pierciey F.J. Bonner M. Biologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease N. Engl. J. Med. 386 2022 617 628 10.1056/NEJMoa2117175 34898139
64 Hoy S.M. Exagamglogene Autotemcel: First Approval Mol. Diagn. Ther. 28 2024 133 139 10.1007/s40291-024-00696-z 38228954
65 Yamada Y. Nucleic Acid Drugs-Current Status, Issues, and Expectations for Exosomes Cancers 13 2021 5002 10.3390/cancers13195002
66 Dhuri K. Bechtold C. Quijano E. Pham H. Gupta A. Vikram A. Bahal R. Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development J. Clin. Med. 9 2020 2004 10.3390/jcm9062004
67 Kulkarni J.A. Witzigmann D. Thomson S.B. Chen S. Leavitt B.R. Cullis P.R. van der Meel R. The current landscape of nucleic acid therapeutics Nat. Nanotechnol. 16 2021 630 643 10.1038/s41565-021-00898-0 34059811
68 Grossman M. Rader D.J. Muller D.W. Kolansky D.M. Kozarsky K. Clark B.J. 3rd Stein E.A. Lupien P.J. Brewer H.B. Jr. Raper S.E. A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia Nat. Med. 1 1995 1148 1154 10.1038/nm1195-1148 7584986
69 Wang L. Muthuramu I. Somanathan S. Zhang H. Bell P. He Z. Yu H. Zhu Y. Tretiakova A.P. Wilson J.M. Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia Mol. Ther. Methods Clin. Dev. 22 2021 1 10 34258325
70 Bissig-Choisat B. Wang L. Legras X. Saha P.K. Chen L. Bell P. Pankowicz F.P. Hill M.C. Barzi M. Leyton C.K. Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model Nat. Commun. 6 2015 7339 10.1038/ncomms8339 26081744
71 Lisowski L. Dane A.P. Chu K. Zhang Y. Cunningham S.C. Wilson E.M. Nygaard S. Grompe M. Alexander I.E. Kay M.A. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model Nature 506 2014 382 386 10.1038/nature12875 24390344
72 Barzi M. Chen T. Gonzalez T.J. Pankowicz F.P. Oh S.H. Streff H.L. Rosales A. Ma Y. Collias S. Woodfield S.E. A humanized mouse model for adeno-associated viral gene therapy Nat. Commun. 15 2024 1955 10.1038/s41467-024-46017-0 38438373
73 Baek G. Choi H. Kim Y. Lee H.C. Choi C. Mesenchymal Stem Cell-Derived Extracellular Vesicles as Therapeutics and as a Drug Delivery Platform Stem Cells Transl. Med. 8 2019 880 886 10.1002/sctm.18-0226 31045328
74 Li Z. Zhao P. Zhang Y. Wang J. Wang C. Liu Y. Yang G. Yuan L. Exosome-based Ldlr gene therapy for familial hypercholesterolemia in a mouse model Theranostics 11 2021 2953 2965 10.7150/thno.49874 33456582
75 Sun L. Xu R. Sun X. Duan Y. Han Y. Zhao Y. Qian H. Zhu W. Xu W. Safety evaluation of exosomes derived from human umbilical cord mesenchymal stromal cell Cytotherapy 18 2016 413 422 10.1016/j.jcyt.2015.11.018 26857231
76 Rezaie J. Feghhi M. Etemadi T. A review on exosomes application in clinical trials: perspective, questions, and challenges Cell Commun. Signal. 20 2022 145 10.1186/s12964-022-00959-4 36123730
77 Ray K.K. Wright R.S. Kallend D. Koenig W. Leiter L.A. Raal F.J. Bisch J.A. Richardson T. Jaros M. Wijngaard P.L.J. Two Phase 3 Trials of Inclisiran in Patients with Elevated LDL Cholesterol N. Engl. J. Med. 382 2020 1507 1519 10.1056/NEJMoa1912387 32187462
