International Journal of

Molecular Sciences
Review

Candidates for Intra-Articular Administration Therapeutics andTherapies of Osteoarthritis
Eriko Toyoda 1,2 , Miki Maehara 1,2 , Masahiko Watanabe 1,212*

Citation: Toyoda, E.; Maehara, M.;

and Masato Sato 1,2, *Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya,
Isehara 259-1193, Japan; etoyoda@tokai-u.jp (E.T.); m-maehara@tsc.u-tokai.ac.jp (M.M.);
masahiko@is.icc.u-tokai.ac.jp (M.W.)
Center for Musculoskeletal Innovative Research and Advancement (C-MiRA),
Tokai University Graduate School of Medicine, 143 Shimokasuya, Isehara 259-1193, Japan
Correspondence: sato-m@is.icc.u-tokai.ac.jp; Tel.: +81-463-93-1121; Fax: +81-463-96-4404

Abstract: Osteoarthritis (OA) of the knee is a disease that significantly decreases the quality oflife due to joint deformation and pain caused by degeneration of articular cartilage. Since thedegeneration of cartilage is irreversible, intervention from an early stage and control throughout
life is important for OA treatment. For the treatment of early OA, the development of a diseasemodifying osteoarthritis drug (DMOAD) for intra-articular (IA) injection, which is attracting attention
as a point-of-care therapy, is desired. In recent years, the molecular mechanisms involved in OA
progression have been clarified while new types of drug development methods based on genesequences have been established. In addition to conventional chemical compounds and protein
therapeutics, the development of DMOAD from the new modalities such as gene therapy andoligonucleotide therapeutics is accelerating. In this review, we have summarized the current status
and challenges of DMOAD for IA injection, especially for protein therapeutics, gene therapy, andoligonucleotide therapeutics.

Watanabe, M.; Sato, M. Candidates
for Intra-Articular Administration

Keywords: disease-modifying osteoarthritis drug; gene therapy; oligonucleotide therapeutics

Therapeutics and Therapies ofOsteoarthritis. Int. J. Mol. Sci. 2021,
22, 3594. https://doi.org/10.3390/
ijms22073594
Academic Editor: Maurizio Battino
Received: 1 March 2021
Accepted: 26 March 2021
Published: 30 March 2021

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Copyright: © 2021 by the authors.
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Attribution (CC BY) license (https://

1. Introduction
Osteoarthritis (OA) of the knee is a disease in which degeneration of articular cartilage
gradually progresses, and the cartilage is eventually worn to expose the subchondralbone. OA significantly decreases the quality of life due to joint deformation and pain.
While most patients with OA are elderly, it has been reported that nearly 40% of people
over the age of 60 years have signs of early OA [1,2]. In addition to aging, OA is adisease involving multiple etiologies such as a history of joint injury, gender, genetic
background, and obesity [3,4]. Since degenerated cartilage does not repair spontaneously,
it is important to prevent the degeneration of cartilage from an early stage. However,current drug treatment for early OA is limited to the administration of analgesics for pain,
viscosupplement such as hyaluronic acid that enhances joint lubricity, and intra-articular(IA) injection of corticosteroids [5]. None of these therapies are considered to have the effectof suppressing the progression of cartilage degeneration and destruction and ultimately,
replacement arthroplasty is the only radical treatment for late-stage OA. Since OA is not alife-threatening disease, minimally invasive treatment at an outpatient level is preferred;
IA injection is most commonly employed for OA. Since OA progresses over a long periodof time, early intervention is important so that symptoms can be controlled throughout
life. The development of a disease-modifying osteoarthritis drug (DMOAD) that can beadministered by IA injection, which is attracting attention as a point-of-care therapeutic,
is desired.

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Int. J. Mol. Sci. 2021, 22, 3594. https://doi.org/10.3390/ijms22073594

