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Nature Reviews Rheumatology
nature reviews rheumatology https://doi.org/10.1038/s41584-023-01067-4
Review article Check for updates
Engineering approaches for
RNA-based and cell-based
osteoarthritis therapies
Carlisle R. DeJulius1,2, Bonnie L. Walton1,2, Juan M. Colazo 1, Richard d’Arcy1, Nora Francini1, Jonathan M. Brunger1
& Craig L. Duvall 1
Abstract
Osteoarthritis (OA) is a chronic, debilitating disease that substantially
impairs the quality of life of aected individuals. The underlying
mechanisms of OA are diverse and arebecoming increasingly
understood at the systemic, tissue, cellular and gene levels. However,
the pharmacological therapies available remain limited, owing to drug
delivery barriers, and consist mainlyof broadly immunosuppressive
regimens, such as corticosteroids, that provide onlyshort-term
palliative benets and do not alter disease progression. Engineered
RNA-based and cell-based therapies developed with synthetic
chemistry and biology tools provide promise for future OA treatments
with durable, ecacious mechanisms of action that can specically
target the underlying drivers of pathology. This Review highlights
emerging classes of RNA-based technologies that hold potential for
OA therapies, including smallinterfering RNA for gene silencing,
microRNA and anti-microRNA for multi-gene regulation, mRNA for
gene supplementation, and RNA-guided gene-editing platforms such
as CRISPR–Cas9. Various cell-engineering strategies are also examined
that potentiate disease-dependent, spatiotemporally regulated
production of therapeutic molecules, and a conceptual framework
is presented for their application as OA treatments. In summary, this
Review highlights modern genetic medicines that have been clinically
approved for other diseases, in addition to emerging genome and
cellular engineering approaches, with the goal of emphasizing their
potential as transformative OA treatments.
Sections
Introduction
Osteoarthritis pathology and
therapy
RNA-based therapies
Genetically engineered cell
therapies
Clinical perspectives and
challenges
Conclusions
1Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA. 2These authors contributed equally:
Carlisle R. DeJulius, Bonnie L. Walton. e-mail: jonathan.m.brunger@vanderbilt.edu; craig.duvall@vanderbilt.edu
Nature Reviews Rheumatology
Review article
coordinate anti-inflammatory and tissue anabolic activities have begun
to be developed to augment the potential of genetic engineering.
Advances in the delivery of synthetic RNA-based or cell-based
therapeutics provide additional strategies that overcome the chal-
lenges of generating disease-modifying treatment modalities. The end
goal of therapies is to influence the activities of cells that occupy and
infiltrate, or are delivered exogenously into, the arthritic joint. To serve
as disease-modifying agents, cells should resolve inflammation, delay
or arrest cartilage loss and other structural changes and contribute to
pain-free joint function, thereby ensuring a better quality of life for the
patient. The precise and flexible targeting offered by RNA-based thera-
peutics make them a straightforward approach to accomplishing this
goal by modulating gene expression of endogenous cell populations.
RNA-guided strategies have also inspired some of the early synthetic
biology approaches to tackling OA, such as the use of CRISPR-Cas9
technology. Now, gene editing is being combined with emergent syn-
thetic biology technologies for programming cells to be therapeutic
engines that implement defined, regenerative programmes in the
context of OA.
In this Review, we explore how advances in non-viral RNA-based
gene modulation and cell-engineering strategies can be applied to
effectively modify the course of OA. We first outline foundational work
on RNA-based therapeutics, including both the chemical modifications
and carrier systems that can potentiate gene expression, silencing and
editing. We highlight contemporary applications of RNA-based thera-
pies in OA and discuss the next generation of designer, cell-based OA
therapies. We provide an overview of the foundational principles of
gene circuit design, and highlight both current and future potential
OA applications of autoregulated cell therapies.
Osteoarthritis pathology and therapy
Severe OA affects all parts of the joint, leading to debilitating pain and
consequent loss of mobility
11
. Individuals with OA commonly exhibit
full-thickness cartilage defects, along with bone remodelling in the
form of osteophytes and subchondral bone thickening. The joint syno-
vial capsule also thickens owing to the proliferation of synoviocytes,
angiogenesis and an influx of immune cells. In addition, disrupted
lymphatic drainage contributes to swelling and stiffness of the joint.
The majority of patients with OA present with idiopathic disease
for which the initiating events are largely unknown. However, joint
injury increases the risk of OA development
12,13
, whereas underlying
factors such as ageing, genetics and obesity are also implicated in
OA
12
. Cellular senescence is thought to be one of the mechanisms that
causes idiopathic OA associated with age
14,15
. With the advancement of
genome-wide association studies, various susceptibility loci have been
identified in patients with OA
16
. Finally, obesity has mechanobiological
implications and also elevates the systemic inflammatory state of an
individual, which can potentiate joint disease17.
A complex interplay between mechanical, cellular and solu-
ble factors drives OA pathogenesis (Fig.1). Broadly, the OA joint
is characterized by elevated levels of inflammatory cytokines and
reactive oxygen species that can activate immune cell recruitment
and skew resident cells towards pro-inflammatory phenotypes.
Senescent cells adopt a senescence-associated secretory phenotype
(SASP) that can be an integral source of soluble factors that drive
OA18. Increased production of proteases, which break down colla-
gens (mediated by matrix metalloproteinases (MMPs)19), and pro-
teoglycans (mediated by a disintegrin and metalloproteinase with
thrombospondin motifs (ADAMTS)20), leads to the destruction of the
Key points
•Osteoarthritis (OA) is a chronic, debilitating disease with limited
treatment options, highlighting the need for disease-modifying,
gene-targeted OA treatments.
•RNA-based genetic medicines enable precise targeting of
OA-associated pathways, but delivery of the necessary therapeutic
components to the joint has historically been challenging.
•Various preclinical and clinical advances have been made in RNA
chemical modiication and the delivery of RNA-based medicines for
broad application across dierent diseases.
•A number of approaches have shown promise in preclinical
models of OA, including the use of small interfering RNA, microRNA,
anti-microRNA, mRNA and CRISPR–Cas9.
•Genetically modiied cells also have potential in OA; synthetic
signalling motifs can be designed to rewire native signalling pathways
or implement speciied responses to user-selected inputs (such as
inlammatory signals).
•RNA-guided and gene circuit-guided technologies can enable the
engineering of cell therapies that recognize and respond to speciic
pathological cues.
Introduction
Osteoarthritis (OA) is a multifactorial, chronic, degenerative joint dis-
ease characterized by cartilage loss, subchondral bone remodelling,
osteophyte formation and synovial inflammation, resulting in joint
pain and loss of function. Despite the considerable burden associated
with OA, treatment options remain limited and disease-modifying
agents are lacking. Gene-specific and engineered cell therapies repre-
sent a new frontier in OA therapy. Genetic medicines can potentially
target the molecular drivers of OA, whereas engineered cells can
provide long-term, feedback-regulated in situ production of thera-
peutic molecules. These technologies have the potential to provide
disease-tailored treatment options that reduce degeneration or even
promote regeneration of the arthritic joint. Innovating effective gene
and cell therapies for OA is met with three major challenges: the iden-
tification of disease-modifying therapeutic targets that holistically
counteract total joint disease; the creation of safe and potent strategies
that can sustainably modulate relevant drug targets; and overcom-
ing cartilage avascularity, synovial fluid recycling and the generally
inhospitable environment within the arthritic joint.
Viral gene therapy, such as with recombinant adeno-associated
viruses, has been leveraged to express therapeutic proteins. Although
this approach has shown preclinical promise in both rodent1,2 and
larger3–7 animal models, viral vectors are challenged in clinical OA
settings by low gene packaging capacities, as well as the potential for
safety issues caused by immunogenicity and unwanted insertional
mutagenesis8–10. Furthermore, the molecular determinants that con-
tribute to OA pathology demand drug specificity and coordinated cell
behaviour to orchestrate regeneration and repair without introducing
adverse effects associated with unregulated transgene expression.
Cells designed with dynamic, conditional and autoregulated abilities to
Nature Reviews Rheumatology
Review article
extracellular matrix that forms the chondrocyte microenvironment.
Ultimately, this process contributes to chondrocyte death and overall
thinning or focal loss of cartilage tissue
21
. Furthermore, matrix frag-
ments and cellular debris released by tissue breakdown can function
as damage-associated molecular patterns that stimulate the innate
immune system, propagating a vicious cycle of inflammation and
degradation22.