78 Ray K.K. Troquay R.P.T. Visseren F.L.J. Leiter L.A. Scott Wright R. Vikarunnessa S. Talloczy Z. Zang X. Maheux P. Lesogor A. Landmesser U. Long-term efficacy and safety of inclisiran in patients with high cardiovascular risk and elevated LDL cholesterol (ORION-3): results from the 4-year open-label extension of the ORION-1 trial Lancet Diabetes Endocrinol. 11 2023 109 119 10.1016/S2213-8587(22)00353-9 36620965
79 Hair P. Cameron F. McKeage K. Mipomersen sodium: first global approval Drugs 73 2013 487 493 10.1007/s40265-013-0042-2 23564617
80 Frank-Kamenetsky M. Grefhorst A. Anderson N.N. Racie T.S. Bramlage B. Akinc A. Butler D. Charisse K. Dorkin R. Fan Y. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates Proc. Natl. Acad. Sci. USA 105 2008 11915 11920 10.1073/pnas.0805434105 18695239
81 Watts G.F. Gaudet D. Altamirano D. Hegele R. Ballantyne C.M. Nicholls S. Chang T. Alagarsamy S. Fu R. San Martin J. Rosenson R.S. ARO-ANG3, an Investigational RNAi Therapeutic, Silences the Expression of ANGPTL3 and Decreases Atherogenic Lipoproteins in Patients With Mixed Dyslipidemia: ARCHES-2 Study Results Circulation 148 2023 A17120 10.1161/circ.148.suppl_1.17120
82 Vasas S. Azizad M. Clifton P. Gaudet D. Goldenberg R. Modesto K. Chang T. Melquist S. Fu R. San Martin J. Ballantyne C.M. ARO-APOC3, an Investigational RNAi Therapeutic, Silences APOC3 and Reduces Atherosclerosis-Associated Lipoproteins in Patients With Mixed Dyslipidemia: MUIR Study Results Circulation 148 2023 A17091 10.1161/circ.148.suppl_1.17091
83 O'Donoghue M.L. Rosenson R.S. Gencer B. López J.A.G. Lepor N.E. Baum S.J. Stout E. Gaudet D. Knusel B. Kuder J.F. Small Interfering RNA to Reduce Lipoprotein(a) in Cardiovascular Disease N. Engl. J. Med. 387 2022 1855 1864 10.1056/NEJMoa2211023 36342163
84 Nissen S.E. Wolski K. Balog C. Swerdlow D.I. Scrimgeour A.C. Rambaran C. Wilson R.J. Boyce M. Ray K.K. Cho L. Single Ascending Dose Study of a Short Interfering RNA Targeting Lipoprotein(a) Production in Individuals With Elevated Plasma Lipoprotein(a) Levels JAMA 327 2022 1679 1687 10.1001/jama.2022.5050 35368052
85 Hofherr A. Schumi J. Rudvik A. Vega R. Ryden-Bergsten T. Hurt-Camejo E. Johanson P. Carlsson B.C. The PCSK9-Targeted Antisense Oligonucleotide AZD8233 Reduces LDL-C, ApoB, and Lp(a) in Patients With Dyslipidemia on Statin Treatment - Data From the Phase 2b ETESIAN Study Circulation 146 2022 A11482 10.1161/circ.146.suppl_1.11482
86 Bergmark B.A. Marston N.A. Bramson C.R. Curto M. Ramos V. Jevne A. Kuder J.F. Park J.G. Murphy S.A. Verma S. Effect of Vupanorsen on Non-High-Density Lipoprotein Cholesterol Levels in Statin-Treated Patients With Elevated Cholesterol: TRANSLATE-TIMI 70 Circulation 145 2022 1377 1386 10.1161/CIRCULATIONAHA.122.059266 35369705
87 Witztum J.L. Gaudet D. Freedman S.D. Alexander V.J. Digenio A. Williams K.R. Yang Q. Hughes S.G. Geary R.S. Arca M. Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome N. Engl. J. Med. 381 2019 531 542 10.1056/NEJMoa1715944 31390500
