The promising novel therapies for familial hypercholesterolemia

Abstract

Background
The incidence of premature atherosclerotic cardiovascular disease in familial hypercholesterolemia (FH) is high. In recent years, novel therapeutic modalities have shown significant lipid‐lowering ability. In this paper, we summarize the recent developments in novel therapies for FH via the treatment of different targets and discuss the characteristics of each targeted therapy. Based on the process of protein synthesis, we attempt to summarize the direct‐effect targets including protein, RNA, and DNA.

Methods
For this systematic review, relevant studies are assessed by searching in several databases including PubMed, Web of Science, Scopus, and Google Scholar. The publications of original researches are considered for screening.

Results
Most drugs are protein‐targeted such as molecule‐based and monoclonal antibodies, including statins, ezetimibe, alirocumab, evolocumab, and evinacumab. Both antisense oligonucleotide (ASO) and small interfering RNA (siRNA) approaches, such as mipomersen, vupanorsen, inclisiran, and ARO‐ANG3, are designed to reduce the number of mRNA transcripts and then degrade proteins. DNA‐targeted therapies such as adeno‐associated virus or CRISPR–Cas9 modification could be used to deliver or edit genes to address a genetic deficiency and improve the related phenotype.

Conclusion
While the therapies based on different targets including protein, RNA, and DNA are on different stages of development, the mechanisms of these novel therapies may provide new ideas for precision medicine.

Full text

J Clin Lab Anal. 2022;36:e24552.  
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1 of 13
https://doi.org/10.1002/jcla.24552
wileyonlinelibrary.com/journal/jcla
1 | INTRODUCTION
Familial hypercholesterolemia (FH) is an autosomal codominant
genetic disease characterized by high serum levels of low- density
lipoprotein cholesterol (LDL- C).1 The disease is diagnosed by the
presence of mutations in low- density lipoprotein receptor (LDLR),
apolipoprotein (ApoB), or proprotein convertase subtilisin/kexin
9 (PC SK9).2 The LDLR variant is the most common cause of FH
and leads to the malfunction of the LDLR and a defect in the re-
moval of LDL- C from blood. Less common are pathogenic variants
in the ApoB gene, which encodes apolipoprotein B100 (ApoB100),
and these variants may be the predominant cause of 5%– 15% of
Received:20April2022
|
Revised:27May2022
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Accepted:28May2022
DOI: 10.1002/jcla.24552
REVIEW ESSAY
The promising novel therapies for familial
hypercholesterolemia
Ruoyu Chen1 | Shaoyi Lin2 | Xiaomin Chen2,3
This is an op en access ar ticle under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provide d the original work is properly cited.
© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.
1SchoolofMedicineofNingboUniver sity,
Ningbo,China
2TheAff iliatedNingboFirstHospital,
SchoolofMedicineofNingboUniver sity,
Ningbo,China
3NingboFirstHospitalAffiliatedtoSchool
ofMedicineofZhejiangUniversity,
Ningbo,China
Correspondence
XiaominChen,NingboFirstHospital
AffiliatedtoSchoolofMedicineof
ZhejiangUniversit y,Ningbo,Zhejiang,
China.
Email: chxmin@hotmail.com
Funding information
NaturalScienceFoun dationofZhejiang
Province,Grant/AwardNumber:
LQ20H020001;NingboHealthBranding
Subjec tFund,Grant/AwardNumber :
PPXK2018-01
Abstract
Background: The incidence of premature atherosclerotic cardiovascular disease in fa-
milial hypercholesterolemia (FH) is high. In recent years, novel therapeutic modalities
have shown significant lipid- lowering ability. In this paper, we summarize the recent
developments in novel therapies for FH via the treatment of different targets and
discuss the characteristics of each targeted therapy. Based on the process of protein
synthesis,weattempttosummarizethedirect-effecttargetsincludingprotein,RNA,
andDNA.
Methods: For this systematic review, relevant studies are assessed by searching in
several databasesincluding PubMed,Web of Science,Scopus,andGoogle Scholar.
The publications of original researches are considered for screening.
Results: Most drug s are protein-targeted su ch as molecule-bas ed and monoclonal
antibodies, including statins, ezetimibe, alirocumab, evolocumab, and evinacumab.
Bothantisenseoligonucleotide(ASO)andsmallinterferingRNA(siRNA)approaches,
suchasmipomersen,vupanorsen,inclisiran,andARO-ANG3,aredesignedtoreduce
thenumberofmRNAtranscriptsandthendegradeproteins.DNA-targetedtherapies
such as adeno- associated virus or CRISPR– Cas9 modification could be used to deliver
or edit genes to address a genetic deficiency and improve the related phenotype.
Conclusion: Whilethetherapies based ondifferent targetsincluding protein, RNA,
and DNA are on d ifferent stage s ofd evelopment, the me chanisms of these novel
therapies may provide new ideas for precision medicine.
KEYWORDS
familial hypercholesterolemia, gene therapy, lipidology, precision medicine