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Currently, corticosteroids and hyaluronic acid are the drugs most commonly administered by IA injection, with the main action being pain control. So far, no DMOADs havebeen developed [6]. Novel treatment options are being developed with the expectation ofcontrolling inflammation and promoting cartilage regeneration in OA. Options include
recombinant protein, gene therapy, platelet-rich plasma, and cell therapy [7]. This review
outlines the current status and challenges that the new biologics bring to treating OA,
especially protein therapeutics, gene therapy, and nucleic-acid therapeutics.
2. Target Biological Pathways for DMOADs
OA is a disease mainly characterized by cartilage degeneration, pathological changes
in entire joint, such as joint capsule thickening, osteophyte formation, subchondral sclerosis, and synovitis. Although the molecular mechanism underlying these pathologicalconditions remains not fully understood, many excellent reviews that discuss the molecularpathology of OA have been published [6,8–12]. As a consensus, prolonged low-grade inflammation is thought to play a pivotal role in OA initiation and progression. In summary,activation of innate immunity by the decomposition products of the joint matrix generated by trauma or mechanical overload causes synovitis. Synovitis induces production ofproinflammatory mediators from synovial cells, immune cells, chondrocytes, or cells insubchondral bone. Risk factors for OA, such as aging, injury history, obesity, and some
genetic backgrounds are thought to trigger or prolong inflammation.
Increased proinflammatory cytokines such as interleukin (IL)1β, tumor necrosis factor
α (TNF-α), IL-6, IL-15, IL-17, and IL-18, and the imbalance of anti-inflammatory cytokines
such as transforming growth factor-β (TGFβ), IL-4, IL-10, and IL-13 suggested contribute
to OA pathogenesis [13–15].
Further, inflammatory joint condition induces alteration of the chondrocyte phenotype,such as cell proliferation, cluster formation, production of both matrix proteins and matrixdegrading enzymes, and chondrocyte hypertrophy and apoptosis [16]. Matrix-degrading
enzymes such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family degrade cartilage component type
II collagen and proteoglycan. Alteration of chondrocyte phenotype considered as regenerative response caused by inflammation. The chondrocytes follow the process of chondrocyte
maturation and resulting matrix remodeling, improper hypertrophy-like maturation andcartilage calcification. Cartilage homeostasis disruption is considered to be involved in
cartilage degeneration and osteophyte formation.
The importance of each factor in OA progression remains unclear. A blockage of theinflammatory signal induced by proinflammatory mediators, a supplementation of antiinflammatory factors, or an alteration of chondrocyte phenotype is considered a promisingtarget for DMOADs.
3. The Advantage of IA Delivery in OA Treatment
For the control of inflammation, protein therapeutics blocking TNFα, IL-1, and IL-6
have been already developed for treatment of rheumatoid arthritis (RA). RA is a systemic
inflammatory disease and also causes joint destruction. Inflammation of RA is generally
more severe than that of OA. Thus, the control of inflammation by these protein therapeutics is of benefit to the RA patients and increases in the occurrence of infections istolerated [17,18].
In contrast to RA, the daily symptoms in OA such as morning stiffness, heat, pain,
and joint effusions are initially comparatively mild [8]. The systemic administration of
immunosuppressive protein therapeutics poses an unacceptable risk for OA patients.

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3 of 17In OA, usually only one or two large joints are affected, and inflammation is limited
to the affected joints. Furthermore, articular cartilage, which is the target tissue for OA
treatment, is an avascular tissue, which makes it difficult for a drug to be distributedby systemic administration. IA drug delivery is relatively easy for the diarthrodial joint,
thereby ensuring that the drug is delivered to the affected area. The merit of IA injectionis that sufficient drug concentration can be achieved at the target site while avoiding thedisadvantages of potential side effects. Thus, IA delivery of DMOADs, including alreadydeveloped immunosuppressive protein therapeutics, has been considered as an option toaddress the issues, adverse effect, and drug distribution.
4. OA Treatment by IA Injection of Protein Therapeutics
As mentioned above, various inflammatory cytokines and cartilage matrix-degrading
enzymes in joint fluid increase. The expression of genes encoding these proteins in synovial
cells and chondrocytes also increases to the extent that suppression of TNFα and IL-1βactions is considered to be a promising target for the treatment of OA. In addition, IA
injection of humoral factors that have cartilage repair and cartilage protection properties isalso of great interest because they may be a DMOAD for OA of the knee (Table 1).
IL-1Ra, which is a natural antagonist of IL-1β, and anti-IL-1β antibody, which neutralizes IL-1β, have already been shown to be effective in RA. Clinical trials targeting
OA have also been conducted. Considering the potent induction of matrix-degrading
enzymes by IL-1β, IL-1Ra may suppress the degradation of articular cartilage. However,in a clinical trial (NCT00110916) of IA injection of IL-1Ra (anakinra) [19], the drug failed toachieve the primary endpoint, a change in the Western Ontario and McMaster Universities
osteoarthritis index (WOMAC) score from baseline to week 4 [20]. It has been pointed outthat there is the possibility the cartilage protective effect had not been exhibited because of
the short half-life of IL-1Ra (4 h) and because patient-relevant pain assessment data wereused as the primary endpoint [21]. In future studies, it is considered necessary to maintainthe IA concentration of IL-1Ra at an effective concentration by improving the dwell time,such as with a controlled release of the recombinant protein. Currently, a clinical trial ofanakinra aimed at preventing OA by administration within 7 days after anterior cruciate
ligament (ACL) injury is scheduled (NCT03968913 [22]). Since it is estimated that 50% of
patients with ACL injury develop OA, the trial plan is to track the progression to OA bymeasuring marker proteins in joint fluid for early inflammation and cartilage damage. The
effect of IL-1Ra could be observed even with a temporary effect.
Table 1. Clinical trials of intra-articular (IA) injection of recombinant protein for osteoarthritis (OA) patients.
Study Name
(ClinicalTrials.gov 1 )Mode of Action
Biologicals

Study Phase
Outcome Measures

Study Identifier
Current Status 1(Completion Year)