88 Tardif J.C. Karwatowska-Prokopczuk E. Amour E.S. Ballantyne C.M. Shapiro M.D. Moriarty P.M. Baum S.J. Hurh E. Bartlett V.J. Kingsbury J. Apolipoprotein C-III reduction in subjects with moderate hypertriglyceridaemia and at high cardiovascular risk Eur. Heart J. 43 2022 1401 1412 10.1093/eurheartj/ehab820 35025993
89 Tsimikas S. Karwatowska-Prokopczuk E. Gouni-Berthold I. Tardif J.C. Baum S.J. Steinhagen-Thiessen E. Shapiro M.D. Stroes E.S. Moriarty P.M. Nordestgaard B.G. Lipoprotein(a) Reduction in Persons with Cardiovascular Disease N. Engl. J. Med. 382 2020 244 255 10.1056/NEJMoa1905239 31893580
90 Lim G.B. ANGPTL3 inhibition for hypercholesterolaemia Nat. Rev. Cardiol. 18 2021 72 10.1038/s41569-020-00483-3
91 Adam R.C. Mintah I.J. Alexa-Braun C.A. Shihanian L.M. Lee J.S. Banerjee P. Hamon S.C. Kim H.I. Cohen J.C. Hobbs H.H. Angiopoietin-like protein 3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance J. Lipid Res. 61 2020 1271 1286 10.1194/jlr.RA120000888 32646941
92 Xu Y.X. Redon V. Yu H. Querbes W. Pirruccello J. Liebow A. Deik A. Trindade K. Wang X. Musunuru K. Role of angiopoietin-like 3 (ANGPTL3) in regulating plasma level of low-density lipoprotein cholesterol Atherosclerosis 268 2018 196 206 10.1016/j.atherosclerosis.2017.08.031 29183623
93 Ying Q. Chan D.C. Watts G.F. Angiopoietin-like protein 3 inhibitors and contemporary unmet needs in lipid management Curr. Opin. Lipidol. 32 2021 210 212 10.1097/MOL.0000000000000747 33900278
94 Yang L. Ma F. Liu F. Chen J. Zhao X. Xu Q. Efficient Delivery of Antisense Oligonucleotides Using Bioreducible Lipid Nanoparticles In Vitro and In Vivo Mol. Ther. Nucleic Acids 19 2020 1357 1367 10.1016/j.omtn.2020.01.018 32160706
95 Gaudet D. Karwatowska-Prokopczuk E. Baum S.J. Hurh E. Kingsbury J. Bartlett V.J. Figueroa A.L. Piscitelli P. Singleton W. Witztum J.L. Vupanorsen, an N-acetyl galactosamine-conjugated antisense drug to ANGPTL3 mRNA, lowers triglycerides and atherogenic lipoproteins in patients with diabetes, hepatic steatosis, and hypertriglyceridaemia Eur. Heart J. 41 2020 3936 3945 10.1093/eurheartj/ehaa689 32860031
96 Huff M.W. Hegele R.A. Apolipoprotein C-III Circ. Res. 112 2013 1405 1408 10.1161/CIRCRESAHA.113.301464 23704213
97 Tromp T.R. Stroes E.S.G. Hovingh G.K. Gene-based therapy in lipid management: the winding road from promise to practice Expert Opin. Invest. Drugs 29 2020 483 493 10.1080/13543784.2020.1757070
98 Khetarpal S.A. Zeng X. Millar J.S. Vitali C. Somasundara A.V.H. Zanoni P. Landro J.A. Barucci N. Zavadoski W.J. Sun Z. A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels Nat. Med. 23 2017 1086 1094 10.1038/nm.4390 28825717
99 Hegele R.A. APOC3 Interference for Familial Chylomicronaemia Syndrome touchREV. Endocrinol. 18 2022 82 83 10.17925/ee.2022.18.2.82 36694895
100 Utermann G. Weber W. Protein composition of Lp(a) lipoprotein from human plasma FEBS Lett. 154 1983 357 361 10.1016/0014-5793(83)80182-3 6219896
101 Gaubatz J.W. Heideman C. Gotto A.M. Jr. Morrisett J.D. Dahlen G.H. Human plasma lipoprotein [a]. Structural properties J. Biol. Chem. 258 1983 4582 4589 6220008