Instead of targeting the underlying aetiology, current clinical man-
agement of OA predominantly focuses on pain reduction using treat-
ments that can introduce adverse effects and do not typically reduce
disease progression
23–27
. Lifestyle modifications and weight loss are
often prescribed; however, compliance is low28, with pain presenting
a barrier to exercise for many patients
29
. Oral NSAIDs can cause gastro-
intestinal, cardiacand renal adverse effects
23,30
, whereas intra-articular
corticosteroids can contribute to cartilage thinning24. Hyaluronic
acid, platelet-rich plasma and cell-based products all exhibit variable
effectiveness25–27. End-stage disease is treated with arthroplasty, an
invasive procedure associated with a considerable economic burden
31
.
Notably, a trend of younger patients undergoing arthroplasty, in light
of the ~20-year lifespan for prostheses, is leading to an increase in costly
revision procedures that further the risk of surgical complications32.
Hence, disease-modifying treatments are sorely needed that can either
delay or reverse progression of OA, with RNA-based therapeutics and
designer cells providing potentially transformative options for OA
patient care.
RNA-based therapies
Advances in nucleic acid chemistry and delivery systems, in addi-
tion to the emergence of RNA-guided gene-editing platforms, are
making an increasing clinical impact in a select set of genetic and
infectious diseases
33–35
. Almost two dozen nucleic acid therapeutics
have reached the market in the past 25 years. The first to be clinically
approved wereantisense oligonucleotides (ASOs), which were fol-
lowed bysmall interfering RNA (siRNA), messenger RNA (mRNA), and
CRISPRguide RNA (gRNA); notably, modulators ofmicroRNA (miRNA
mimics and anti-miRNA) are now also moving towards clinical approval.
Although ASOs often incorporate alternative chemistries, these com-
pounds are based predominantly on DNA. ASOs have been successfully
developed for gene expression inhibition and exon skipping, and the
chemistry and disease applications of ASOs (including in OA) have
been extensively reviewed elsewhere36,37. In this Review, we focus on
more recently emerging, RNA-based therapeutics that can be developed
to upregulate or downregulate any single gene (using siRNA, mRNA
or CRISPR-Cas9 approaches) or families of genes (using miRNA or
anti-miRNA) (Fig.2). The possibility of synthesizing virtually any
desired sequence endows nucleic acid-based therapeutics with high
Cartilage
defects
Macroscale biomaterials can
be inserted to deliver
therapeutics and cells
The lymphatic system drains synovial
luid, rapidly clearing locally
delivered drugs and cells.
The large size of microcarriers
makes them resistant to clearance,
enabling sustained local release
Positively charged macromolecules
and nanocarriers can bind to and
deeply penetrate cartilage
Drug delivery considerationsPathophysiological changes
Synovial
thickening
Chondrocyte
death
Cytokines and
proteases
Osteophyte
formation
Unique, targetable molecular
features of cartilage:
Negatively charged GAGs
Dense type II collagen network
Chondrocytes
•
•
•
Angiogenesis
Iniltration and/or
proliferation of cells
The dense, negatively charged
cartilage is impenetrable to
some drugs
Systemically delivered
nanocarriers and RNA
conjugates can pass through
leaky vessels to iniltrate the joint
Fig. 1 | Pathophysiological features of OA and considerations for delivering
RNA-based and cellular therapeutics. OA is characterized by joint remodelling
mediated by cartilage catabolism, bone spur (osteophyte) formation and
thickening of the synovium. Other features include the upregulation of pro-
inflammatory cytokines and proteases, as well as blood vessel formation. Targets
for disease-modifying OA drugs exploit these features through the development
of size-based and charge-based RNA carriers that localize to the arthritic joint
and deploy therapeutic payloads. Cells can also be genetically engineered
to express OA-targeting moieties, such as antibodies against cartilage
constituents or cytokine receptors, and delivered to the joint to mediate
therapeutic effects. Intra-articular localization of RNA-based therapeutics can
be achieved through biomaterial encapsulation (such as the use of hydrogel- or
scaffold-based systems) or via systemic delivery through leaky vessels. GAGs,
glycosaminoglycans.
Nature Reviews Rheumatology
Review article
potential for improving patient outcomes in new clinical applications,
including in OA.
Applying therapeutic RNAs in vivo
RNAs were first broadly applied in fundamental, in vitro studies using
transfection reagents that package and deliver the RNA molecules into
cells. Despite the potential of RNA to selectively modulate gene expres-
sion, its chemical properties have historically limited its therapeutic
use in vivo38,39. RNA is inherently unstable and susceptible to rapid
nuclease degradation in vivo, in both the extracellular and the intra-
cellular spaces. RNA is also quickly cleared from the body via kidney
filtration, and in the context of OA, synovial fluid flow rapidly clears
the joints of therapeutic macromolecules. Furthermore, the presence
of free, extracellular RNAs can activate the innate immune system
through multiple pathways40,41, potentially exacerbating persistent
inflammation in OA
22
. At the cell level, the large size, anionic charge and
hydrophilicity of RNA contribute to poor cell uptake and low penetra-
tion in the densely packed, negatively charged cartilage extracellular
matrix. Inside the cell, escape from endolysosomal trafficking that
leads to lysosomal degradation represents another formidable barrier
to bioactivity. Overcoming these challenges is crucial to realizing the
potential of RNA medicines and has necessitated the development of
•
•
•
Delivery challenges of RNA-based
therapeutics
High molecular weight
Hydrophilic
Negatively charged
RNA theraputic
Chondrocyte or
synoviocyte
Nucleus
Cytosol
Endosome
Ribosome
Transcription
Endosome
escape
No endosome escape
leads to degradation
Extracellular space
(i.e. synovial fluid)
a siRNA b miRNA c Anti-miRNA d Cas9 gRNA e mRNA
Double-stranded
Approximately 20 nt
Triggers mRNA
degradation
Gene-speciic
silencing
•
•
•
•
Double-stranded
Approximately 20 nt
Typically inhibits mRNA
translation
Multi-gene partial
suppression
•
•
•
•
Single-stranded
Approximately 20 nt
Sterically inhibits miRNA
Multi-gene activation via
relieved suppression
•
•
•
•
Single-stranded
Approximately 100 nt
Guides Cas9 RNP to
genomic DNA
Site-directed HDR
or NHEJ
•
•
•
•
Single-stranded
Approximately 1,000 nt
Translated to protein
Protein-speciic
augmentation
•
•
•
•
b miRNA
d Cas9 gRNA
c Anti-miRNA
e mRNA
a siRNA
Cleaved and
degraded mRNA
Inhibition of
miRNA
Protein
expression
RISC RISC
RISC RISC
RISC
Inhibited translation
or degraded mRNA
Site-speciic
NHEJ or HDR
Fig. 2 | Key mechanisms of therapeutic RNA. RNA therapeutics can function as
disease-modifying osteoarthritic drugs by suppressing or augmenting protein
expression. Unmodified RNA is limited by nuclease instability and entrapment
within endosomal vesicles, necessitating the use of modifications and/or
drug delivery vehicles. In the context of OA, the major cell types targeted are
chondrocytes and synoviocytes. a, Upon cytosolic delivery, small interfering
RNAs (siRNAs) are incorporated into a multi-protein complex called the RNA-
Induced Silencing Complex (RISC) that mediates target mRNA degradation
(in a process known as RNA interference). b, microRNAs can also participate
in RISC-mediated RNA interference, and can bind mRNA with partially
complementary sequences, resulting in broader suppression than siRNAs.
microRNA can also promote mRNA translation in certain contexts (not shown).
c, Anti-microRNA sterically inhibits endogenous microRNA to prevent microRNA-
mediated activity. d, CRISPR-Cas9 ribonucleoprotein complexes function in the
nucleus to achieve site-specific editing of genomic DNA, with targeting achieved
via the gRNA. Gene editing can be used to knock out, knock in, silence or base edit
the genomic DNA. e, Ultimately, mRNAs (delivered therapeutically or transcribed
by the cell) are translated to proteins by ribosomes in the cytoplasm. HDR,
homology-directed repair; NHEJ, non-homologous end-joining.
Nature Reviews Rheumatology
Review article
innovative chemical modifications and specialized delivery systems,
as reviewed in this section.