Biologic Therapy to Prevent
Osteoarthritis After ACL Injury

Inhibit IL-1β
IL-1 Receptor antagonist
r-metHuIL-1ra(Anakinra)

Early Phase 1
cytokine level/Knee pain andfunction/marker level

NCT03968913 [22]Not yet recruiting

Study of Safety, Tolerability,
Preliminary Efficacy ofIntra-articular LNA043 Injectionsin Patients with Articular
Cartilage Lesions and Knee
Osteoarthritis

Assist cartilage repair.
A modified human angiopoietin-like
3 proteinPhase 2
MRI/AEs/protein
level/antibodies/Others

NCT03275064 [23]2017~Recruiting

A Study to Investigate the Safety
and Effectiveness of Different
Doses of Sprifermin in
Participants with Osteoarthritis of
the Knee (FORWARD)

Assist cartilage repair
Fibroblast growth
factor 18(Sprifermin)

Phase 2
MRI/WOMAC/PGA/mJSW/
Protein level

NCT01919164 [24]Completed (2019)has results

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Table 1. Cont.
Study Name
(ClinicalTrials.gov 1 )Mode of Action
Biologicals

Study Phase
Outcome Measures

Study Identifier
Current Status 1(Completion Year)

Dose Finding Study of BoneMorphogenetic Protein 7 (BMP-7)
in Subjects with Osteoarthritis(OA) of the Knee

Assist cartilage repair
Bone Morphogenetic Protein 7(BMP-7/OP-1)

Phase 2WOMAC

NCT01111045 [25]Completed (2011)

To Determine the Safety,
Tolerability, Pharmacokinetics andEffect on Pain of a Single
Intra-articular Administration of
Canakinumab in Patients with
Osteoarthritis in the Knee

Inhibit IL-1β
humanized monoclonal antibody tointerleukin-1β(Canakinumab)

Phase 2
AEs/VAS/WOMAC/Others

NCT01160822 [26]Completed (2010)
has results

Treatment of Knee Osteoarthritis
with Intra-Articular Infliximab

Inhibit TNFαchimeric monoclonal antibody to
TNF-α(Infliximab)

Phase 4
Cellular infiltrates/Effusion/WOMAC/Others

NCT01144143 [27]Completed (2011)
has results

Study of Intra-articular DLX105
Applied to Patients with Severely
Painful Osteoarthritis of the Knee

Inhibit TNFαa single-chain (scFv) antibody
fragment against TNF-α (DLX105)

Phase 1/2
AEs/VAS/WOMAC

NCT00819572 [28]Completed (2010)

1 https://clinicaltrials.gov (accessed on January 2021), the most recent phase and status study of the same drug candidate. ACL, anterior

cruciate ligament; WOMAC, Western Ontario and McMaster osteoarthritis index (WOMAC) pain subscale; MRI, quantification orassessment of cartilage by MRI; AEs, incidence of adverse events; PGA, Patient’s Global Assessment; mJSW, Minimal Joint Space Width in
the Medial and Lateral Compartments as Evaluated by X-ray; VAS, Visual Analogue Scale to assess the intensity of knee pain.

The IL-1β neutralizing antibody, canakinumab, is effective against juvenile idiopathic
arthritis [29]. A clinical trial of IA injection of canakinumab in OA patients was conducted
in 2012 (NCT01160822 [26]). Further study of this drug for the treatment of OA has not been
conducted. Interestingly, in the Canakinumab Anti-inflammatory Thrombosis Outcomes
Study, a clinical trial in atherosclerosis patients involving 10,061 participants, patients
received drug subcutaneously once every 3 months. Study results report that inhibition of
IL-1β with canakinumab may substantially reduce rates of total hip arthroplasty/total knee
replacement and ameliorate osteoarthritis-related symptoms [30]. Although verification isnecessary, these results suggested that persistent inhibition of IL-1β might suppress OA
progression. If subcutaneous administration of canakinumab is safe and tolerated, it may
be an option for DMOAD.
Clinical trials have been conducted to determine the efficacy of a TNFα neutralizing
antibody (infliximab) and a single-chain (scFv) antibody fragment against TNF-α, both ofwhich inhibit the action of TNFα. For infliximab, the suppression of synovial inflammation
had not been observed. For antibody fragment, no results have been posted and subsequentstudies have not been performed.
An alternative to suppression of inflammation as an approach to DMOADs, IA injection of protein factors that promote chondrocyte differentiation and inhibit cartilage
hypertrophy, has been carried out in anticipation of cartilage protection and promotionof regeneration. Human recombinant bone morphogenetic protein/osteogenic protein-1(BMP-7/OP-1) is a factor that has been reported to suppress chondrocyte hypertrophy andprotect cartilage. It had been used to promote bone regeneration [31]. Clinical trials of
BMP-7 in OA showed that in participants who received BMP-7, there was a trend toward
greater symptomatic improvement than with a placebo [25,32]; however, rhBMP-7 havebeen withdrawn from the market because of lingering safety concerns; vertebral osteolysis,
ectopic bone formation, radiculitis, or cervical soft tissue swelling [33], and further study
for OA treatment have not been conducted.
FGF18 has been confirmed as both inducing cartilage differentiation and having
a protective action [34,35]. A clinical trial of IA injection of rhFGF18 (Sprifermin) was
conducted, but no clear improvement was observed in the pain evaluation [24,36,37].
However, a dose-dependent increase in cartilage thickness has been observed, and there
are high expectations for its potential as a DMOAD [38–40].