102 Tsimikas S. Fazio S. Ferdinand K.C. Ginsberg H.N. Koschinsky M.L. Marcovina S.M. Moriarty P.M. Rader D.J. Remaley A.T. Reyes-Soffer G. NHLBI Working Group Recommendations to Reduce Lipoprotein(a)-Mediated Risk of Cardiovascular Disease and Aortic Stenosis J. Am. Coll. Cardiol. 71 2018 177 192 10.1016/j.jacc.2017.11.014 29325642
103 Philippidis A. CASGEVY Makes History as FDA Approves First CRISPR/Cas9 Genome Edited Therapy Hum. Gene Ther. 35 2024 1 4 10.1089/hum.2023.29263.bfs 38231658
104 Yang L. Chen J. A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing Trends Biochem. Sci. 45 2020 874 888 10.1016/j.tibs.2020.06.003 32616331
105 Anzalone A.V. Koblan L.W. Liu D.R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime Nat. Biotechnol. 38 2020 824 844 10.1038/s41587-020-0561-9 32572269
106 Zuccaro M.V. Xu J. Mitchell C. Marin D. Zimmerman R. Rana B. Weinstein E. King R.T. Palmerola K.L. Smith M.E. Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos Cell 183 2020 1650 1664.e15 10.1016/j.cell.2020.10.025 33125898
107 Haapaniemi E. Botla S. Persson J. Schmierer B. Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response Nat. Med. 24 2018 927 930 10.1038/s41591-018-0049-z 29892067
108 Kosicki M. Tomberg K. Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements Nat. Biotechnol. 36 2018 765 771 10.1038/nbt.4192 30010673
109 Yang B. Yang L. Chen J. Development and Application of Base Editors CRISPR J. 2 2019 91 104 10.1089/crispr.2019.0001 30998092
110 Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Nature 533 2016 420 424 10.1038/nature17946 27096365
111 Gaudelli N.M. Komor A.C. Rees H.A. Packer M.S. Badran A.H. Bryson D.I. Liu D.R. Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage Nature 551 2017 464 471 10.1038/nature24644 29160308
112 Tong H. Wang X. Liu Y. Liu N. Li Y. Luo J. Ma Q. Wu D. Li J. Xu C. Yang H. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase Nat. Biotechnol. 41 2023 1080 1084 10.1038/s41587-022-01595-6 36624150
113 Ye L. Zhao D. Li J. Wang Y. Li B. Yang Y. Hou X. Wang H. Wei Z. Liu X. Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells Nat. Biotechnol. 2024 1 10 10.1038/s41587-023-02050-w 38191665
114 Wang L. Xue W. Zhang H. Gao R. Qiu H. Wei J. Zhou L. Lei Y.-N. Wu X. Li X. Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations Nat. Cell Biol. 23 2021 552 563 10.1038/s41556-021-00671-4 33972728
115 Landrum M.J. Lee J.M. Benson M. Brown G. Chao C. Chitipiralla S. Gu B. Hart J. Hoffman D. Hoover J. ClinVar: public archive of interpretations of clinically relevant variants Nucleic Acids Res. 44 2016 D862 D868 10.1093/nar/gkv1222 26582918
116 Chen P.J. Liu D.R. Prime editing for precise and highly versatile genome manipulation Nat. Rev. Genet. 24 2023 161 177 10.1038/s41576-022-00541-1 36344749
117 Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 576 2019 149 157 10.1038/s41586-019-1711-4 31634902
118 Seidah N.G. Benjannet S. Wickham L. Marcinkiewicz J. Jasmin S.B. Stifani S. Basak A. Prat A. Chretien M. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation Proc. Natl. Acad. Sci. USA 100 2003 928 933 10.1073/pnas.0335507100 12552133