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FH cases, leading to reduced binding of LDL- C to LDLR. Gain- of-
function (GOF) mutations in the PC SK9 gene account for 1% of re-
ported FH cases, such mutations increase the destruction of LDLR
on the hepatocyte sur face and accordingly result in an increase in
levels of circulating LDL- C.3
The global morbidity rate of FH is between 1:200 and 1:300 with
limited therapies.4 Patients with Heterozygous FH (HeFH) in China
usually have a 3 .5- to 16- fold in creased risk of corona ry arter y disease
(CAD) in comparison with non- FH patients. If untreated, FH would
lead to premature atherosclerosis and an increased risk of cardiovas-
cular events.5 The decrease in LDL- C levels has been explicitly shown
to mitigate cardiovascular risk.6,7 The goal of the first- line treatments
is to limit cholesterol and saturated fats via a healthy way of living.8
However, lifestyle alone is insufficient for maintaining the normal av-
erage blood LDL- C levels in patients with FH. Consequently, the use
of lipid- lowering therapy is vital. The pharmacological options now
available include statins, bile acid sequestrants, fibrates, and choles-
terol absorption inhibitors.9,10 Although advances have been made in
potential therapies and treatment of plasma lipids shows improve-
ment, cer tain patients with FH fail to meet the treatment targets
for LDL- C.11 In addition, it is sometimes very difficult for traditional
pharmacotherapy to be ef fective for patients with homozygous FH
(HoFH), unless pharmacotherapy is coupled with LDL apheresis.
Although LDL apheresis or orthotopic liver transplantation has been
considered to be an optional therapeutic method for patients with
HoFH, the complex and risky operation, severely limited availability
of suitable donors, surgical risks, and perpetual use of immunosup-
pressive drugs after transplantation poses great challenges.12,13
Currently, the majority of clinical therapies for patients with FH
are small- molecule- or antibody- based approaches. It is important
to develop new lipid- lowering therapies with novel mechanisms of
action and favorable side effect profiles.14 We assume that the ex-
pansion of novel FH therapy options may have the potential to bring
about considerable economic and social benefits.
In this review, we propose novel therapies for FH according to
thedifferenttargets—proteins,mRNAs,andDNA.Inmolecularbiol-
ogy, such therapies depend on the different stages of central dogma:
the transcriptionofDNAinto mRNA and the translationof mRNA
into proteins.15
2 | PROTEIN- TARGETED THERAPEUTICS
In the clinic, the most commonly used drugs are small compounds.
These drugs interact with target proteins to influence biological
functions.
2.1 | Statins
Protein- targeted therapeutics such as statins can inhibit receptors
or enzymes in the cytoplasm or on the cytomembrane. Statins are
small- molecule lipid- lowering drugs that act on proteins in nanogram
to microgram.16
Statins are hydroxymethylglutaryl coenzyme A (3- hydroxy- 3-
methyl-glutaryl-CoA, HMG-CoA) reductase inhibitors. In clinical
practice, statins are the first- line treatments for patients with FH in
terms of pharmacotherapeutic management.17 Statins inter fere with
cholesterol biosynthesis by restricting the key step in the biosynthe-
sisofisoprenoidsandsterols,andthisprocessiscatalyzedbyHMG-
CoA.18 In a randomized controlled trial (RCT ) of patients with HeFH,
dailyadministrationof80-mgatorvastatinresulted,onaverage,ina
50% decrease in LDL- C levels from baseline. In addition to reducing
LDL- C levels, statins also mitigate the risk of cardiovascular disease
(CVD) and improve certain outcomes in prognosis.7,1 9
Although the options for lipid- lowering therapy have increased
markedly since statins were introduced for clinical applications in
1987,only10%–25% ofdiagnosedpatients havereceived appropri-
ate therapy, and many individuals have failed to achieve their LDL- C
goals.20 At present, most drugs interact with proteins, and they often
bind to nontarget proteins or produce adverse effects via unnoticed
interactions.21 Some patients are intolerant to statins and are easily
affected by side effects such as myalgias and weakness.22Further-
more, statins upregulate the expression of not only LDLR but also
PCSK9, which may limit the pharmacological ef fects of lowering
LDL- C levels. This is the “paradoxical effect” of statin management.23
2.2 | Ezetimibe
Studies show that adding other lowering agents to the statins may
further lower LDL- C with few side effects. In the clinic, the Food
and Drug Administration (FDA) approved the ezetimibe in 2002.
Ezetimibe, a cholesterol absorption inhibitor, is used as an adjunct to
statin forfurtherreduceLDL-C.ItworksbyblockingtheNiemann-
Pick C1-Like (NPC1L1) receptor, which is a multipass membrane
protein, expressed in the small intestine and liver. The NPC1L1
plays an important role in the process in cholesterol movement
into the enterocyte.24 Individuals with the non- GOF mutation of
the NPC1L1genehave lower levels ofLDL-Candacorresponding
risk of ASC VD.25 In addition, the ezetimibe upregulates LDLR in the
liver to reduce the circulating LDL- C; it is an independent mechanism
to statins.26Manyresearchexploredthe relationshipbetweenthe
additive effects of statins and ezetimibe. It provided an additional
23.4% reduction in LDL- C compared with single statins therapy.
What is more, the drug combination reduced high- sensitivity C-
reactive protein (CRP).27
Nowadays,whiletherehasundertreatmentofFH,mainlytreat-
ment priorities for drugs are statins, ezetimibe, and bile acid bind
resins.28
2.3 | Alirocumab and evolocumab
Protein- targeted therapeutics also include antibody- based PCSK9
inhibitor drugs such as alirocumab and evolocumab, which block
proteins in plasma at microgram to milligram.29 PCSK9 is an intrinsic
protein that modulates the amount of LDLR and is very impor tant in