Synthesis and chemical modiication of therapeutic RNA. Chemical
modification of RNA can improve the delivery, safety and efficacy of
RNA therapeutics in vivo (Fig.3a). The value of backbone modifications
was originally established in the field of ASOs. The production of short,
chemically modified oligonucleotides, including siRNA42, miRNA,
anti-miRNA
43
and gRNA
44
, is achieved usingsolid-phase synthesis,
a process that has facilitated nucleic acid drug discovery and the
large-scale manufacturing of clinically applied oligonucleotides. A vast
array of chemical modifications has been discovered that can enhance
the chemical and metabolic stability and reduce the immunogenicity
of RNA therapeutics, but only a few of these modifications are thus
far included in clinically approved therapeutics. Modifications can be
placed at the ribose, phosphate linkage or base, and their applicability
is generally dependent on the class of nucleic acid and mechanism of
action. However, modifications at the 2′-hydroxyl and of the phosphate
linkage are of general importance. For example, some modifications,
including O-methyl (2′OMe) and fluorine (2′F), can confer resistance
to endonuclease activity45. Exonuclease resistance can also be further
improved via substitution of canonical phosphodiester backbone
linkages with phosphorothioate bonds
46
. 2′OMe is also essential to
inhibit RNA activation of innate immune reactions (such as activation
via Toll-like receptors), increasing the safety, on-target specificity
and translational potential of oligonucleotides40,41. Further examples
of investigated chemical functionalities includelocked nucleic acid
(LNA) chemistry, which can increase the binding stability of an RNA
molecule or introduce favourable asymmetry to increase the potency
of the molecule47. Additional backbone modifications have been suc-
cessfully developed in the context of ASOs, including neutrally charged
morpholino derivatives (so-called phosphorodiamidate morpholino
oligomers)36 and peptide nucleic acids48. These chemically modified
compounds are wellsuited to applications that require high bind-
ing affinities, but are less suited to applications such asRNA interfer-
ence (RNAi), owing to their non-natural backbone. Finally, to enhance
pharmacokinetics and cellular uptake, particularly of siRNA, miRNA
and anti-miRNA, cholesterol, fatty acids or targeting ligands such as
N-acetylgalactosamine (GalNAc) can also be conjugated to the end(s)
of the strand49,50.
Delivery of mRNA is a viable strategy for supplementation
of expression of a target gene, but this strategy requires different
manufacturing considerations because the longer size of mRNAs
renders solid-phase synthesis impractical, relegating mRNA produc-
tion to in vitro transcription of template plasmid DNA
51
. As a result,
site-selective modification of mRNA is more challenging than that
of other RNA classes, but it remains possible to incorporate modi-
fied nucleobases. In particular, pseudouridine, 2-thiouridine and
5-methylcytidine can be added during mRNA transcription to increase
the enzymatic and thermal stability of the mRNA molecule and reduce
its ability to activate the innate immune system in vivo52. These modi-
fications improve the level and duration of expression of the proteins
encoded by the mRNA52. Notably, base modifications have also been
tested for other oligonucleotide therapeutics, but are only clinically
approved for mRNA53.
RNA delivery vehicles. Packaging RNAs in drug delivery vehicles is
another strategy to improve their resistance to degradation and capac-
ity to penetrate cells. Encapsulating RNAs within polymeric, peptide or
lipid-basednanoparticles, microparticles or hydrogels can protect the
RNAs from nucleases, enhance their retention at delivery sites (such
as, within joints) and/or promote cytoplasmic delivery of the RNA by
aiding their escape from endosomes. In the context of OA, carrier sys-
tems can also be tuned to promote cartilage penetration and binding or
preferential targeting to specific cell types within the joint (Fig.1). Most
nanoparticle vehicles for RNA delivery incorporate cationic or ioniz-
able molecules (for example, polymers, peptides and lipids) to facilitate
complex formation with negatively charged RNA; these molecules can
also be structurally optimized to promoteendosomal escape upon
cell internalization through pH buffering and/or membrane fusion or
disruption. Resistance to aggregation is another important considera-
tion for RNA-delivery systems and is commonly achieved through the
use of ‘stealth’ polymer coatings such as poly(ethylene glycol) (PEG).
Surface chemistry and the size of particulate carrier systems can
also be used to tune joint retention, uptake by specific cell types and
cartilage penetration. Targeted binding to cartilage, synovium or cell
type-specific surface receptors can be achieved using elements such as
aptamers
54
, antibodies
55,56
or peptides
57
that facilitate specific ‘lock and
key’, molecularly defined interactions. Particle size can also be utilized to
maximize joint retention, with microparticles >10 µm considered large
enough to avoid synovial drainage and phagocytosis58,59. Notably, the
size and charge of the particle are also important determinants of joint
distribution and hence are crucial factors to consider when aiming for
selective targeting of specific tissues or cell types within the joint.
For example, nanoparticles less than 10 nm have been shown to most
efficiently penetrate the full thickness of cartilage60, especially when
positively charged. By contrast, micrometre-sized delivery vehicles
typically accumulate in the synovium
61,62
, enabling locally sustained
release of cargo into the synovial fluid and potential delivery of the
cargo via diffusion throughout the joint. These seemingly juxtaposed
functions can be combined via nano-in-micro formulations to achieve
sustained local delivery of cartilage-penetrating nanoparticles59,61.
Synthetic polymeric platforms are widely used for RNA delivery
systems owing to their tuneable charge, surface chemistry, stability and
size. As noted above, cationic moieties are incorporated to enable the
polymer to form complexes with RNA molecules (known aspolyplexes
or micelleplexes depending on the nature of the block copolymer63);
commonly used polymers for this purpose include linear or branched
poly(ethylene imine)64, polyamidoamine dendrimers54,65, poly(2-
(dimethylamino)ethyl methacrylate) copolymers
55,61,66
and chitosan
67
(Fig.3b). Another useful function of cationic RNA polyplexes is their
ability to bind and penetrate cartilage through electrostatic attraction
to negatively charged glycosaminoglycans65. These polymers are also
weak bases and have a pH-buffering capacity in the acidic endolys-
osomal environment, helping to facilitate endosome disruption
and cytosolic delivery.
Peptides are another class of cationic molecules used for elec-
trostatic complexation of RNA cargos. Peptide systems have various
advantages, including in the ease of synthesis, precise sequence control
and simplicity of the formulations. Similar to synthetic polymers, pep-
tide systems can be designed to include cationic, pH-responsive and/or
endosome disrupting elements. The most straightforward approach
is to complex RNA molecules with repeating polypeptides such as
polylysine
68
and polyarginine
69
. Nature-derived cell-penetrating pep-
tides, such as HIV-1 TAT and its derivatives, also contain a high amount of
arginine and lysine
70
, helping to complex RNA and increase cell uptake.
Glutamic acid and histidine residues have pH responsiveness within
a physiologically relevant range, which can be leveraged in peptide
Nature Reviews Rheumatology
Review article
a Common RNA modifications
Base
O
OH
O
P
O
O O
O
Modiication sites
RibosePhosphate
linkage
Base
O
OH
O
P
O
S O
O
Phosphorothioate 2’ O-methyl Locked nucleic acid 2' deoxy-2' fluoro
Exonuclease
resistance
NHHN
O
O
OH
OO
P
O
O O
O
Pseudouridine
Increased enzymatic and thermal
stability and reduced immunogenicity
Base
O
O
O
P
O
O O
O
Endonuclease resistance and
reduced immunogenicity
Base
O
F
O
P
O
O O
O
Endonuclease
resistance
Base
O
O
O
P
O
O O
O
Increased binding
stability and potency
Increased pharmacokinetics
and cellular uptake
Lipid or ligand conjugate
b Nanoparticle polyplex or micelleplex delivery
Provides colloidal
stability
Examples of RNA-complexing cationic polymers
p5RHH peptide
Non-polar Polar/uncharged Polar/basic
PAMAM dendrimer
VLT T GLPALISWI R RRHRRHC
Cationic
pH-responsive
Amphipathic
•
•
•
Chitosan
O
HO
NH3
+
OH
OH
O
H
n
PEI
H2
N
n
PDMAEMA
n
O O
NH
NH3
HN
NH
H
N
O
O
O
O
N
NH
N
O
HN
N
NH3
NH3
NH3
HN
NH
HN
N
H
O
O
O
O
N
HN
N
O
NH
H3N
H3N
H3N
H3N
N
NH
O
O
RNA molecule
c Nanoparticle lipidic delivery
C16 PEG2000 ceramide
Lipid nanoparticle
PEG lipid
Helper lipid
Phospholipid
Phospholipid
Ionizable
lipid
Helper
lipid
RNA molecule
Liposome or exosome
Cholesterol
Dlin-MC3-DMA
Provides colloidal stability
Improves system
rigidity
Condenses RNA and facilitates
endosomal escape
HO
H
H
H
HHO
H
HN
O
O
O
O
O
O
45
6
5
O
NH
O
7
7
RNA molecule
Cl
Possible stealth polymer
coating (i.e., PEG)
Nature Reviews Rheumatology
Review article
delivery systems to trigger membrane fusion
71
or to promote cytoso-
licdelivery via theendosomal proton sponge effect72. The p5RHH peptide,
derived from the bee venom peptide melittin73 (Fig.3b) has been exten-
sively studied for oligonucleotide delivery owing to its combinatorial
cell penetrating and endosomal escaping activity74–76. Peptides can also be
integrated to provide additional functions, such as chondrocyte affinity77
or enabling protease-triggered cargo delivery78,79, which augment
the specificity of polymeric or lipidic drug delivery vehicles57,80.