119 Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M.-C. Hamelin J. Varret M. Allard D. NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol J. Biol. Chem. 279 2004 48865 48875 15358785
120 O'Connell E.M. Lohoff F.W. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in the Brain and Relevance for Neuropsychiatric Disorders Front. Neurosci. 14 2020 609 10.3389/fnins.2020.00609 32595449
121 Shapiro M.D. Tavori H. Fazio S. PCSK9: From Basic Science Discoveries to Clinical Trials Circ. Res. 122 2018 1420 1438 10.1161/circresaha.118.311227 29748367
122 Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice J. Lipid Res. 44 2003 2109 2119 10.1194/jlr.M300203-JLR200 12897189
123 Horton J.D. Cohen J.C. Hobbs H.H. Molecular biology of PCSK9: its role in LDL metabolism Trends Biochem. Sci. 32 2007 71 77 10.1016/j.tibs.2006.12.008 17215125
124 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. 37 2005 161 165 10.1038/ng1509 15654334
125 Abifadel M. Varret M. Rabès J.-P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia Nat. Genet. 34 2003 154 156 12730697
126 Cohen J.C. Boerwinkle E. Mosley T.H. Jr. Hobbs H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease N. Engl. J. Med. 354 2006 1264 1272 16554528
127 Rao A.S. Lindholm D. Rivas M.A. Knowles J.W. Montgomery S.B. Ingelsson E. Large-scale phenome-wide association study of PCSK9 variants demonstrates protection against ischemic stroke Circ. Genom. Precis. Med. 11 2018 e002162
128 Koren M.J. Sabatine M.S. Giugliano R.P. Langslet G. Wiviott S.D. Ruzza A. Ma Y. Hamer A.W. Wasserman S.M. Raal F.J. Long-Term Efficacy and Safety of Evolocumab in Patients With Hypercholesterolemia J. Am. Coll. Cardiol. 74 2019 2132 2146 10.1016/j.jacc.2019.08.1024 31648705
129 Schwartz G.G. Steg P.G. Szarek M. Bhatt D.L. Bittner V.A. Diaz R. Edelberg J.M. Goodman S.G. Hanotin C. Harrington R.A. Alirocumab and Cardiovascular Outcomes after Acute Coronary Syndrome N. Engl. J. Med. 379 2018 2097 2107 10.1056/NEJMoa1801174 30403574
130 Pérez de Isla L. Díaz-Díaz J.L. Romero M.J. Muñiz-Grijalvo O. Mediavilla J.D. Argüeso R. Sánchez Muñoz-Torrero J.F. Rubio P. Álvarez-Baños P. Ponte P. Alirocumab and Coronary Atherosclerosis in Asymptomatic Patients with Familial Hypercholesterolemia: The ARCHITECT Study Circulation 147 2023 1436 1443 10.1161/circulationaha.122.062557 37009731
131 Räber L. Ueki Y. Otsuka T. Losdat S. Häner J.D. Lonborg J. Fahrni G. Iglesias J.F. van Geuns R.-J. Ondracek A.S. Effect of Alirocumab Added to High-Intensity Statin Therapy on Coronary Atherosclerosis in Patients With Acute Myocardial Infarction: The PACMAN-AMI Randomized Clinical Trial JAMA 327 2022 1771 1781 10.1001/jama.2022.5218 35368058
132 Ding Q. Strong A. Patel K.M. Ng S.L. Gosis B.S. Regan S.N. Cowan C.A. Rader D.J. Musunuru K. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing Circ. Res. 115 2014 488 492 10.1161/circresaha.115.304351 24916110
133 Rothgangl T. Dennis M.K. Lin P.J.C. Oka R. Witzigmann D. Villiger L. Qi W. Hruzova M. Kissling L. Lenggenhager D. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels Nat. Biotechnol. 39 2021 949 957 34012094