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Angiopoietin-like 3 (ANGPTL3) has been identified as an inducer of chondrogenesis
and cartilage repair. Clinical trials of IA injection of a modified form of ANGPTL3 areunder way [41].
Provided that articular cartilage repair is expected, chondrocytes that respond to thesefactors or progenitor cells capable of cartilage differentiation need to be present or supplied
to the degenerated cartilage. Chondroprogenitor cells have been reported to be present insynovial cells, cartilage surface cells, and periosteal cells, and are expected to contribute to
joint protection [42,43]. Encouraging results with rhFGF18 [38–40] imply that the ability torespond to regenerative stimuli may remain in the joints of OA patients.
The development of protein therapeutics has been accelerated and many other proteins have been shown to be effective in animal OA models (Table 2). Protein engineering is increasingly used aiming at enhancing the efficacy and improving biodistribution
and bioavailability.
Table 2. Protein therapeutics candidates in OA animal models with reported efficacy.
Protein

OA Animal Models

References

Rat TMJ-MIA model[44]

Rabbit collagenase injection model

[45]

GH

Growth hormone

HB-IGF-1

Humanized insulin like growth factor-1
fusion protein with a heparin-binding
domain for targeting to cartilage

Rat MMx model[46]

FzD7 CRD

Recombinant-Frizzled 7-cysteine-richdomain designed to inhibit
Wnt3a/β-catenin signaling

Mouse DMM model[47]

rhMidkine

rhMidkine

Mouse DMM model[48]

IL4-10 FP

A fusion protein, the biological activity of
IL-4 and IL-10 are preserved

Canine groove model[49,50]

Mouse tibial compression OA injury model[51]

Atsttrin

An engineered protein composed of threetumor necrosis factor receptor(TNFR)-binding fragments ofprogranulin (PGRN)

Rat noninvasive ACL rupture model
mouse ACLT model[52]

rhGDF5rh growth differentiation factor-5

Rat MMx model[53]

rhPRG4rh lubricin

Yucatan minipigs DMM model[54]

Rat ACLT model[55]

CRB0017

recombinant monoclonal antibodies directedagainst ADAMTS5

STR/ort[56]

Sclerostin

Efficacy reported in an experimental animal model by IA injection in last 10 years in PubMed. TMJ-MIA, monosodium iodoacetate injection
to temporomandibular joint; rh, recombinant human; MMx, the medial meniscus was resected; DMM, destabilizing the medial meniscus
surgery; ACLT, anterior cruciate ligament transection; STR/ort, spontaneous osteoarthritis mouse.

5. Gene Therapy for OA Treatment by IA
In recent years, gene therapy with viral vectors containing plasmid deoxy ribonucleic
acid (DNA) or messenger ribonucleic acid (mRNA) that incorporates genes with therapeutic
effects has been put into practical use. Gene therapy exerts its effects through intracellular
nucleic-acid transfection and translation into protein. In the case of gene therapy with
mRNA, it does not need to be transcribed intracellularly, protein can be produced directlyfrom the mRNA. When aiming for continuous expression, mRNA transfection is considered
to be inferior to gene transfer by vectors. Since mRNA is of very low stability, drug delivery
system (DDS) techniques that incorporate mRNA into cells while avoiding degradation
are necessary. Lipid nanoparticles (LNPs) are currently the mainstream DDS for mRNAtransfection but they are difficult to target to particular cell types, other than hepatocytes,

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because most LNPs are trapped by the liver when administered systemically. IA injection of
the LNP-encapsulated mRNA may maintain protein bioavailability and action on synovial
cells or chondrocytes in the target joint before the LNPs circulate to the whole body. Genetherapy with mRNA administration could be suitable for transient gene expression aimed
at controlling inflammation after trauma and suppressing chondrocyte apoptosis.
Currently, research is being conducted on therapeutic methods in which vectors andplasmids are administered to cells in joints to produce factors with anti-inflammatory andcartilage repair effects instead of IA administration of recombinant proteins. Since OA
is not a life-threatening disease, a vector with a higher safety profile than those used forcancer treatment is required. Table 3 shows the clinical studies of IA gene therapy for OA
conducted to date. The viral vectors currently in clinical trials were adeno-associated virus(AAV) vectors, helper-dependent adenovirus (HDAd) vectors, and plasmids.
AAV is a nonpathogenic virus that can be transfected into cells in a tissue-specificmanner, depending on their serotype, and can transfer genes into nondividing cells. Theiruse is expanding due to those characteristics. The introduction of AAV vectors into joints
has been investigated in rat [57], rabbit [58], and equine [59] species, and is introduced
into chondrocytes. Since adult chondrocytes usually do not proliferate and are rarely
replaced, it is expected that long-term transgene expression will be possible, if genes canbe introduced into chondrocytes [60]. If anti-inflammatory proteins can be continuously
supplied through gene therapy, it may be possible to modify OA progression. In thiscontext, gene therapy utilizing an IL-1Ra gene-containing vector has major implications for
OA treatment based on the results obtained with protein administration. Viral transfectionof the potent anti-inflammatory cytokines, IL-10, and IFNβ is also being investigated.
Table 3. Clinical trials of intra-articular delivery of gene therapy for OA patients.
Study Name
(ClinicalTrials.gov 1 )Mode of Action
Biologicals