Lipid nanoparticles and nanoscale liposomes comprise another
popular class of RNA delivery systems (Fig.3c). Liposomes were first
applied for mRNA delivery in 1978 (ref. 81), and cationic lipids were
recognized as a strategy to improve RNA transfection in 1987 (ref. 82).
Since that time, enormous effort has been dedicated to optimizing
lipid-based nanomaterials by tailoring these formulations for a variety
of cargo and cell types83. Notably, intra-articular injection of lipid nano-
particles and liposomes can also help to lubricate the joint84, potentially
offering additional therapeutic benefit in OA over their drug delivery
function alone. As with polymers and peptides, cationic or ionizable
lipids such as DLin-MC3-DMA, 1,2-dioleoyl-3-dimethyaminopropane
and SM-102 are employed in lipid nanoparticles to condense RNA and
facilitate endosomal escape83. Lipid nanoparticles and liposomes both
incorporatehelper lipids, typically cholesterol, to improve the rigidity
of the system, and PEG lipids, such as C16 PEG2000 ceramide, can be
added to improve the colloidal stability.
In summary, multiple strategies using various technologies are
now available that can potentiate the efficacy of RNA medicines in
OA, including RNA chemical modification and packaging into carrier
systems. The customizability of RNAs towards any desired gene target
offers unique opportunities for strategies that precisely address spe-
cific OA disease stages or patient-specific aetiologies. The historical
hurdles that have blocked RNA clinical use — namely, in vivo degra-
dation, immune activation and intracellular delivery — have been
overcome, as evidenced by the multiple siRNA drugs
85,86
and mRNA
vaccines
33
that are now approved for various indications. However,
limited progress has been made in the clinical application of these
technologies in OA. The studies summarized in the next section, and
in Table1, provide an insight into how RNA chemistry and delivery
systems have been applied to encourage neomatrix synthesis and/or
attenuate matrix-degrading enzymes, ultimately promoting cartilage
regeneration, or at least abrogating cartilage degeneration, in OA. Find-
ings from these preclinical studies contribute to the advancement of
RNA medicines towards clinical translation in OA therapy.
RNA therapeutics in OA
siRNA. siRNAs are double-stranded RNA molecules (~19–27 nucleo-
tides) that block protein translation of a target gene through a process
called RNAi (Fig.2a). The double-stranded RNA molecule is unwound
intracellularly, and the antisense strand is loaded into the RNA-induced
silencing complex (RISC). The antisense strand contains a sequence
that is complementary to the target mRNA, targeting the RISC to the
specific mRNA molecule to facilitate RISC-mediated mRNA cleav-
age. Patisiran (Alnylam Pharmaceuticals), a siRNA lipoprotein nano-
particle, became the first FDA-approved siRNA therapeutic in 2018
(refs.85,86), which was a major clinical breakthrough in the field of RNA
therapeutics; patisiran-mediated knockdown of transthyretin in the
liver provided notable benefits to patients with previously intracta-
ble polyneuropathy caused by hereditary transthyretin-mediated
amyloidosis
86
. Subsequently, a carrier-free, chemically modified siRNA
conjugate was developed that harbours a terminal trivalent GalNAc
ligand to promote liver hepatocyte uptake, and thisconstruct was
applied to achieve efficacy in the treatment of acute hepatic porphyria
(givosiran, approved in 2019)
35
. Thus far, however, the clinical use of
siRNA has been limited to hepatic gene targets; therefore, new delivery
mechanisms are warranted for extra-hepatic applications.
p5RHH peptide is simple to formulate into polyplexes that can
potentiate endosomal escape and exhibit low toxicity. This system has
hence been extensively studied for siRNA delivery74–76. One landmark
OA study in the murine model of knee joint impact injury demonstrated
that delivery of p5RHH–siRNA complexes that target the NF-κB pathway
can improve the cartilage response to injury through the reduction of
chondrocyte apoptosis, preservation of autophagy and suppression
of β-catenin signalling
74
. Additionally, the nanoparticles can penetrate
the intermediate zone of human cartilage, probably owing to their
small size and positive charge74. Other researchers have used alterna-
tive surface modifications of siRNA nanoparticle formulations for
cell targeting, tissue binding and joint retention purposes. Exam-
ples include delivery systems that incorporate the integrin-targeting
peptide Arg-Gly-Asp (RGD)
87
, a chondrocyte affinity peptide
88
, and a
type II collagen-binding antibody55. In the latter example, incorpora-
tion of a type II collagen-binding antibody increased the retention,
silencing potency and durability of endosomolytic polyplexes loaded
with siRNA against Mmp13 (ref. 55). Mmp13 knockdown in a mouse
model of post-traumatic OA (PTOA) preserved the articular cartilage
morphology of the mice and reduced synovial hyperplasia and osteo-
phyte formation. These tissue level effects were related to a broad
suppression of pro-inflammatory gene expression in the joint
55
. In a
complementary approach, researchers have also integrated endoso-
molytic polymer-based micelleplexes loaded with Mmp13 siRNA into
size-engineered poly(lactic-co-glycolic acid) (PLGA) microplates to
create a local nano-in-micro siRNA depot for sustained siRNA delivery61.
This strategy required only a single injection of the system for potent
and long-lasting (at least 4 weeks) target knockdown and joint protec-
tive effects, demonstrating the benefit of size-engineered polymer
depots for resisting synovial fluid clearance.
Towards carrier-free therapeutic application of RNAi, lipophilic
siRNA conjugates that contain stabilizing chemical modifications
Fig. 3 | Key RNA drug delivery strategies and modifications. The major
goals for developing RNA drug delivery systems are to protect the molecule
from degradation, improve circulation half-life and/or tissue residence time,
and increase cell uptake and endosome escape. The two major strategies are
direct chemical modification and packaging in polymeric or lipidic vehicles.
a, RNA drugs are commonly modified at the phosphate linkage, ribose, base
and/or terminus to enhance the pharmacokinetics and/or stability of the RNA,
increase resistance to exonuclease or endonuclease activity and/or to reduce the
immunogenicity of the molecule. b, Nanoparticle polyplexes or micelleplexes
utilize polymers or peptides with cationic, pH-responsive and/or amphipathic
properties to electrostatically package RNA into nanoparticles for intracellular
delivery. c, Lipid nanoparticle, liposome and exosome composition can
also be tuned for RNA packaging and delivery, and can incorporate ionizable
lipids, stabilizing lipids and helper lipids. Example structures of commonly
used RNA modifications, polymers, peptides and lipids are shown, although
many others have been studied. PAMAM, poly(amidoamine); PDMAEMA,
poly((2-dimethylamino)ethyl methacrylate); PEG, poly(ethylene glycol);
PEI, polyethylenimine.