134 Musunuru K. Chadwick A.C. Mizoguchi T. Garcia S.P. DeNizio J.E. Reiss C.W. Wang K. Iyer S. Dutta C. Clendaniel V. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates Nature 593 2021 429 434 34012082
135 Chadwick A.C. Evitt N.H. Lv W. Musunuru K. Reduced blood lipid levels with in vivo CRISPR-Cas9 base editing of ANGPTL3 Circulation 137 2018 975 977 29483174
136 Qiu M. Glass Z. Chen J. Haas M. Jin X. Zhao X. Rui X. Ye Z. Li Y. Zhang F. Xu Q. Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3 Proc. Natl. Acad. Sci. USA 118 2021 e2020401118
137 Zuo Y. Zhang C. Zhou Y. Li H. Xiao W. Herzog R.W. Xu J. Zhang J. Chen Y.E. Han R. Liver-specific in vivo base editing of Angptl3 via AAV delivery efficiently lowers blood lipid levels in mice Cell Biosci. 13 2023 109 37322547
138 Lee R. Denizio J. Mizoguchi T. Dutta C. Clendaniel V. Garrity R. Cox N. Glass Z. Chamarthi H. Braun M. An investigational in vivo base editing medicine targeting ANGPTL3, VERVE-201, achieves potent and LDLR-independent liver editing in mouse models Eur. Heart J. 44 2023 ehad655
139 Kasiewicz L.N. Biswas S. Beach A. Ren H. Dutta C. Mazzola A.M. Rohde E. Chadwick A. Cheng C. Garcia S.P. GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy Nat. Commun. 14 2023 2776 37188660
140 Horie T. Ono K. VERVE-101: a promising CRISPR-based gene editing therapy that reduces LDL-C and PCSK9 levels in HeFH patients Eur. Heart J. Cardiovasc. Pharmacother. 10 2024 89 90 10.1093/ehjcvp/pvad103 38142221
141 Grinstein J.D. Bittersweet Symphony: Verve’s Pause on VERVE-101 Narrows LNP-Delivery Strategy https://www.genengnews.com/topics/genome-editing/bittersweet-symphony-verves-pause-on-verve-101-narrows-lnp-delivery-strategy/ 2024
142 Coppinger C. Movahed M.R. Azemawah V. Peyton L. Gregory J. Hashemzadeh M. A Comprehensive Review of PCSK9 Inhibitors J. Cardiovasc. Pharmacol. Therapeut. 27 2022 10742484221100107 10.1177/10742484221100107
143 Karatasakis A. Danek B.A. Karacsonyi J. Rangan B.V. Roesle M.K. Knickelbine T. Miedema M.D. Khalili H. Ahmad Z. Abdullah S. Effect of PCSK9 Inhibitors on Clinical Outcomes in Patients With Hypercholesterolemia: A Meta-Analysis of 35 Randomized Controlled Trials J. Am. Heart Assoc. 6 2017 e006910 10.1161/jaha.117.006910
144 Eckel R.H. Lipoprotein lipase N. Engl. J. Med. 320 1989 1060 1068 2648155
145 Romeo S. Yin W. Kozlitina J. Pennacchio L.A. Boerwinkle E. Hobbs H.H. Cohen J.C. Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans J. Clin. Invest. 119 2009 70 79 19075393
146 Musunuru K. Pirruccello J.P. Do R. Peloso G.M. Guiducci C. Sougnez C. Garimella K.V. Fisher S. Abreu J. Barry A.J. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia N. Engl. J. Med. 363 2010 2220 2227 20942659
147 Dewey F.E. Gusarova V. Dunbar R.L. O’Dushlaine C. Schurmann C. Gottesman O. McCarthy S. Van Hout C.V. Bruse S. Dansky H.M. Genetic and pharmacologic inactivation of ANGPTL3 and cardiovascular disease N. Engl. J. Med. 377 2017 211 221 28538136
148 Ge H. Cha J.Y. Gopal H. Harp C. Yu X. Repa J.J. Li C. Differential regulation and properties of angiopoietin-like proteins 3 and 4 J. Lipid Res. 46 2005 1484 1490 15863837