Study Phase
Outcome Measures

Study Identifier
Current Status 1(Completion Year)

Safety of Intra-Articular
Sc-rAAV2.5IL-1Ra in Subjects
with Moderate Knee OA(AAVIL-1Ra)

Inhibit IL-1β
sc-rAAV2.5IL-1Ra

Phase 1
AEs

NCT02790723 [61]2019~Recruiting

Study to Evaluate the Safety
and Tolerability of FX201 in
Patients with Osteoarthritis of
the Knee

Inhibit IL-1β
humantakinogene hadenovec
IL-1Ra (FX201)

Phase 1
AEs/biodistribution

NCT04119687 [62]2020~Recruiting

Efficacy and Safety of XT-150
in Osteoarthritis of the KneeSupply IL-10
plasmid DNA with a variant of
human IL-10 transgene (XT-150)

Phase 2
KOOS/WOMAC/Others

NCT04124042 [63]2020~Recruiting

A Single Dose Clinical Trial to
Study the Safety of ART-I02 inPatients with Arthritis

Supply IFN-β
Recombinant AAV type 2/5
containing a hIFN-b gene
(ART-I02)

Phase 1
AEs/clinical scores
distribution/immune
response/Others

NCT02727764 [64]Active (2022)

1 https://clinicaltrials.gov (accessed on January 2021), the most recent phase and status study of the investigational drug. AEs, incidence ofadverse events; KOOS, Osteoarthritis Outcome Score; WOMAC, Western Ontario and McMaster Osteoarthritis Index pain subscale.

A clinical trial of protein therapeutics of IL-1Ra showed that IL-1Ra rapidly disappeared from the joint. However, clinical trials of FX201 (humantakinogene hadenovec)
aiming to express IL-1Ra in the joint are under way (NCT04119687 [62]). FX201 is an
HDAd vector based on human serotype 5 (Ad5) that is designed to express IL-1Ra under
the control of an inflammation-sensitive promoter. HDAd is a vector that lacks all viralgenes except the regions required for viral genome replication and packaging to increase
the safety of adenovirus vectors. It is a nonintegrating, nonreplicating vector that can
incorporate relatively long genes. IA expression of IL-1Ra using HDAd in mice and rats

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7 of 17has been reported [65]. It has also been reported that cotransfection with the HDAd vector
containing the gene coding for lubricin exhibited improved protection against OA thansingle IL-1Ra coding HDAd vector transfection [66].
Similarly, aiming for IA expression of IL-1Ra, sc-rAAV2.5rIL-1Ra (a self-complementing,recombinant adeno-associated viral vector carrying IL-1Ra complementary DNA) has been
investigated in a clinical trial (NCT02790723 [61]). The conventional AAV vector is singlestranded, and gene expression occurs after the complementary strand is synthesized in thecell to become double-stranded. In the process of self-complementing, the positive andnegative strands are connected and packaged into a viral capsid to quickly become doublestranded, and hence gene expression occurs rapidly [67]. It is inferior to conventional AAV
in that large-sized genes cannot be introduced. Preclinical studies have confirmed that
more than 90% of the post-IA vector in rats remains in the joint and that IL-1Ra levels
are also elevated [68]. Furthermore, improvement in total joint pathology following genetherapy has been reported in an equine OA model [69].
IL-10 is a potent anti-inflammatory cytokine and is thought to affect OA. IL-10 suppresses the production of IL-1β, IL-6, and TNF-α and induces the expression of IL-1Ra [70].
In addition, it suppresses matrix metallopeptidases (MMPs) expression. IL-10 is expected
to be able to broadly suppress the inflammatory response and cartilage destruction seen
in OA [71]. A plasmid DNA-based therapy (XT-50) that expresses a long-acting human
IL-10 variant has been developed. The variant (hIL-10var) is inserted under the control of a
C-X-C motif chemokine ligand 10 promoter and the promoter is expected to regulate IL-10
expression according to the onset of inflammation in the joint [72]. Administration of XT150 to canine joints has reportedly increased IA IL-10 levels and reduced pain [73]. Clinicalstudies of a plasmid vector (XT-150) on OA have been conducted (NCT04124042 [59]).
IFNβ also regulates and suppresses the production of TNFα, IL-1β, and IL-6, and an
AAV vector (ART-I02) that expresses IFNβ is being studied in rheumatoid arthritis and
OA. ART-I02 is designed to express IFNβ under the control of a nuclear factor κB (NF-κB)
promoter, which acts as a transcription factor during the induction of many inflammatory
cytokines. More than 90% of the virus stayed in the joint after IA administration of ART-I02
and protein expression was confirmed even after 7 weeks [74]. In the administration of ARTI02 for collagen-induced arthritis in rhesus monkeys, a beneficial effect on joint swelling,histological inflammation, and bone erosion scores has been reported [75]. ART-I02 is also
undergoing clinical research targeting OA (NCT02727764 [60]).
Introduction of PRG4/lubricin, which plays an important role in cartilage integrity,
was investigated. Using 10mabHDV-PRG4, HDAd vector conjugated to an α-10 integrin
monoclonal antibody, the increase of transduction into chondrocytes and productionof lubricin have been reported [76]. Introduction of IGF-I by AAV vector has been also
investigated [77]. Since the protein can be continuously expressed, gene therapy considered
one of the options for improving the biodistribution and bioavailability. Using gene therapy,
it is possible in principle to express a range of receptors and transcription factors, andthus, it may be possible to use gene therapy to change cellular properties such as thedifferentiation potential of chondrocytes. The safety and usefulness of gene therapy in
OA treatment is yet unknown. It is expected that the knowledge of the production ofneutralizing antibodies and the efficiency of introduction into target cells by IA in humanbodies will be clarified from the ongoing clinical trials in the future.