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LDL-Cmetabolism.Moreover,PCSK9issecretedfromhepatocytes
into blood and binds to LDLR , which targets LDLR for lysosomal
degradation.30 The loss- of- function (LOF) mutation of PCSK9, which
was found to decrease LDL- C levels, reinforces the fact that PCSK9
could be a target for therapy.31 The two antibodies currently in use
against PCSK9 are fully human IgG subtypes that modify this path-
way by binding to circulating PCSK9 and preventing it from binding
LDLR.32 This process results in a lack of PCSK9 and leads to a large
accumulationofLDLRon the membranes oflivercells. Ultimately,
the removal of LDL particles is accelerated, and circulating LDL- C
levels are substantially reduced.33 Subcutaneous injection of PCSK9
inhibitors bound all newly secreted PCSK9 in the serum within hours
of administration, and the effect lasted for the next few days.34,35
However, this approach results in a large accumulation of the
compound in the blood, with an average 10- fold increase. For some
patients, the total concentrations would be increased 20- fold.36 As
part of the immune complex, clearance of PCSK9 may be slow, and
the return of circulating PCSK9 to the liver would be inhibited. This
may stimulate counterregulatory transcriptional pathways that en-
hance PCSK9 synthesis and secretion.
2.4 | Evinacumab
Traditional lipid- lowering therapies, such as statins and PCSK9 in-
hibitors, work by upregulating LDLR expression, but there is virtu-
ally no activity in individuals with two null alleles, such as those with
HoFH. Evinacumab is a monoclonal antibody against angiopoietin-
like protein 3 (ANGPTL3); it has received FDA approval in the
United States and can confer a potential benefitinHoFH.37 Both
ANGPLT3LOFvariantsandpharmacologicinhibitionofANGPTL3
can lower LDL- C levels without LDLR activity.37ANGPTL3isa
secreted hepatic glycoprotein that disrupts the clearance of cir-
culating LDL- C by inhibiting lipases such as endothelial lipase and
lipoprotein lipase, which hydrolyze triglycerides and phospho-
lipids in circulating lipoproteins.38 Genome- wide association and
exome sequencing studies have found correlations bet ween the
LOFgeneticvariantsofANGPTL3andareduction inplasmaLDL-
C, high- density lipoprotein cholesterol (HDL- C), and triglyceride
levels for cardiovascular prevention.39– 41 Prior to these findings,
acombinedhypolipidemia phenotype was observed inANGPTL3
knockout mice.42 In a phase 3 trial, the LDL- C level was decreased
from baseline in the evinacumab group compared with the placebo
group, which ledtoa 49.0%differenceinLDL-C levels anda47%
differ ence in triglyce ride levels bet ween the grou ps at 24 weeks,
regardless of the degree of their LDLR function.37 Evinacumab was
firstapprovedintheUSAonFebruary11,2021,assupplementary
therapy for LDL- C reduction for adult and pediatric patients with
HoFHwhowereolderthan12 years,andithasreceivedapositive
responseintheEuropeanUnion.38
However, the use of antibody- based drugs may create problems
such as patient compliance, problems with the injection technique,
and so on. For evinacumab, the main side effects include nasophar-
yngitis, influenza- like illness, headache, and infusion- site pruritus.37
Therefore, more options are needed because of the interactions and
mechanisms of these drugs.
In summar y, discrimination between the primary t argets and off-
targets is necessar y in the development of protein- targeted drugs.
The related phenotypes, including the curative effects and side ef-
fects, are dependent on the activation or inhibition of target pro-
teins to a large extent.
3 | RNA- TARGETED THERAPEUTICS
Small molecules that affect proteins by inhibiting enzyme function
or receptor activity have no therapeutic effec t on proteins without
enzyme activity. In addition, it may be difficult to achieve adequate
concentrations of humanized monoclonal antibodies in circulation
because of tolerance and cost considerations.43
RNA-targeted therapeutics seemtobe anovelandelegantap-
proach. It can act to regulate genes by directly targeting the nucleic
acids that encode the proteins by interfering with the translation of
mRNAintoprotein.Furthermore,RNA-targetedtherapeutics,which
represent a drug discovery platform involving oligonucleotides,
are akin to small- molecule therapeutics and include small interfer-
ing RNA (siRNA), antisense oligonucleotide (ASO) (Figures 1 and
2), oligonucleotide-induced alternative splicing, anti-miRs, miRNA
mimics,andmRNAupregulation.44 They are synthetic and small, do
not integrate into the host genome, and have limited duration and
activity.45
ASOsorsiRNAcanbindtothemRNAofdisease-causinggenes
and block their activation.46,47 Notably, N-acetylgalactosamine
(GalNAc) modification ofASOs orsiRNAbytheasialoglycoprotein
receptor could facilitate selective uptake into hepatocytes and
FIGURE 1 Antisenseoligonucleotide-basedapproaches:ASO
utilizesasingle-strandedRNaseHmechanism

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ultimately significantly increase hepatic uptake.48 Therefore, these
approachescouldpreciselyandselectivelymatchthespecificmRNA
intargetedorgansorcells.Ingeneral, thenewgenerationof RNA-
targeted drugs has enhanced potency, thereby reducing plasma drug
levels, improving efficacy, and possibly reducing side effects.49
3.1 | Antisense oligonucleotides (ASOs)
Sincethe1970s,ASOshavefrequentlybeenusedforsilencinggene
expressionandcanlowerthelevelsofspecificmRNAsinasafeand
selective manner.50 ASOs are single- stranded molecules composed
ofmodifiedDNA.ThemiddlebasesareDNA,whilethoseoneither
sideareusuallymodifiedDNAthat hasbeencorrected, often con-
taining 2′methoxyethyl.Anunspecifiednumberofphosphorothio-
ate moieties often replace the phosphodiester backbone, while the
nucleic acid bases are not modified. The chemical modifications con-
tribute to increased plasma stability and high affinity and binding
tomRNA. Thisisthe keyreasonthatASOscanbeused asadrug,
unlike unmodified native oligonucleotides, which degrade promptly
in circulation.43 After subcutaneous injection, ASOs bind to circulat-
ing proteins, reach the liver, enter the cells, and then reach the nu-
clear membranes for ef ficacy. Because of their amphipathicity, ASOs
can be dissolved in normal saline and be injected subcutaneously
with no carrier. The basic premise of the use of ASOs is the abil-
ity to administer shor t- and single- stranded nucleic acid sequences
that bindtocomplementar yRNAsubstrates. Then, the ASOs bind
tospecificsensemRNAsincellswithinthenucleusorcytoplasmby
Watson– Crick base pairing; finally, the resulting complex competi-
tively inhibits translation or is degraded (Figure 1).51 ,52 ASOs have
rapid plasma uptake and a short distribution life (<1 h) but have a
relatively long intracellular life (2–4 weeks), consequently having
a long- lasting effect. They are then cleaved to short fragments by
exonucleases and endonucleases.53 Second- generation ASOs are
composedofsequence-specificduplexesofthe targetmRNA.The
duplexesserveasasubstrateforRNaseH,whichisanendogenous
mechani sm in cells for cleavi ng ASO-bound RN A, result ing in the
degradation of target mRNA and ultimately in a decrease in the
translation of the corresponding protein.54, 55
3.1.1 | ApoB100-specificantisense
oligonucleotides:Mipomersen
Mipomersen(Kynamro)isasecond-generation20-nucleotideASO
that binds to A poB100 mRNA in t he liver and cr eates a subst rate
for intrahepatic endonucleases. Then, the transcripts of ApoB100
are degraded, thereby reducing plasma LDL- C concentrations.56
ApoB100 is o ne of two isoforms fro m the RNA transc ript of the
human ApoB gene, which has great importance in lipoprotein me-
tabolism. And, ApoB100 is a vital structural component of ver y
low- density lipoprotein ( VLDL) and LDL, which are synthesized by
the human liver. In the early phase of atherosclerosis, dysfunctional
arterial endothelial cells allow LDL to enter the endothelial layer
and then adhere to intimal proteoglycans by way of ApoB, where it
accumulates gradually in the intimal layer.57,5 8 The genetic basis of
non- ApoB family hypobetalipoproteinemia (FHBL) suggests the pos-
sibility of ApoB as an attractive target for lipid- lowering therapy.59
FHBL is an autosomal dominant disease characterized by low LDL- C
and ApoB levels, which may be due to the mutation of ApoB.59
Mipomersenincludes10 2′-O-methoxyethylmodifications,and
there are five modifications at each end of the oligonucleotide that
increase the affinit y of target binding, prolong the half- life in the
target organ, and resist degradation by nucleases.60 In the center,
the phosphorothioate nucleotides promote RNase H-mediated
cleavage.55 The plasma concentrations of mipomersen peak within
4 h after ad ministrati on. Mipomer sen is metabo lized in tissues as
a chain- shortened metabolite, which is initially accomplished by
endonucleases and produces oligonucleotides that have no further
pharmacological effects. The smaller oligonucleotides are further di-
gested by exonucleases and excreted mainly in the urine.61 Studies
have shown that in healthy adults, mipomersen could increase the
fractional catabolic rate of ApoB in both VLDL and LDL, thereby
decreasing the levels of lipoproteins that contain ApoB.62 Phase I
and II studies showed that mipomersen produced dose- dependent
decreases in all ApoB- containing lipoproteins. In phase III trials, mi-
pomersen was effective in patient s with HoFH and patients with
HeFH.63 In a pri mary phas e III study, patien ts older tha n 12 years
who were clinically diagnosed with or genetically confirmed to have
HoFH received the maximum tolerated dose of a lipid- lowering
drug andwereassignedtoreceivemipomersen 200 mg or placebo
FIGURE 2 siRNA-basedapproaches:siRNAtechnologyemploys
a double- stranded RISC mechanism