Nature Reviews Rheumatology
Review article
Table 1 | RNA therapeutics applied in OA
Target Delivery system RNA modiication(s) Key results Ref.
siRNA
Ror2 None 0.5% atellocollagen siRNA
conjugate Mouse model of menisco-ligament injury; Ror2 knockdown
protected mice from instability-induced OA with improved structural
outcomes and sustained pain relief, without apparent adverse
effects or organ toxicity
215
Abat p5RHH peptide Not speciied Mouse model of menisco-ligament injury; Abat knockdown lowered
OARSI scores, subchondral sclerosis, and synovial thickness and
cellularity
216
Yap None 2OMe + 5chol ACLT mouse model of OA; Yap knockdown mitigated OA
development by reducing aberrant subchondral bone formation and
cartilage degradation
89
Mmp13 None Proprietary Accell siRNAs
(Dharmacon) DMM mouse model of OA; Mmp13 knockdown reduced cartilage
loss (as indicated by the OARSI score) 92
Mmp13 None Proprietary Accell siRNAs
(Dharmacon) DMM mouse model of OA; Mmp13 knockdown reduced OARSI score
and delayed cartilage degeneration 93
Mmp13
and
Adamts5
None Proprietary Accell siRNAs
(Dharmacon) DMM mouse model of OA; Mmp13 and Adamts5 knockdown
reduced the amount of cartilage degeneration and lowered the
OARSI score
91
Ihh Lipid nanoparticle Not speciied ACLT rat model of OA; Ihh knockdown provided chondroprotection
and attenuated cartilage loss 217
Hif2a PEI polyplex and
chondrocyte-homing peptide 2OMe ACLT or MCLT mouse model of OA; Hif2a knockdown reduced
cartilage degeneration and alleviated synovitis 88
Ncf1 PLGA nanoparticle Not speciied Mouse model of MIA-induced OA; Ncf1 knockdown attenuated
oxidative stress and decreased cartilage damage 218
Nkb p5RHH peptide Not speciied Mechanical loading in mice; Nkb knockdown reduced synovitis and
chondrocyte apoptosis 74
Postn p5RHH peptide ProprietarySilencer siRNA
(Ambion) DMM mouse model of OA; Postn knockdown reduced OARSI score,
MMP13 expression and phosphorylation of p65 75
Jmjd3 p5RHH peptide Not speciied ACLT mouse model of OA; Jmjd3 knockdown increased SOX9
and type II collagen signal, reduced OARSI score and reduced the
expression of COX2 and MMP13
76
Mmp13 Poly[EG-block-(DMAEMA-co-BMA)]
micelleplex conjugated to an
anti-type II collagen antibody
2OMe and 2F Mechanical loading in mice; Mmp13 knockdown reduced OARSI
score, synovitis and osteophyte formation, with broad suppression
of pro-inlammatory gene expression
55
Mmp13 Poly[DMAEMA-block-
(DMAEMA-co-PAA-co-BMA)] polyplex
loaded in PLGA microplates
2OMe and 2F Mechanical loading in mice; Mmp13 knockdown reduced OARSI
score and alleviated synovitis 61
Itgb3 Poly(NIPAM) nanogel ProprietarySilencer siRNA
(ThermoFisher) DMM mouse model of OA; Itgb3 knockdown attenuated cartilage
erosion, IPFP inlammation and subchondral trabecular bone
remodelling
87
Cdkn2a PLGA nanoparticle Not speciied Mouse model of partial MMx-induced OA; knockdown of Cdkn2a
(encoding (p16INK4a) reduced pain (as indicated by pain behaviour),
bone remodelling and cartilage expression of MMP13
219
mRNA
Dnmt3b p5RHH polyplex Proprietary mRNA (TriLink
Biotechnologies) Mouse model of menisco-ligament injury; DNMT3b production
reduced subchondral bone sclerosis, cartilage degeneration
(as measured by OARSI scoring), synovitis, pain sensitivity,
weight-bearing changes and in vivo MMP13 expression
220
WNT16 p5RHH polyplex Proprietary mRNA (TriLink
Biotechnologies) Human cartilage explant; WNT16 production increased the
production of lubricin and decreased chondrocyte apoptosis 109
Runx1 PAsp(TET)-block-PEG micelleplex None MCLT mouse model of OA; RUNX1 production decreased the OARSI
score and osteophyte formation and augmented chondrocyte
markers of anabolism and proliferation
108
Il1ra PAsp(DET)-block-PEG micelleplex None Rat model of MIA-induced TMJ OA; IL-1RA production provided
sustained pain relief and reduced cartilage degradation (as assessed
by the Mankin score)
110
Nature Reviews Rheumatology
Review article
have gained traction as a strategy to promote cell uptake of the siRNA
therapeutic. siRNAs harbouring cholesterol
89,90
or a proprietary, com-
mercially available conjugate
91–93
have demonstrated efficacy after
intra-articular injection in mouse models of PTOA and inflammatory
arthritis against a variety of targets. An early proof-of-concept appli-
cation of this emerging technology showed that targeting Mmp13 and
Adamts5 with proprietary siRNA conjugates reduced cartilage damage
and histological disease scores in a mouse model of PTOA91.
Target Delivery system RNA modiication(s) Key results Ref.
miRNA
miR-224-5p ROS-scavenging urchin-like ceria
nanoparticles Not speciied DMM mouse model of OA; miR-224-5p supplementation had
beneicial effects on joint-space narrowing, osteophyte formation,
subchondral bone microstructure, synovial hyperplasia and
neovascularization
221
miR-140
mimic Exosomes and chondrocyte afinity
peptide Not speciied DMM rat model of OA; miR-140 supplementation reduced the OARSI
score and the expression of MMP13 and ADAMTS5 80
miR-29b-5p Peptide nanoiber hydrogel Agomir structure:
phosphorothioate, 2OMe and
3chol
ACLT rat model of OA; miR-29b-5p supplementation reduced
synovitis and the OARSI score and inhibited the expression of
senescence-related and catabolic-related markers
100
Anti-microRNA
miR-141
and
miR-200c
PAMAM–PEG dendrimer and
chondrocyte-speciic aptamer Not speciied DMM mouse model of OA; Inhibition of miR-141 and miR-200c
reduced the OARSI score, osteophyte formation, MMP13 expression
and pain behaviour
54
miR-449a None LNA DMM rat model of OA; miR-449a inhibition reduced the OARSI and
Mankin scores and increased the levels of type II collagen and ACAN 102
miR-221-3p Fibrin and hyaluronic acid hydrogel miRCURY LNA miRNA Power
Inhibitor that incorporates
phosphorothioates and mixed
DNA and LNA bases that have
been optimally placed for
potency (Qiagen)
Bovine cartilage explant defect model of OA; miR-221-3p inhibition
increased the levels of glycosaminoglycans and type II collagen 103
miR-181-5p None miRCURY LNA miRNA Power
Inhibitor that incorporates
phosphorothioates and mixed
DNA and LNA bases that have
been optimally placed for
potency (Qiagen)
Rat model of puncture-induced facet joint OA and DMM mouse
model of OA; miR-181-5p inhibition reduced the OARSI score and the
expression of MMP13 and other markers of OA
105
miR-365 Carbon nanotube nanoparticle
loaded in yeast cell wall microparticle None DMM mouse model of OA; miR-365 inhibition reduced the systemic
expression of various cytokines, the expression of Mmp13 in the joint
and the OARSI score
106
CRISPR–Cas9 system
Mmp13 Exosome (containing chondrocyte
afinity peptide)-mediated delivery of
CRISPR–Cas9 components and gRNA
plasmid
Not speciied DMM rat model of OA; knockout of Mmp13 decreased the OARSI
score and the expression of MMP13 and increased the levels of type
II collagen and ACAN
137
Ngf,
Il1b and
Mmp13
AAVa-mediated delivery of CRISPR–
Cas9 components and gRNA plasmid Not speciied Mouse model of partial MMx-induced OA; triple knockout of Ngf, Il1b
and Mmp13 reduced pain and structural damage and was superior
to targeting any of the independent genes alone
222
Ctnnb1 AAVa-mediated delivery of CRISPR–
Cas9 components and gRNA plasmid Not speciied Mouse model of needle-induced coccygeal disc degeneration;
knockout of Ctnnb1 (encoding β-catenin) preserved the cartilage
matrix, reduced disc calciication and lowered the severity of disc
generation (as indicated by the Thompson grade score)
223
MMP13 Lipofectamine CRISPRMAX-mediated
delivery of CRISPR–Cas9 RNP Not speciied Human articular chondrocytes; knockout of MMP13 decreased the
secretion of MMP13 and increased the levels of type II collagen 136
aViral systems are excluded from this table, with the exception of those that delivered CRISPR–Cas9 components, to demonstrate the potential of this newly emerging technology for OA. 2F,
2luorine; 2OMe, 2 O-methyl; 3chol, 3 cholesterol; 5chol, 5 cholesterol; AAV, adeno-associated virus; ACAN, aggrecan; ACLT, anterior cruciate ligament transection; ADAMTS5, a disintegrin
and metalloproteinase with thrombospondin motifs 5; BMA, butyl methacrylate; COX2, cyclooxygenase-2; CRISPR, clustered regularly interspaced short palindromic repeats; DMAEMA,
2-(diethylamino)ethyl methacrylate; DMM, destabilization of the medial meniscus; EG, ethylene glycol; gRNA, guide RNA; hif2a, hypoxia-inducible factor 2α; Ihh, Indian hedgehog; IL-1RA,
interleukin-1 receptor antagonist; Jmjd3, Jumonji domain-containing protein-3; LNA, locked nucleic acid; MCLT, medial cruciate ligament transection; MIA, monoiodoacetate; MMP13, matrix
metalloproteinase 13; MMx, medial meniscectomy; mRNA, messenger RNA; Ngf, nerve growth factor; NIPAM, N-isopropylmethacrylamide; OA, osteoarthritis; OARSI, Osteoarthritis Research
Society International; PAA, propyl acrylic acid; PAMAM, poly(amidoamine); PAsp(DET), poly(N-(N-(2-aminoethyl)-2-aminoethyl)aspartamide); PAsp(TET), poly(N-(N-(N-(2-aminoethyl)-2-
aminoethyl)-2-aminoethyl)aspartimide); PEG, poly(ethylene glycol); PEI, polyethylenimine; PLGA, poly(lactic-co-glycolic acid); RNP, ribonucleoprotein; ROS, reactive oxygen species; RUNX1,
runt-related transcription factor 1; siRNA, small interfering RNA; SOX9, SRY-box transcription factor 9; TMJ, temporomandibular joint; WNT16, Wnt family member 16; Yap, yes-associated protein.
