Recent progress in gene therapy for familial hypercholesterolemia treatment

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

Introduction

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 adjustments^9,10,11 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.²⁹

Figure 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.^52,53

Table 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

^aADA-SCID: severe combined immunodeficiency due to adenosine deaminase deficiency.

^bLCA: Leber Congenital Amaurosis.

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

^dCALD: cerebral adrenoleukodystrophy.

^eHSC: 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.^70,71,72

Table 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

^aFCS: Familial Chylomicronemia Syndrome.

^bCVD: 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).¹³⁴

Table 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

^aNHPs: 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.¹⁵⁸

Figure 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.