149 Raal F.J. Rosenson R.S. Reeskamp L.F. Hovingh G.K. Kastelein J.J.P. Rubba P. Ali S. Banerjee P. Chan K.C. Gipe D.A. Evinacumab for Homozygous Familial Hypercholesterolemia N. Engl. J. Med. 383 2020 711 720 10.1056/NEJMoa2004215 32813947
150 Raposo C.D. Canelas A.B. Barros M.T. Human Lectins, Their Carbohydrate Affinities and Where to Find Them Biomolecules 11 2021 188 10.3390/biom11020188
151 Morell A.G. Gregoriadis G. Scheinberg I.H. Hickman J. Ashwell G. The role of sialic acid in determining the survival of glycoproteins in the circulation J. Biol. Chem. 246 1971 1461 1467 5545089
152 Pricer W.E. Jr. Ashwell G. The binding of desialylated glycoproteins by plasma membranes of rat liver J. Biol. Chem. 246 1971 4825 4833 4327329
153 Sarkar M. Liao J. Kabat E.A. Tanabe T. Ashwell G. The binding site of rabbit hepatic lectin J. Biol. Chem. 254 1979 3170 3174 429341
154 Steer C.J. Ashwell G. Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes J. Biol. Chem. 255 1980 3008 3013 6244303
155 Spiess M. The asialoglycoprotein receptor: a model for endocytic transport receptors Biochemistry 29 1990 10009 10018 10.1021/bi00495a001 2125488
156 Stockert R.J. The asialoglycoprotein receptor: relationships between structure, function, and expression Physiol. Rev. 75 1995 591 609 10.1152/physrev.1995.75.3.591 7624395
157 Pricer W.E. Jr. Hudgin R.L. Ashwell G. Stockert R.J. Morell A.G. A membrane receptor protein for asialoglycoproteins Methods Enzymol. 34 1974 688 691 10.1016/s0076-6879(74)34090-6 4449482
158 Roggenbuck D. Mytilinaiou M.G. Lapin S.V. Reinhold D. Conrad K. Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-specific autoimmunity Auto. Immun. Highlights 3 2012 119 125 10.1007/s13317-012-0041-4 26000135
159 D'Souza A.A. Devarajan P.V. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications J. Contr. Release 203 2015 126 139 10.1016/j.jconrel.2015.02.022
160 Nioi P. Sigurdsson A. Thorleifsson G. Helgason H. Agustsdottir A.B. Norddahl G.L. Helgadottir A. Magnusdottir A. Jonasdottir A. Gretarsdottir S. Variant ASGR1 associated with a reduced risk of coronary artery disease N. Engl. J. Med. 374 2016 2131 2141 27192541
161 Xu Y. Tao J. Yu X. Wu Y. Chen Y. You K. Zhang J. Getachew A. Pan T. Zhuang Y. Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression JCI Insight 6 2021 e147038 10.1172/jci.insight.147038
162 Xie B. Shi X. Li Y. Xia B. Zhou J. Du M. Xing X. Bai L. Liu E. Alvarez F. Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans PLoS Genet. 17 2021 e1009891 10.1371/journal.pgen.1009891
163 Xiao X. Song B.-L. SREBP: a novel therapeutic target Acta Biochim. Biophys. Sin. 45 2013 2 10 10.1093/abbs/gms112 23257291
164 Zhu Y. Lin X. Zhou X. Prochownik E.V. Wang F. Li Y. Posttranslational control of lipogenesis in the tumor microenvironment J. Hematol. Oncol. 15 2022 120 10.1186/s13045-022-01340-1 36038892
165 Wang J.Q. Li L.L. Hu A. Deng G. Wei J. Li Y.F. Liu Y.B. Lu X.Y. Qiu Z.-P. Shi X.J. Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion Nature 608 2022 413 420 10.1038/s41586-022-05006-3 35922515