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CHEN Et al .
weekl y.64LDL-Clevelsdecreasedbyameanpercentageof24.7%in
all subjec ts after 6 months.65 In randomized trials and the open- label
extension phase, long- term mipomersen treatment of FH reduced
the levels of atherogenic lipoproteins and led to a decrease in car-
diovascular events.66
However, mipomersen is approved only for HoFH, and the re-
sponse varies widely among patients. Loss of ApoB is also associated
with on- target adverse effects such as liver steatosis, possibly be-
cause all ApoB- containing lipoproteins are targeted and there may
be no mechanism by which the liver clears excess fat. The use of
mipomersen is complicated by its side effects. For instance, hepato-
toxicity, injection- related side effects, hepatic fat, and the high cost
of the medication are also limiting factors.67
3.1.2 | ANGPTL3-specificantisense
oligonucleotides: Vupanorsen
ANGPTL3has been assessed asapotentialtargetfor loweringthe
levels of LDL- C and triglycerides for CVD prevention.68 Vupanorsen
(AKCEA-ANGPTL3-LRx) is an N-acetyl galactosamine-conjugated
(GalNAc3-modified)ASOtargetedtotheliverthatselectivelyinhib-
itsthesynthesisoftheANGPTL3protein.GalNAc3-modifiedASOs
can target the ASO to the asialoglycoprotein receptor on liver cells,
and it has a treatment effect similar to that of unconjugated ASOs,
although at a 20– 30- fold lower dose. Therefore, it could reduce
systemic exposure risk and be administered at significantly lower
doses and intervals.69 In a phase 2 study, 105 patients had fasting
triglycerides >150 mg/dl(>1.7 mmol/L),type2diabetes,andhepatic
steatosis at baseline. The patients received vupanorsen for 6 months
bysubcutaneousinjectionof40or80 mgmonthlyor20 mgweekly.
The vupanorsen groups achieved a statistically significant dose-
dependent reduction in triglyceride levels (44%) compared with
the placebo group, but no changes in liver fat or HbA1c were ob-
served.Inaddition,thereductionsinapolipoproteinC-III(58%),re-
sidualcholesterol(38%),totalcholesterol(19%),non-HDL-C(18%),
HDL-C(24%),apolipoproteinB(9%),andLDL-C(7%)werealsodose
dependent, and the frequent side effects were generally mild and
occurred at the injection site.70
Vupanorsen is also distinct from evinacumab, which is a mono-
clonal ant ibody targe ted to ANGPTL 3. Vupanorsen a cts in liver
cells, but evinacumab acts in plasma. Whether this would cause
a difference in effect is still not clear. Evinacumab appears to
be deficient regarding compliance, cost, and an inability to self-
administer.Thisisduetothehighintravenousdosages(15–20 mg/
kg) or subcutaneous injections required each week.71 An increas-
ing number of epidemiologic, genetic, and genome- wide associ-
ation stu dies suggest tha t reducing plasma AN GPTL3 levels by
inhibitinghepaticANGPTL3synthesiswillbenefitapolipoprotein
B levels and improve the metabolic measurements related to dys-
lipidemia and atherosclerosis.72 There was no correlation of the
effect with the concentration; rather, it is related only to complete
deficiency.73 Patients who are homozygous for LOF mutations in
the ANGP TL3 gene showed a dditional met abolic benef its, such
as lower circulating free fatty acid levels and better insulin sen-
sitivit y. In addition, vupanorsen could reduce ApoC- III levels in
respons e to the reductio n in ANGPTL3 leve ls. It is well known
that triglyceride levels may be related, but vupanorsen reduces
plasmatriglyceridesby targetinghepaticANGPTL3independent
of apoC- III.74
According to available studies, the adverse effects of vupan-
orsen are generally mild and include flu- like reactions and adverse
effects at the injection site.70 Further studies are needed to explore
the efficacy and safety of different doses of vupanorsen to maximize
target engagement in a population of individuals with elevated non-
HDL- C and triglyceride levels receiving statin treatment . In conclu-
sion, the reduction in triglyceride and atherogenic triglyceride- rich
lipoprotein levels by vupanorsen may suggest a novel strategy for
decreasing the CV risk for patients with FH.
3.2 | Small interfering RNA(siRNA) molecules
Small interferingRNA(siRNA)issmalldouble-strandedRNAmol-
ecules that are critical regulators of eukaryotic genome expression
and function. siRNA post-transcriptionally influences the mRNA
of target genes, ultimately degrading them and preventing trans-
lation.75TheydegradetargetmRNAbybindingwiththeRNA-
induced silencing complex (RISC), after which the antisense strand
ofsiRNAisloadedintotheRISCthroughWatson–Crickbasepair-
ing for seq uence-specif ic cleavage of th e mRNA.76 The RISC has
endonucleaseactivityto specificmRNA ,andthenaftercleavage,
thecellularexonucleasesdegradetheresultingmRNAfragments.77
Thecomplexof thesiRNAstrandandRISCcanthenbereusedto
targetothercomplementarymRNAs,therebyprovidingmoretran-
scriptsfor RNA silencingandmaintaining a long durationof effi-
cacy for an extended period of several months. (Figure 2) s iRNA
has activity in somatic and germline lineages of various eukaryotic
species.Small noncodingRNA regulatesvariousimportantstages
of genome function. Some examples are chromatin, chromosomal
segregation, transcription, RNA processing, RNA stability, and
translation.46 These molecules are also involved in protecting the
genome from invasive nucleic acids.78 In most mammalian cells, long
dou ble-s tra ndedRNAcannotbeusedtosi lenceasp ecificgenebe-
cause it provokes the interferon response to defend against viruses
and then prompts the shutdown of protein synthesis. In contrast,
siR NAc ane vad etheinterferonresponseinm amm alsandgenerate
specific and efficient gene silencing.79
3.2.1 | Inclisiran(ALN-PCSscPCSK9)
As shown in previous studies, PCSK9 inhibitors, such as evolocumab
and alirocumab, have become promising new therapeutic ap-
proachesforLDL-Creduction.Inrecentyears,siRNAhasalsobeen
used to inhibit PCSK9.80