Table 1 (continued) | RNA therapeutics applied in OA
Nature Reviews Rheumatology
Review article
siRNA conjugates have also been engineered to enable systemic
delivery of the drug, rather than intra-articular injection, which
could avoid the risks associated with local injections, such as tissue
damage and pain
94
. Additionally, systemic administration could be a
more amenable route than intra-articular injection in the treatment
of patients with multi-joint OA. One example of this approach is the
design of siRNA conjugates that incorporate lipids that bind to and
‘hitchhike’ on endogenous serum albumin. This strategy improves
the pharmacokinetic properties of the siRNA conjugate and pro-
motes its accumulation at sites of inflammation, a concept originally
explored for tumour delivery
95,96
In forthcoming work, an optimized
albumin-binding siRNA conjugate with phosphorothioate and 2′OMe
and 2′F modificationsshowed robust accumulation in arthritic joints,
potent Mmp13 silencing and therapeutic efficacy both in mouse models
of PTOA and in rheumatoid arthritis
97
. The relative simplicity of siRNA
conjugates compared with nanocarriers or microcarriers is beneficial
from a translational perspective, and the rapid preclinical advance-
ment of siRNA conjugates into disease applications outside of the liver
suggests that this approach has potential for application in the clinical
management of OA49.
miRNA and anti-miRNA. Exogenously delivered siRNAs hijack the
natural machinery utilized by miRNAs. miRNAs are non-coding, regu-
latory RNAs that are expressed and processed by Dicer into short,
Glossary
Agomir
A synthetic, stabilized RNA molecule
designed to mimic a mature microRNA.
Chemical stabilizations on the antisense
strand include phophorothioate bonds
at the 5 and 3 ends, full-length 2 OMe
ribose modiications, and 3 cholesterol
strand end conjugation.
Anti-microRNA
Short (19–22 base-pairs),
single-stranded synthetic RNA that
binds microRNA to block binding to
target mRNA.
Antisense oligonucleotide
A short oligonucleotide that changes
gene expression by base-pairing
with RNA and triggering dierent
post-hybridization mechanisms.
Base editors
A genome-editing technology
capable of changing DNA sequences
without generating a double-stranded
break; contains a Cas9 nickase
fused to nucleoside deaminase for
the chemical modiication of bases
located within complementary
sequences to a complexed gRNA
sequence; typically used for single
nucleotide changes.
Colloidal stability
For nanoparticles, refers to their
resistance to aggregation.
Endosomal escape
Nanomoaterials typically enter
cells via endosomes (intracellular
traicking vesicles), but are usually
targeting the cellular cytosol or
nucleus. Therefore, a mechanism
to exit the endosome after uptake is
essential for activity.
Endosomal proton sponge
eect
Refers to the phenomenon in which
pH-responsive materials, including
certain polymers and lipids, accumulate
protons in the endosome, resulting
in swelling and eventual rupture of
endosomes.
Guide RNA
RNA that loads into the CRISPR proteins
and directs the ribonuclear protein
to a complementary nucleic acid
sequence; in the case of the CRISPR–
Cas9 ribonuclear protein complex, this
process leads to a double-stranded
break in the target nucleic acid strand.
Helper lipids
Lipids that are incorporated into lipid
nanoparticles or liposomes to improve
stability and luidity.
Hydrogels
Three-dimensional polymeric networks
that absorb a large amount of water;
can be adapted to a wide range of
mechanical properties, polymer
compositions and fabrication methods.
Indel
A term referring to an insertion and/or
deletion of nucleotides from a DNA
sequence.
Locked nucleic acid
A synthetic structural modiication to
RNA ribose in which a methylene bridge
‘locks’ the nucleoside in a particular
conformation, increasing its stability;
this modiication can also increase
target-binding ainity.
Messenger RNA
Long (hundreds to thousands of
base-pairs), single-stranded RNA that is
translated to proteins.
Micelleplexes
Drug delivery vehicles formed
from block copolymers that have
amphiphilic properties (that is, contain
both hydrophobic and hydrophilic
blocks) and cationic properties, which
can self-assemble for nucleic acid
delivery.
Microparticles
Materials that are typically >1m; can
be spherical, porous or templated to
a desired shape; typically formulated
from polymers.
MicroRNA
Short (19–22 base-pairs), double-
stranded non-coding RNA that binds
mRNA and aects translation of
these RNAs, either through targeted
degradation or through increased
translation. Typically aects multiple
related messenger RNAs.
Nanoparticles
Materials that are typically 1–500nm
in diameter and tend to be spherical;
can be formulated from inorganic
materials (such as gold) or organic
materials (such as polymers, lipids
or peptides).
Orthogonality
Capacity of an engineered signalling
platform to function independently of
native or additional artiicial pathways
owing to having only minimal or no
mutual, overlapping components.
Polyplexes
Drug delivery vehicles formed from
cationic polymers with or without
an additional hydrophilic block for
complexing nucleic acids.
Prime editors
A genome-editing technology capable
of changing DNA sequences without
generating a double-stranded break;
contains a Cas9 nickase fused to a
reverse transcriptase that complexes
with a prime editing gRNA complex
to insert desired DNA sequences with
site speciicity; results in precise DNA
modiications such as an insertion or
deletion.
RNA interference
Inhibition of protein translation via
binding or degrading messenger RNA.
Small interfering RNA
Short (19–22 base-pairs),
double-stranded synthetic RNA that
binds mRNA with high speciicity and
targets mRNA for degradation, resulting
in inhibition of protein translation.
Solid-phase synthesis
A strategy for chemically synthesizing
oligonucleotides in which nucleosides
are immobilized on solid supports, and
the strand is constructed one base at
a time.
Nature Reviews Rheumatology
Review article
double-stranded RNAs that silence gene expression. However, miRNAs
typically target the 3′ untranslated region (3′ UTR) of mRNA and are par-
tially complementary to (and hence partially block) multiple mRNAs
43
.
miRNAs can mediate RNAi through suppressing mRNA translation,
promoting deadenylation or causing mRNA degradation98 (Fig.2b).
miRNAs do not typically regulate any one gene as potently as siRNAs,
but instead have more subtle effects across families of genes that can
cumulatively result in functional outcomes. Anti-miRNAs bind to target
miRNAs via complementary base-pairing, blocking miRNA interactions
with mRNA targets (Fig.2c). Synthetic miRNA mimics and anti-miRNA
oligonucleotides, respectively, can be delivered therapeutically to sup-
plement or block the function of a specific miRNA. Currently, no miRNA
or anti-miRNA drugs are clinically approved, though some candidates
have entered clinical trials for various applications, including cancer
and hepatitis C
99
. A potential challenge faced by miRNA drugs and
anti-miRNA drugs is that, unlike siRNA, miRNAs do not act on a single
genetic target, posing difficulty in predicting and/or characterizing
the on-target and off-target effects of the drugs.