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6. Oligonucleotide Therapeutics as a Candidate for OA Treatment by IA Injection
In recent years, the development of oligonucleotide therapeutics that introduce oligo
nucleic acids such as antisense and small interfering RNA (siRNA) into cells has progressed.
The major oligonucleotide therapeutics are double-stranded RNA, siRNA [78], micro RNA(miRNA) [79], and single-strand RNA antisense [80]. Antisense is classified into splicingcontrolled antisense that interferes with pre-mRNA splicing [81], RNA-degrading antisensethat collaborates with RNaseH to degrade mRNA, and miRNA-inhibiting antisense that
inhibits miRNA function [80]. In the past, in vivo administration of nucleic acid has had
problems of intracellular delivery and in vivo stability. Over the past decade, modified
nucleic acids have been developed that have improved nuclease resistance and increased
stability in the body. By chemically modifying the nucleic acid, the binding property to thetarget sequence and the efficiency of uptake into cells have also been improved [82–85]. So
far, antisense and siRNA have been put into clinical use [86–95].
For OA treatment, attempts are being made to suppress cartilage destruction bycontrolling the production of protein molecules involved in pathological conditions suchas cytokines and signal transduction molecules using oligonucleotide therapeutics. In
recent years, many comprehensive analyses of miRNAs and mRNAs in OA joints havebeen carried out, and the networks of mRNAs and miRNAs involved in the progression
of OA have been clarified [96–98]. Many miRNAs involved in OA and chondrogenesis
have been reported [99–101]. For genes involved in promoting OA, there is the possibility
that OA progression could be suppressed by degrading specific mRNAs with siRNA or
antisense RNA and suppressing protein production. In the case where miRNAs thattarget proteins associated with cartilage destruction in OA are reduced, administrationof oligonucleotide therapeutics may be able to supplement the anabolic miRNAs andsuppress OA progression. Conversely, using an antisense RNA to inhibit miRNAs thatare increased in OA, it may be possible to restore cartilage protective protein production,
which is suppressed by these miRNAs and control OA progression.
Table 4 shows oligonucleotides that exhibited in vivo efficacy in experimental OA
models, those are limited in rodent models. Application of oligonucleotide therapeutics for
IA injection is still underway. So far, no oligonucleotide therapeutics for DMOADs is inclinical trials yet.

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Table 4. Oligonucleotide therapeutics candidates 1 with in vivo efficacy via IA injection.
Mode of Action

Oligonucleotide

Target Gene(s)mRNA inhibition

miRNA supplement

miRNA
inhibitors
antisense oligonucleotide

References

Recover SIRT1/modify IL-6/STAT3 pathway/prevent OA in mouse
DMM model[102]

miR-203

Recover Erα/decrease cartilage degradation in postmenopausal OA rats[103]

miR-21-5p

Recover FGF18/attenuate the severity of OA in the mouse DMM model[104]

miR-34-5a

Protect cartilage in the DMM and high-fat diet/DMM mice[105]

miR-146b

Recover α2-macroglobulin/
prevent OA in mouse DMM model[106]

miR-181a-5p

Attenuate cartilage destruction, hypertrophic, apoptotic/cell death, and type II
collagen breakdown markers in mouse DMM model[107]

miR-128a

Recover ATG12/slow articular tissue destruction in rat ACLT model[108]

miR-449a

Recover SIRT 1/Prevent cartilage degradation in rat DMM model[109]