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Inclisiran(ALN-PCSsc PCSK9) isalong-acting,synthetic siRNA
moleculethattargetsmRNA ofPCSK9tocounteractitandreduce
the production of intracellular PC SK9, resulting in a durable and sub-
stantial reduction in LDL- C levels.81,82 The inclisiran molecule con-
sists ofaguidestrandandpassengerstrand.Thetwo RNAstrands
are complementary. Once inclisiran molecules are incorporated into
hepatocytes, the guide strand binds the RISC and hybridizes with
thecomplementarymRNAofPCSK9,causingdegradation.83 Finall y,
translation of the PCSK9 protein would be limited, resulting in a re-
duction in serum LDL- C levels.84Notably,aspreviouslymentioned,
when degradation of the transcript is initiated, the complex is deliv-
ered to the liver where it remains active for a long period, interfering
wi thmor em RN A .85 In phase 2 and pha se 3 trials, inclisi ran was found
to decrease LDL- C levels by approximately 50% when administered
subcutaneously ever y 6 months. Inclisiran was well tolerated, and
there were no severe adverse events; however, injection- site side
effects were common compared to placebo (2.6% vs. 0.9% in the
ORION-10 trialand4.7% vs.0.5%in theORION-11trial),although
they were basically mild and did not last long.82 Interestingly, pa-
tients with diabetes showed no additional effects after treatment
withPCSK9-targetedsiRNA-drivenstrategies.86 In December 2020,
inclisiranwasfirstapprovedintheEUforuseinadultpatientswith
primary hypercholesterolemia, including HeFH and nonfamilial hy-
percholesterolemia or mixed dyslipidemia, by subcutaneous injec-
tion twice yearly as a dietary supplement. Inclisiran is intended to be
used in combination with statins or with statins combined with other
lipid- lowering treatments in patients who cannot meet LDL- C tar-
gets on the maximum tolerated statin doses. Inclisiran can be used
separately or in conjunc tion with other approaches for patients who
have statin intolerance or contraindications.87
3.2.2 | ARO-ANG3(ANGPTL3mRNA)
PreviousresultsindicatethatANGPTL3isanotablenewtherapeutic
target .Inadditiontomonocl onalantibodiesandA SOs ,siR NAthera-
piestargetingANGPTL3arealsobeingdeveloped.
The siRNA ANGPTL3 mRNA (ARO-ANG3) is a GalNAc3-
conjugated siRNA that effectively and persistently inhibits the
transcription ofANGPTL3 mRNAin liver cells.ThephaseI/IIclin-
icalstudyofARO-ANG3 (NCT03747224)isongoing.Inthecontrol
group, ARO -ANG3 tre atment for 16 weeks at do ses of 100, 200,
and 30 0 mg injected sub cutaneously r educed circulati ng levels of
ANGPT L3 by 96%, triglycerid es by 72%, and LDL-C b y 50%.88 In
the FH grou p, ARO-A NG3 reduce d ANGPTL3 l evels by 62%–92%
at week 16 in a dose- dependent manner. LDL- C levels were reduced
by23%–37%,andTGlevelswerereducedby25%–43%atdosesof
100,200,and300 mginjectedsubcutaneously.Fornon-FHpatients,
ANGPTL3levelswerereducedby85%,and LDL-Clevelswerere-
ducedby28%,comparabletothosepatient swithFH.
AsofMay2020,nodrug-relatedsideeffectsordiscontinuations
werefound.Mostadverseeventsweremild.Themostcommonad-
verseeventsofARO-ANG3treatmentarerespiratorytractinfection
(RTI) in 30% of participants and adverse events at the injection site
in 13% of participants.89
In general, whether the aforementioned siRNA treatment
method would be an effective approach for targeting ANGPTL3
needs further observation.
4 | DNA- TARGETED THERAPEUTICS
AlthoughRNA-targetedtherapiesareusedselectivelytosilenceand
interferewithgeneexpression,DNA-targetedtherapyisusuallyin-
tended to achieve the opposite effect. This technique involves intro-
ducing functional gene copies to restore the function of a mutated
gene.
Accordingtopreviousstudies,certainDNAsequencesofagene
have been observed to change lipoprotein levels, such as the LDLR
variant, which is the most common cause of FH.90 Some kinds of
variants are considered pathogenic, such as missense and nonsense
variants, insertion and deletion mutations, splicing mutations, and
large-scale DNA copy number variation (CNV).91 LDLR mutations
could influence various processes involved in LDL- C uptake and
metabolism and are the key driver of LDL- C clearance without sub-
stitutes in vivo.92Approximately 75%of human LDLR isexpressed
in the liver and determines LDL- C removal from the bloodstream.93
Patients with HoFH have biallelic LOF mutations in LDLR, and or-
thotopic liver transplantation effectively resolves the HoFH pheno-
type; however, the adverse ef fects and risks of transplantation and
long- term immunosuppression are limitations.94 Consequently, pa-
tients with HoFH are considered ideal candidates for gene delivery
and mutation correction.
Accumulating evidence supports the concept that overexpres-
sion of a corrected copy of a gene with an LOF mutation, such as
LDLR, by retrovirus,13 adenovirus, or adeno- associated virus (AAV)
can effectively ameliorate the phenotype.94 The first clinical trial
of gene replacement therapy was performed in five patients with
HoFH and used a recombinant murine retroviral vector that con-
tained LDLR cDNAtotransfect thecellsfromresectedlivertissue
ex vivo, after which the genetically modified cells were injected into
patients through a por tal vein catheter.13 Although the process is
safe and well tolerated, the challenges of the technique and inva-
siveness are present, and the inefficiency of the genetic recombina-
tion of retroviral vectors is the major barrier causing relatively low
ef fic ac y.95 Since then, in vivo preclinical research has star ted to ex-
plore efficient and safe vectors for delivering genetic cargo directly
to liver cells.96 Various methods for gene delivery have been studied
in different kinds of animal models of hypercholesterolemia with
varying degrees of success.97
AAV vectors have low immunogenicity and no pathogenicity in
humans, so AAV vectors could be suitable for gene delivery therapy.
AAV is a branch of the Parvoviridae family, and it has different se-
rotypes with different organ- specific tropisms.98,99Morethan90%
of LDL- C catabolism is mediated by LDLR in the liver. Thus, AAV se-
rotype8(AAV8)hasbeendeveloped,whichisahighlyhepatotropic