166 Braun C.J. Boztug K. Paruzynski A. Witzel M. Schwarzer A. Rothe M. Modlich U. Beier R. Göhring G. Steinemann D. Gene Therapy for Wiskott-Aldrich Syndrome—Long-Term Efficacy and Genotoxicity Sci. Transl. Med. 6 2014 227ra33 10.1126/scitranslmed.3007280
167 Hacein-Bey-Abina S. Von Kalle C. Schmidt M. McCormack M.P. Wulffraat N. Leboulch P. Lim A. Osborne C.S. Pawliuk R. Morillon E. LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1 Science 302 2003 415 419 10.1126/science.1088547 14564000
168 Manno C.S. Pierce G.F. Arruda V.R. Glader B. Ragni M. Rasko J.J. Ozelo M.C. Hoots K. Blatt P. Konkle B. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response Nat. Med. 12 2006 342 347 10.1038/nm1358 16474400
169 Mingozzi F. Meulenberg J.J. Hui D.J. Basner-Tschakarjan E. Hasbrouck N.C. Edmonson S.A. Hutnick N.A. Betts M.R. Kastelein J.J. Stroes E.S. High K.A. AAV-1–mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells Blood 114 2009 2077 2086 10.1182/blood-2008-07-167510 19506302
170 Suoranta T. Laham-Karam N. Ylä-Herttuala S. Strategies to improve safety profile of AAV vectors Front. Mol. Med. 2 2022 1054069 10.3389/fmmed.2022.1054069
171 Yin H. Kanasty R.L. Eltoukhy A.A. Vegas A.J. Dorkin J.R. Anderson D.G. Non-viral vectors for gene-based therapy Nat. Rev. Genet. 15 2014 541 555 10.1038/nrg3763 25022906
172 Wang C. Pan C. Yong H. Wang F. Bo T. Zhao Y. Ma B. He W. Li M. Emerging non-viral vectors for gene delivery J. Nanobiotechnol. 21 2023 272 10.1186/s12951-023-02044-5
173 Jackson A.L. Linsley P.S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application Nat. Rev. Drug Discov. 9 2010 57 67 10.1038/nrd3010 20043028
174 Crooke S.T. Molecular Mechanisms of Antisense Oligonucleotides Nucleic Acid Therapeut. 27 2017 70 77 10.1089/nat.2016.0656
175 Judge A.D. Sood V. Shaw J.R. Fang D. McClintock K. MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA Nat. Biotechnol. 23 2005 457 462 10.1038/nbt1081 15778705
176 Han G. Noh D. Lee H. Lee S. Kim S. Yoon H.Y. Lee S.H. Advances in mRNA therapeutics for cancer immunotherapy: From modification to delivery Adv. Drug Deliv. Rev. 199 2023 114973 10.1016/j.addr.2023.114973
177 Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Nat. Biotechnol. 31 2013 822 826 10.1038/nbt.2623 23792628
178 Enache O.M. Rendo V. Abdusamad M. Lam D. Davison D. Pal S. Currimjee N. Hess J. Pantel S. Nag A. Cas9 activates the p53 pathway and selects for p53-inactivating mutations Nat. Genet. 52 2020 662 668 10.1038/s41588-020-0623-4 32424350
179 Ihry R.J. Worringer K.A. Salick M.R. Frias E. Ho D. Theriault K. Kommineni S. Chen J. Sondey M. Ye C. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells Nat. Med. 24 2018 939 946 10.1038/s41591-018-0050-6 29892062
180 Grünewald J. Zhou R. Garcia S.P. Iyer S. Lareau C.A. Aryee M.J. Joung J.K. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors Nature 569 2019 433 437 10.1038/s41586-019-1161-z 30995674
181 Zhou C. Sun Y. Yan R. Liu Y. Zuo E. Gu C. Han L. Wei Y. Hu X. Zeng R. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis Nature 571 2019 275 278 10.1038/s41586-019-1314-0 31181567