Various miRNA mimics have shown therapeutic potential in pre-
clinical models of OA. For example, miR-140 (ref. 80) and miR-29b-5p
100
are recognized disease-modifying OA drug candidates owing to their
ability to suppress catabolic and/or senescence-related genes. One
promising approach investigated the delivery of a 2′OMe-stabilized and
phosphorothioate-stabilized, cholesterol-modified miR-29b-5p (known
as an ‘agomir’ structure) via a self-assembling peptide hydrogel. This sys-
tem, which combined the advantages of RNA chemical stabilization with
the use of an intra-articular sustained release depot
100
, improved joint
histological scores in a rat model of PTOA by reducing the expression
of miRNA-targeted genes, including Mmp13, Cdkn2a and Cdkn1a.
Overactivation of several specific miRNAs has been impli-
cated as a disease mediator and/or biomarker of OA101. The anti-
chondrogenic102–104 or pro-inflammatory and pro-catabolic105,106 activity of
miRNAs can be targeted with anti-miRNAs. For example, inhibition
of miR-449a, a negative regulator of hBMSC chondrogenesis and a
promoter of IL-1β-induced cartilage destruction, with an LNA-based
anti-miRNA, improved the histological scores of rats with PTOA by
increasing the levels of aggrecan and type II collagen
102
. Similarly, inhi-
bition of the pro-catabolic miR-181a-5p with LNA-modified anti-miRNA
improved the OARSI score and reduced the levels of MMP13, COL10 and
cleaved caspase 3 in PTOA models105. Overall, stabilized miRNAs and anti-
miRNAs represent emerging technologies for OA therapy through the
coordinated regulation of disease-relevant gene expression.
mRNA. The goal of mRNA administration is to promote temporary
in vivo production of therapeutic proteins (Fig.2d). mRNA is sin-
gle stranded and can vary in length from hundreds to thousands of
nucleotides. Upon cytosolic delivery, mRNA is translated by cellular
ribosomes into the encoded protein. The transient nature of mRNA
suggests that mRNA administration has a potentially high safety profile,
making it an increasingly popular therapeutic strategy and vaccine
modality107. A major milestone in the clinical translation of mRNA
was the FDA approval of the lipid nanoparticle mRNA-1273, a vaccine
against SARS-CoV-2 (ref. 33). In addition to other vaccination applica-
tions, mRNA is being tested clinically for a variety of diseases, including
cancer, heart disease and autoimmune disorders51.
In OA, mRNA offers the advantage of promoting the expres-
sion of pro-anabolic proteins or transcription factors that have
cartilage-restoring properties for late-stage disease. For example,
the delivery of Runx1 mRNA in PEG-b-polyamino acid nanomicelles
suppressed disease progression and increased the expression of mark-
ers of anabolism and proliferation in chondrocytes in a mouse model
of OA 108. mRNA platforms have also been explored for their ability to
block critical pro-inflammatory pathways in OA via promoting the
expression of natural antagonists, including proteins that reduce
signalling through the β-catenin/Wnt3a
109
and IL-1 pathways
110
. For
example, delivery of IL-1 receptor antagonist (Il1ra) mRNA in polyamino
acid nanomicelles in a rat model of temporomandibular joint OA sup-
pressed the expression of various inflammatory cytokines, improved
histological scores and provided pain reliefs110.
mRNA can also be leveraged for therapeutic gene editing by deliv-
ering and encoding CRISPR–Cas9 components (as discussed in the next
section). This avenue is promising, as it bypasses the known risks of
viral-vector delivery approaches (such as viral-vector-mediated inser-
tional mutagenesis) that increase the potential for off-target effects
on the genome111. Co-delivery of mRNA encoding Cas enzymes and
cognate gRNA sequence(s) can provide an opportunity to overcome
this risk by providing rapid onset and transient expression profiles.
RNA-guided CRISPR–Cas9 systems. In addition to the aforemen-
tioned RNA-based strategies for altering gene expression by modu-
lating endogenous RNAs, RNA-guided CRISPR systems can precisely
modify DNA and RNA targets in living cells (Fig.2e). The originally
described CRISPR–Cas9 system comprises the Cas9 nuclease, which
induces a double-strand break in DNA, as well as a sequence-specific
gRNA that directs the Cas9 enzyme to the targeted cleavage site112–114.
In eukaryotic cells, double-strand breaks are repaired by a process
known as non-homologous end-joining; the inaccurate nature of
this repair process typically leads to the insertion and/or deletion
of nucleotides (known as indels). Indels consequently introduce
premature stop codons or generate frameshift mutations that ren-
der the protein non-functional, effectively knocking out the tar-
get gene. Alternatively, double-strand breaks can be resolved by
template-guided, homology-directed repair. In homology-directed
repair, the inclusion of a donor DNA template enables targeted inte-
gration of a DNA sequence at a defined genomic locus (the basis of
CRISPR–Cas9 gene-editing strategies). Clinical investigation of in vivo
and ex vivo nuclease-based CRISPR–Cas9 gene editing is currently
underway for several applications, including for the treatment of
Leber congenital amaurosis 10 (ref. 115), transthyretin amyloidosis116,
transfusion-dependent β-thalassaemia
117
and sickle cell disease
117
, and
for the engineering of chimeric antigen receptor (CAR)T cells
118
. Ongo-
ing trials suggest that such approaches have acceptable safety profiles
in these applications, with recent clinical approval in the UK
119
, followed
by FDA approval in December 2023 (ref. 120), for CRISPR–nuclease
treatment of haemoglobinopathies
34
. Progression into clinical testing
and the recent FDA approval represent major milestones for this rela-
tively new technology, positioning the field for expansion to new appli-
cations, including OA. Notably, safety concerns, including off-target
editing121–123, the immunogenicity of bacterial Cas9 proteins124,125, and
the risks associated with unintended insertions at sites of genomic
double-strand breaks126, must continue to be carefully monitored.
Delivery strategies for Cas9-mediated gene therapy must accom-
modate constraints of the large physical size of Cas9 (127 kDa and
160 kDa for Staphylococcus aureus Cas9 and Streptococcus pyogenes
Cas9, respectively), as well as the highly negatively charged phos-
phate gRNA backbone. In vivo methods that have been explored for
Cas9-mediated gene therapy include viral vectors, mRNA delivery
and packaging of CRISPR-Cas9 ribonucleoprotein (RNP) complexes
Nature Reviews Rheumatology
Review article
in synthetic delivery vehicles127. The discussion here is focused on the
promise of non-viral lipid128–130, polymer131,132 and gold133,134 nanoparti-
cles, which have the potential to circumvent both manufacturing and
immunogenicity limitations of viral vectors135.
In an in vitro proof-of-concept study that applied CRISPR–Cas9
targeting to reduce the expression of MMP13 in human chondro-
cytes, a CRISPR–Cas9 RNP system was used to create non-homologous
end-joiningindels in the target gene encoding this protease (MMP13)136.
Achieving an editing efficiency of over 60%, the 3D spheroid cultures
of edited human chondrocytes contained higher accumulated levels of
cartilage matrix proteins after 1 week than cultures of non-edited chon-
drocytes. In a more advanced in vivo therapeutic approach, research-
ers have also created hybrid liposome and cell-derived exosome
structures that integrate a chondrocyte-targeting peptide to deliver
MMP13-targeting CRISPR–Cas9 components to chondrocytes137. Appli-
cation of this system in vivo diminished the progression of surgically
induced OA in rats. A porous silicon and polymer hybrid nanoparticle
system that delivers a MMP13-targeting CRISPR–Cas9 RNP complex
can similarly reduce the expression of Mmp13 by ~60% in a mouse
model of PTOA66.
Taken together, these studies establish the potential therapeu-
tic utility of CRISPR-Cas9 RNP delivery systems to edit cells of the
joint in situ as a route to suppressing OA pathogenesis. RNA-guided
gene editing also has strong potential to be applied to create designer
cells exvivo that can then be delivered into the joint, as an alterna-
tive to editing endogenous cells. The following section will explore
the cutting-edge application of CRISPR and other strategies for the
engineering of improved, next-generation cell therapies.
Genetically engineered cell therapies
Initial studies of cell-based therapies for application in OA sought to
leverage the capacity of cells (typically mesenchymal stromal cells or
autologous chondrocytes) to promote the synthesis of new cartilage
tissue, either in situ or by the ex vivo tissue engineering of constructs
intended for subsequent implantation in vivo. However, these con-
ventional cell-based therapies produce fibrocartilage rather than a
hyaline matrix
27,138
and had inconsistent outcomes owing to variability
in the cell types, donors and recipients tested139. Genetic engineering
strategies through viral
140,141
or non-viral
142–145
means have been used to
enhance the function of cell therapies by promoting their constitutive
expression of factors that combat OA. Considering the complexity of OA
pathology, an attractive approach made possible by synthetic biology
is to engineer ‘intelligent’ musculoskeletal cell therapies that can exert
therapeutic functions via autoregulated synthetic gene circuits146. Suc-
cessfully engineered cells should be able to integrate multiple cytokine,
mechanical and matrix-related inputs to respond in a dynamic way to
modulate OA pathogenesis.