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vector.ArecombinantlivertropicAAV8vectornamedAAV8.TBG.
hLDLR is under development. This vector carries a human wild- type
(WT ) LDLR transgene under the control of a liver- specific promoter
called thyroxin- binding globulin. Recombinant LDLR- expressing
AAV8vectors have high efficiencyingenetransfer andstable he-
patocytetransductionforupto180 days(comparedtoothertissues
in which transduction is 1000- fold higher), and they significantly
improve lipid levels. In HeFH or HoFH humanized mouse animal
models,AAV8genetherapyleadssignificantreductionintotal and
LDL- C with safety.100 Studies have shown the efficiency of AAV vec-
tor transduction in human hepatocytes is lower than that in murine
hepatocytes.101Nonetheless,inthetreatmentofhemophiliaB,re-
combinantFIX-expressingAAV8inducedsignificantlyhigherserum
FIX levels inmicethan inhuman patients.Nevertheless,despite a
reconversionrateofonly1%–7% inpatientswithhemophilia com-
pared to individuals with a normal level, this vector could show sig-
nificantclinicalefficacythatlastedmorethan 3 years.102 The case
ofAAV8isrelatedtoitsusein HoFH.Thisis becauseHoFHindi-
viduals carrying LDLR variants with residual LDLR activity greater
than 2% (LDLR- defective) have a much better prognosis than indi-
viduals with LDLR variants with residual LDLR activity <2% (LDLR-
negative).103 According to the preclinical studies in preparation for a
phase 1clinicaltrial, AAV8.TBG.hLDLRhadgood safety.104,105 The
phase I and IItrials ofAAV-basedliver-directedA AV8.TBG.hLDLR
aiming to assess the safety and effic acy of the treatment for HoFH
are currently underway (NCT02651675; AAV8-mediated LDLR
Gene Replacement in Subjects With HoFH). Subjects in that study
arefollowedfor5 years.TheFDAhasapprovedtwoA AV-mediated
therapies. Other research in this area will allow further treatment
applications to be developed, and the results of the trials for HoFH
will generate new ideas for treating FH.
Genome editing can also permanently repair existing genes in-
stead of introducing a WT gene with gene replacement therapy.
Recently, treatment strategies involving the clustered- regularly
interspaced- short- palindromic- repeat/CRISPR- associated gene 9
(CRISPR– Cas9) system, an efficient gene- editing complex, have
begun to appear. TheRNA-guidednuclease cleavesthestrands of
DNAatthetargetsite.Then,theCRISPR–Cas9systemprovidesen-
gineereddonorDNAforinsertionintothetargetsitewhenthecell's
standardDNArepairmechanismtriestoinitiateDNArepair.106
CRISPR/Cas9 therapy has been explored for most genes associ-
ated with the regulation of lipid homeostasis and the genes targeted
in RNA therapeutics. Although gene editingmay not be poisedto
be adopted in clinical practice in the foreseeable future, achieve-
ments with this technique in animal models are notable.107,108 The
first HeFH mouse model created by in vivo somatic gene editing,
whichdoesnotpassmutationstothenextgeneration,usedA AV8-
delivered CRISPR/Cas9 and disrupted the LDLR gene in mice within
6 weeks, resultingin severe hypercholesterolemiaand atheroscle-
rosis.109Moreover,geneeditingofPCSK9byCRISPR–Cas9isac-
complished in animals through different delivery methods and has
revealed a clinically meaningful reduction of >30% in LDL- C levels.
This finding seems to suggest that permanent silencing of disease-
causing genes has tremendous therapeutic potential.110,111 A vari-
ant of CRISPR–Cas9 na med base editi ng does not nee d the DNA
double-strandbreak.InLDLR(−/−)mice,thismethodisusedtodis-
rupttheANGPTL3gene,andthelipidlevelsofthemicewerelargely
reduced without off- target mutagenesis.111
In another trial using the CRISPR– Cas9 system, T cells from
HoFH individuals were effectively reprogrammed, and then plu-
ripotent stem cells were induced.112 The study showed that LDLRs
are present in the cell membrane, providing a potential therapeu-
tic approach for FH. Another study described an ef ficient approach
for the simultaneous base editing and reprogramming of fibroblasts
using a CRISPR– Cas9 adenine base.113 In this study, the approach
generated gene- edited human- induced pluripotent stem cells iso-
lated from the skin biopsies of patients carrying pathogenic point
mutations and showed restoration of LDLR activity after gene mod-
ification. The approach is presented as being highly efficient while
significantly reducing the time of the in vitro cell culture, thereby
reducing the risk of in vitro changes. This may provide a solid basis
for further experiments in vivo.
In recent studies, somatic cell gene editing has been performed
in vivo by using the CRISPR/Cas9 system transduced via A AV to
treat FH induced by the LDLR mutation using germline editing in
mouse models.114 In this study, AAV- CRISPR /Cas9 was used to mod-
ify the point mutation in hepatocy tes and was delivered subcutane-
ouslyintoLDLRE208XmicecarryinganFH-relatedgenemutation.
Studies have shown that AAV- CRISPR/Cas9- mediated homologous
directed repair of LDLR gene correction can partly salvage LDLR
gene expression in vivo and efficiently improve atherosclerosis
phenotypes such as cholesterol, triglyceride, and LDL- C levels.114
Therefore, we can infer that the CRISPR– Cas9 system may play an
important role in the recovery of LDLR mutations or defect in many
othergenes,includingPCSK9,ApoB,andANGPTL-3,andwouldbea
broadly useful therapeutic approach for FH.
5 | CONCLUSIONS AND PERSPECTIVES
Despite significant advances in the treatment of FH, compound
HeFH and HoFH remain serious genetic disorders that affect the
human qua lity of life. For thes e patients, convent ional treatment may
not be very effective. Residual LDLR activity is critical for a thera-
peutic response, so restoration of LDLR activit y or achievement of
an obvious lipid- lowering effect is a challenging task. Furthermore,
novel therapies with mechanisms of action independent of this
pathway are being explored. Gene- based treatments offer great
potential and possibilities to regulate key points in lipid metabolism
withhighspecificityand efficiency.Noveltherapiesprovidemeth-
ods to address hitherto undruggable targets. While the long- term
safety and efficacy of such strategies still need more exploration,
these novel platforms are in different phases of clinical development
(Tables 1 and 2), which may trigger a paradigm shift in the treatment