Hif-2αPrevent cartilage degeneration in ACLT/DMM mice

[110]

Mmp13

Delay cartilage degradation in mouse DMM model[111,112]

miR-141/200c

miRNA inhibitor

Outcomes

Yap

Ameliorate OA development and reduce subchondral bone formation in
ACLT mice

[113]

Thr

Reduce angiogenic activities in subchondral bone ameliorated cartilage
degradation in mouse DMM model

[114]

FoxC1

Decrease β-catenin, ADAMTS-5, fibronectin, MMP3, and MMP13/decrease
cartilage destruction in mouse DMM model[115]

miR-210 mimic

Not mentioned

Upregulate Col2a1 expression in the meniscus cells and VEGF and FGF2
expression in the synovial cell/enhance repair of the meniscus and prevent
cartilage degeneration in rat DMM model[116]

miR-26a/26b mimic

Fut4

Promote chondrocytes proliferation and inhibit apoptosis/attenuate OA
progression in rat ACLT-MMx model[117]

miR-145 mimic

Mkk4

Suppress the expression of MMP-3 and MMP-13, as well as p-MKK4, p-c-Jun,and p-ATF2/reduce cartilage destruction in rat MCLT-DMM model[118]

miR-140 mimic

Not mentioned

Reduce pathological scores and MMP-13 and ADAMTS-5 expression in rat
ACLT-MMx model[119]

siRNA for target genes

1 Efficacy reported in an experimental animal model by IA injection. DMM, destabilizing the medial meniscus surgery; ACLT, anterior cruciate ligament transection; ACLT-MMx, ACL was transected, and the

medial meniscus was completely resected; MCLT-DMM, medial collateral ligament transection and DMM.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW

1

9 of 17

Efficacy reported in an experimental animal model by IA injection. DMM, destabilizing the medial meniscus surgery;

ACLT,
Int. J. Mol.
Sci. anterior
2021, 22,cruciate3594 ligament transection; ACLT-MMx, ACL was transected, and the medial meniscus was completely
resected; MCLT-DMM, medial collateral ligament transection and DMM.

10 of 17

7. Discussion

7. Discussion

The molecular mechanism of cartilage degeneration in OA and an understanding of
The molecularcartilage
degeneration
in OA
an understanding
target moleculesthat aremechanism
the key to OAofcontrol
are rapidly
increasing.
Onceanda targetmoloftargetmolecules
that
are
the
key
to
OA
controlarerapidly
increasing.
Once a target
ecule is identified, it is possible to obtain DMOAD candidates by various drug discovery
molecule
it is possible
obtainand
DMOAD
candidates
by various drug dismethods
suchisasidentified,protein therapeutics,
genetotherapy,nucleic-acidtherapeutics
coverysuch astherapeutics,
gene therapy,
andfornucleic-acidtherapeutics(Figure1).methods
Each approachhasprotein
pros andcons and appropriate
modality
DMOADs dif(Figure1). Eachhas pros andconsandandtheappropriate
modalityfers
dependingon theapproach
expected mechanismof action
target cells (Table5). for DMOADs

differs depending on the expected mechanism of action and the target cells (Table 5).

Figure 1. Current and potential targets of DMOADs for IA injection. (A) Current drug targets
of protein therapeutics candidates for OA aim to block the interaction between proinflammatory
cytokines and their receptors (I) or to supply cartilage protective proteins to the joint (II). The attempts
of gene therapy candidates aim to supply proteins, which have anti-inflammatory (III) or cartilage
repair effects (IV) instead of IA administration of recombinant protein. (B) OA progression may becontrolled by reducing the protein production involved in pathological conditions through expression
translation suppression by miRNA (V), gene silencing by siRNA (VI), or mRNA cleavage by antisense(VII). MicroRNA inhibitors inhibit OA promoting miRNAs (VIII) and may restore the expression of
OA protecting proteins and may contribute to suppress OA progression.

Int. J. Mol. Sci. 2021, 22, 3594

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Table 5. Summary of new approaches for disease-modifying osteoarthritis drugs (DMOADs).
Protein Therapeutics

Gene Therapy

Oligonucleotide
Therapeutics

Mechanism of action

Supply the required protein

Transduce target gene in cells
Gene expression andtranslation is needed

Transfer to target cells
Modulate the function or thefate of target mRNA

Application range of targets

For proteins that act
extracellularly

Limitation in size of gene

Oligonucleotide sequencecan be designed without off
target effect

Delivery and distribution

Distribute to whole joint by IA

AAV vector provide cell typespecific gene transfer

Drug delivery system fortarget need to be established

Short
Rapidly excreted fromthe joint

Vector: Continuous
expression can be expected
when transfected cells andvector retainedmRNA: transient
protein production

Effect will continue as long asoligonucleotide remain
in cytosol

Control dosage and time

PossibleAmount of protein depend on
transfection efficiency, hostcell activity and etc.
Promotor design provide
regulated induction of protein

Possible

Relative manufacturing cost

High
Biological manufacturing

High
Biological manufacturing

Low
Chemical synthesis”

Technical establishment and
Safety concerns

Established
Predictable

Approved mainly on life
threatening disease
Remain unknown risks?