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TABLE 1 NovelandemergingplatformsforFH
Classify (platform) Name Tar ge t Mechanism of actin Biochemical effect Stage Dose
Protein
Enzyme and substrate Statin HMG-CoA HMG-CoAcompetitive
combination
Reduce LDL- C Approved daily po
Enzyme and substrate Ezetimibe NPC1L1 NPC1L1inhibition Reduce LDL- C and hs- CRP combined statins Approved daily po
Antigen antibody Alirocumab, evolocumab PCSK9 Antigen antibody
reaction
Reduce LDL- C and Lp(a) Approved sc, once or twice monthly
Antigen antibody Evinacumab ANGPTL3mAb Antigen antibody
reaction
Reduce LDL- C and apolipoprotein B Approved 15 mg/kgivmonthly
RNA
ASO Mipomersen(Kynamro) APOB Anti- APOB antisense Reduce LDL- C Approved 200 mgsconceweekly
ASO Vupanorsen(ANGPTL3-LRx) ANGPTL3 Anti-ANGPTL3
antisense
Reduce TG, non- HDL- C Phases II 40-80 mgscmonthly
siRNA Inclisiran Anti- PCSK9
siRNA
PCSK9mRNA
degradation
Reduces LDL- C Approved 300 mgsct wiceyearly
siRNA ARO-ANG3 ANGPTL3 ANGPTL3mRNA
degradation
Reduces LDL- C phases I On study
DNA
AAV 8 AAV8.TBG.hLDLR(RGX-501)
RegenXbio, RGX- 501
LDLR LDLR gene therapy Reduces LDL- C Phases I
and II
Single iv infusion
CRI SPR/Cas9 /ANGPTL3/PCSK9 PCSK9 gene CRISPR/
Cas9 editing
/Preclinical /
Abbreviations: iv, intravenous; po, per os; sc, subcutaneous.

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TABLE 2 TheselectedandlatestclinicaltrialsofnovelandemergingplatformsforFH
Name Clinical trials Population Duration Treatment arms Primary endpoint Effic acy Safety
Alirocumab ODYSSEY HoFH
(NCT03156621)123
HoFH subjects
(N = 69)
12 weeks 150 mgevery2 weeks Percent change
in LDL- C from
baseline
LDL-C(−35.6%) Similar to placebo
Evolocumab OSLER-1(NCT01439880)124 FH subjects
(N = 1255)
1 year and
additional
4 years
420 mgmonthly Percent change
in LDL- C from
baseline
LDL-C(−56%,57%,
56%, 56%)
Similartoplacebo,5.7%
discontinuation
Mipomersen
(Kynamro)
NCT0 060737366 HoFH subjects aged
12 yearsand
older (N = 51)
26 weeks 20 0 mgweekly Percent change
in LDL- C from
baseline
LDL-C(−24.7%) Injection- site reaction
(76%vs24%inplaco)
Vupanorsen
(ANGPTL3-
LRx)
TRANSLATE–TIMI70
(NCT04516291)125
Non-
HDL-C ≥ 100 mg/
dl and
triglycerides 150
to500 mg/dlon
statin therapy
(N = 51)
24 weeks 80 mg,120 mg,or
160 mgevery
4 weeks,or60 mg,
80 mg,120 mg,
or160 mgever y
2 weeks
Percent change in
non– HDL- C from
baseline
Non–HDL-C22.0%
60 mg2weekly,
27.7%80 mg2
weekly
Injection- site reaction
(33.3%), ALT or AST
>3×ULN(44.4%)
Inclisiran ORION-1082 L D L - C > 1.8 mmol/L
(N = 1561 )
540 days 28 4 mgonday1,day
90, and every
6 months over a
periodof5 40 days
Percent change
in LDL- C from
baseline
LDL-C(−52.3%) Injection- site adverse
events (2.6% vs. 0.9%
in placo)
Inclisiran ORION-1182 ASCVD or A SCVD
risk equivalent
(N =1617 )
540 days 28 4 mgonday1,day
90, and every
6 months over a
periodof5 40 days
Percent change
in LDL- C from
baseline
LDL-C(−49.9%) Injection- site adverse
events(4.7%vs.0.5%
in placo)
ARO-ANG3 NCT03747 2241 26 Healthy and
dyslipidemia
individuals
(N = 93)
16 weeks 100 mg,200 mg,
300 mg
/ANGPTL3(−96%),
TG(−72%),LDL-C
(−50 %)
Similar to placebo

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of patients with FH, especially those who suffer from the severe
clinical symptoms of HoFH or are intolerant to conventional lipid-
lowering treatment.
Monoclo nal antibodie s against PC SK9 are a landmar k therapy
for patients with FH. They are combined with other lipid- lowering
treatment approaches to lower LDL- C levels, and less frequent dos-
ing provides greater convenience. In addition to PCSK9 suppression
based on antibodies, a variety of RNA-focused approaches have
been attempted. Gene- targeted treatments specifically intervene at
the transcriptional level, for instance, by impeding the produc tion
ofproteinsvia siRNA andASOsat themRNAlevel.If theDNAse-
quence is clearly noted to af fect the level of a lipoprotein that causes
certa in disorders , it may be a target fo r an ASO or siRNA , which
willintegratewiththemRNAofthespecificgene.Smallmolecules
are usually found in plasma and cells at similar concentrations, while
antibodies are found only in plasma and other extracellular compart-
ments.ASOsandsiRNAs are designedto reachhigher concentra-
tions in liver cells than in plasma.49
One of the important advantages of ASOs in comparison with
siRNA is th e markedly hig her affin ity for the t argeted mo lecule.80
ASOs presumablyactin thenucleus, whiletheactivityofsiRNAis
confined to the cytoplasm,115 which may decrease off- target hy-
bridization and side effects. However, due to the relatively short
duration of exposure in most clinical trials, the long- term effec ts of
ASOsandsiRNAsarebasicallyunknown.Thus,moreinvestigations
are needed.
In contrast to protein- targeted therapy, gene- based therapy is
sometimes not feasible. Early trials with ASOs targeting PCSK9 were
terminated due to renal tubular toxicity.116 Another option is to in-
crease the production of a specific protein by introducing specific
coding sequences such as LDLR via gene editing.
CRISPR/Cas9 is a very versatile approach with some contro-
versies. This technique produces double- strand breaks, which are
usually restored by nonhomologous end joining but are just as
likely to be repaired by faulty repair strategies.117 Alternatively, the
homology- directed repair is accomplished by careful nucleotide se-
quencereconstructionwith homologouschromosomal DNAorby
exogenous s ingle-stran d template DNA to i ntroduce a spe cific in-
corporation sequence.118 In addition, it is vital to distinguish somatic
gene edit ing and germline ge ne editing. In 2018 , the birth of th e
world's first twingirlsto havetheirgermcellseditedcausedshock
and outrage around the world.119 Germline editing refers to mak-
ing changes to the genome in the embryonic stage, which would be
passed on to future generations. This approach may create troubling
ethical issues.117
One alternative, exosomes, are intracellular vesicles that play a
significant role in intercellular communication.120 Studies have grad-
ually shown that exosomes may be promising treatment vectors,
andtheycaneffectivelyconveymRNA,miRNA,andplasmidDNAto
specific cells. Intracellular vesicles are naturally nanosized and easy
to use and have no cytotoxicit y or immunogenicity compared with
viruses.121TheycouldencapsulateabundantLDLRmRNAandex-
press LDLR in donor liver cells. In the recipient cells, encapsulated
mRNAissta bil izedand fun ctional.M ore over,thephenot ypesofani-
mal models may eventually be reversed using exosomes.122
In summar y, it is plausible to postulate that whether gene- based
therapeutics inhibit or enhance the targeted gene, appropriate drug
affinity, and resistance to nucleases are the key points that need to
be addressed and may help improve the efficiency and frequency
of medication, which requires further research and development in
chemical therapy. This would be an important step toward the prog-
ress of precision medicine. While novel therapies have entered clini-
cal use, problems such as high cost and the specific needs of various
subpopulations may arise. For rare and devastating diseases, gene
therapy may play a decisive therapeutic role. However, there are sig-
nificant ethical challenges related to the abuse of gene- modifying
techniques. As an example, the use of CRISPR/Cas9 technology for
the deliberate editing of human embryos caused much controversy.
This reminds us that we should remain vigilant to ensure the smooth
development of gene therapy in a scientific and ethical manner. To
sum up, it indicates that further research on different therapeutics
for different targets will be helpful in providing new ideas for the
individual treatment of various diseases.
CONFLICT OF INTEREST
We do not have any conflict of interest.
DATA AVAIL ABILI TY STATEMENT
Data sharing is not applicable to this article as no datasets were gen-
erated or analyzed during the current study.
ORCID
Ruoyu Chen https://orcid.org/0000-0002-5210-7702
Xiaomin Chen https://orcid.org/0000-0003-1498-0452
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