Approved mainly in specificgenetical disorder
Remain unknown risks?

Retention time

Since protein therapeutics do not penetrate the cell membrane, this approach is applicable to target molecules such as cell surface receptors and secretory factors. Protein
therapeutics can be expected to have high specificity for target molecules. Many drugs
have been put into practical use and drug discovery procedures have been established.
Although IA injection can deliver the protein therapeutics directly, even macromolecules
such as proteins and hyaluronic acid are excreted from the joints in few hours or a day [120].
Technological advances such as modification of amino acid sequence [121], chemical modification [122], and development of sustained-release carrier [7,120,123] will contribute to thedevelopment of DMOADs. For oligonucleotide therapeutics, once the gene sequence of thetarget molecule is known, a method for quickly designing drug candidates has almost been
established. Once the method of transfection into cells is established, the existing method
can be used for oligonucleotide therapeutics for other targets [124]. Since oligonucleotide
therapeutics are based on gene sequences, they are expected to act specifically on the target.
They can be manufactured by chemical synthesis making it easy to set standards andcontrol quality. Oligonucleotide therapeutics act as siRNA and can hybridize only to thetarget mRNA to reduce specific proteins’ synthesis. Oligonucleotide therapeutics aimed
at supplementing or inhibiting miRNAs may act on multiple proteins. After being taken
up into cells, their effect lasts for a certain period of time [110,125]. Oligonucleotide therapeutics can be inexpensively produced by chemical synthesis as distinct from biologically
produced recombinant proteins and viral vectors. This is important for a candidate for thetreatment of OA, which may be used for long periods in large numbers of patients. Usinggene therapy, both intracellular and extracellular molecules can be expressed, either transiently or in response to a specific signal depending on the design of the vector [126]. Thereis also the possibility of the practical application of vectors that express siRNA [127–129],
miRNA [130,131], and antisense RNA [132]. The ability to localize and supplement proteinsthrough oligonucleotide therapeutics for long-term is considered to be one of the greatInt. J. Mol. Sci. 2021, 22, 3594

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advantages of gene therapy. However, so far, gene therapy has rarely been put to practicaluse in areas other than cancer and serious congenital diseases [133]. In those who haveantibodies to viral vectors, there are concerns about reduced introduction efficiency andadverse events. In addition, because viral vectors or plasmid is biologically manufactured,
it is difficult to control the quality standard, and the manufacturing cost is considered
to be higher than that of oligonucleotide therapeutics. Gene therapy for OA may makesignificant progress if the use of gene therapy in high-severity diseases accumulates andsafety concerns diminish.
There are several obstacles for development of DMOADs. Generally, surgically induced OA models have been used to evaluate in vivo efficacy. These models reflect some
aspect of post traumatic OA in human, however heterogeneous nature of human OA should
be considered. It has been reported that 12% of the overall prevalence of symptomatic OA
is attributable to post-traumatic OA [134]. The pathophysiology of disease models usedin preclinical research may be distinct from that of the majority of patients with OA. It
should be considered to select appropriate experimental model, such as naturally occurringor genetically modified spontaneous models, surgically or chemically induced model or
non-invasive models, which can replicate the targeted etiology in human OA [135]. For
heterogeneity of human OA, the new classification for distinguishing different phenotype
of OA is proposed [136]. It is expected that more precise OA classification will enable
the selection of appropriate target patients for clinical trials according to the mechanismof action of the test drug [137]. Meanwhile establishment of reliable outcome measures,
which can follow the course of disease and effect of intervention, are desired. The great
potential of functional or compositional MRI for noninvasive assessment of tissue-structurechanges in OA has been reported [138]. The selection of suitable experimental models,target patients, and appropriate outcome measure would be crucial for the successful
development of new therapies.
8. Conclusions
OA progresses gradually over a long period of time and degeneration and damage toarticular cartilage are irreversible. It is important to intervene in cartilage degenerationfrom an early stage and control its progression. Thus, IA injection of DMOAD at point-ofcare may be one of the treatment options. In addition to conventional chemical compounds
and protein therapeutics, DMOAD may be created from state-of-the-art gene therapy andoligonucleotide therapeutics.
Author Contributions: Conceptualization, E.T. and M.S.; writing—original draft preparation, E.T.
and M.M.; writing—review and editing, E.T. and M.S.; supervision, M.W. and M.S.; funding acquisition, E.T., M.W. and M.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by AMED under Grant Numbers 20bk0104101h0001, JSPS
KAKENHI Grant Number JP20K07297 and TERUMO LIFE SCIENCE FOUNDATION.
Data Availability Statement: No new data were created or analyzed in this study.
Acknowledgments: The image of Antibody and plasmid is from TogoTV (©2016 DBCLS TogoTV/CC-BY-4.0)
Conflicts of Interest: The authors declare no conflict of interest.

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