Cell design concepts
Engineered, ‘designer’ cells have the potential to overcome the histori-
cal shortcomings of cell-based OA therapies by performing regenerative
functions that are both feedback regulated and localized to the arthritic
joint. Designer cell therapies commandeer the underlying modular
composition of signalling proteins and networks
147,148
and rewire the sig-
nalling pathways to elicit specific cellular responses to non-canonical
inputs. Native signalling architectures contain sensing and actuating
domains that dictate the cellular responses to particular microenvi-
ronmental inputs
149
(Fig.4a). For example, in some signalling pathway
archetypes, a receptor (that is, sensing module) binds to a cognate
ligand, leading to a conformational change that potentiates activity of
a transcription factor or phospho-regulator (that is, actuation or pro-
cessing modules). These actuators then induce a change in the intracel-
lular state, such as gene transcription
150–152
or protein degradation
153,154
.
Using technologies such as CRISPR-based platforms, chimeric protein
engineering and novel protein design, synthetic biologists can recon-
figure existing or engineer de novo sensor, processing and actuation
modules to produce artificial signalling networks149. These modules
can be integrated within endogenous signalling pathways or developed
orthogonally to control the cell response (behaviour) to a particu-
lar input. Thus, native signal transmission modules can be rewired
to couple specific inputs of interest (for example, signals relating to
inflammation and matrix damage) to pre-determined, selected outputs
(for example, attenuation of inflammation and production of growth
factors to repair the matrix). Strategies for designing cell behaviours
often involve either redirecting outputs of native receptors or develop-
ing orthogonal receptors that interface with engineered gene circuits
for custom, tailored input-to-output relationships. A subset of artifi-
cial receptor designs that enable the detection of markers associated
with OA pathology are illustrated in Fig.4, and relevant study details
are listed in Table2. A comprehensive discussion of sensor–actuator
platform design characteristics is beyond the scope of this Review,
and interested readers are referred to specialized reviews on the topic
elsewhere149,155,156.
Synthetically regulated cell therapies
In this section, we consider next-generation strategies for redirecting
the behaviours of cells in the context of an OA joint by using gene-editing
techniques to wire custom-selected input-to-output relationships,
with the goal of developing cells that mediate feedback-controlled,
localized therapeutic effects in the OA joint.
Redirecting outputs from native signalling pathways. The tradi-
tional method of reconfiguring natural cell signalling pathways is to
interface existing receptors with artificial gene circuits through the
use of engineered transcriptional response elements. This strategy is
widely used to generate reporter systems of cell signalling pathways,
such as the NF-κB, NFAT and JAK–STAT signalling pathways, and can also
be used to manipulate the expression of therapeutic transgenes157–160.
These and similar systems have been deployed extensively in arthritis
therapies to dynamically govern the expression of anti-inflammatory
proteins161–164. For example, one study has applied CRISPR–Cas9 edit-
ing in stem cells to insert an anti-inflammatory transgene (in this case,
the gene encoding either IL-1RA or soluble TNF receptor 1) into an
inflammation-inducible locus (in this case, the mouse Ccl2 locus),
reprogramming the cells to respond to IL-1 and TNF-mediated inflam-
mation in an autoregulated fashion165 (Fig.4b). The endogenous
promoter of Ccl2 could dynamically govern the expression of either
transgene to achieve rapid, cytokine dose-dependent modulation
of transgene expression. This strategy yielded an approach to engi-
neering cells that mediates sustained, long-term, feedback-controlled
release of anti-inflammatory mediators. Such engineered cells have
been used pre-clinically in the K/BxN serum transfer-induced model
of arthritis to antagonize disease, illustrating the promise of designer
cell-based therapies to mitigate OA progression166,167.
CRISPR-based technologies with functions outside of the canoni-
cal Cas9 nuclease double-strand break-inducing activity exist and can
be applied to silence, rather than redirect, native signalling within
cells. For example, researchers have leveraged the RNA-guided CRISPR
Nature Reviews Rheumatology
Review article
interference (CRISPRi) platform to silence the expression of IL-1 and
TNF receptors in adult-derived stem cells, as well as in cells from the
intravertebral nucleus pulposus
168,169
. Given the association between
OA and dysregulated IL-1 and TNF signalling, this approach holds
promise as a therapy for OA. The CRISPRi platform relies on persistent
expression of a catalytically dead form of Cas9 (dCas9) that is fused
TRADD
RIP
NF-κB
NF-κB JNK
NFκB NF-κB
NF-κB
MAPK
NFAT
TRAF2 MEKK3/6
NIK
IKK
TNF
TNFR 1 Actuator
Processor
T cell functions
T cell-mediated
apoptosis
MATRIX platform MESA platform
Soluble VEGF
VEGF
scFV
IL-2
dCas9
T cell
uPAR
CD28 Granzyme B
Perforin
Cytokines
CD3ζ
Anti-uPAR scFv CAR
Nucleus
Nucleus Nucleus
Sensor
Cytoplasm
Extracellular space
Cytoplasm
Extracellular space
Nucleus
sTNFR1
CRISPR–Cas9 editing
CRISPR–Cas9gRNA
b Rewired endogenous signallinga Native signalling domains
d Orthogonal gene circuitsc Engineered receptors
Designer promoter sequences
IL-1
IL-1RA
IL-1R1 complex
TRPV4
Ca2+
Dynamic loading
sTNFR1
Calcium signalling
Downstream
NF-κB activation
Synthetic NF-κB
responsive promoter
PTGS2 promoter IL1RA
IL1RA
TF-responsive promoter
PTGS2
upregulation
Ccl2 promoter 5’ UTR C cl2 exon 1
Ccl2 exon 1
Ccl2 promoter 5’ UTR sTNFR1 transgene
sTNFR1 IL2 gene
TRADD
TRAF2
NF-κB
IκB
Senescent cell ×
VP64
VP64
TNFR1
Protease
sTNFR1
Synthetic
transcription
factor
γ-secretase
and ADAMTS
Cognate scFV
Ligand-capturing
substratum
Soluble
ligand
Synthetic Notch
receptor
TNF
Fig. 4 | Synthetic gene circuit designs for OA-specific engineered cell
therapies. Artificial sense/response platforms for OA cell therapy can regulate
biologic drug production in a feedback-controlled manner. a, Endogenous
signalling pathways contain sensing, actuation and processing domains.
In the pro-inflammatory TNF signalling pathway, the TNF detection domain
(sensor domain) recognizes pro-inflammatory TNF to initiate signalling; the
TNF ‘death domain’ (actuator) recruits secondary messengers that activate
downstream transcription factors (processors) that translocate to the nucleus
to regulate gene expression. b, Technologies such as CRISPR–Cas9 (ref. 165)
or designer promoter sequences174 can be used to rewire endogenous
pro-inflammatory signalling for automated production of anti-inflammatory
products. On the left, CRISPR–Cas9-mediated insertion of a transgene leads to
upregulated production of sTNFR1 in response to TNF signalling. On the right,
the introduction of a designer promoter sequence results in IL-1RA production
(and IL-1 antagonism) following TRPV4-mediated calcium signalling induced by
mechanical loading. c, Engineered receptors, such as chimeric antigen receptors
(CARs), can contain sensing motifs that recognize pathological markers. For
example, the recognition of senescent markers by the anti-uPAR scFv CAR T cell
receptor179 invokes a signalling cascade that leads to the expression of various
T cell effector genes and T cell-mediated apoptosis of the senescent cell.
d, Orthogonal receptors and gene circuits can be designed to respond to particular
fixed ligands (using the MATRIX platform)197 or soluble ligands (using the MESA
platform)226. On the left, the binding of a synthetic Notch receptor to ligands
displayed on a biomaterial surface leads to cleavage and release of a synthetic
transcription factor that induces the expression of sTNFR1 (antagonizing TNF
signalling). On the right, VEGF binding to the synthetic MESA receptor causes
receptor dimerization, protease cleavage and subsequent release of a dCas9–VP64
fusion protein containing a gRNA complementary to IL2, leading to IL-2 secretion.
IFNγ, interferon γ; IκB, inhibitor of κB; IKK, IκB kinase; JNK, Jun N-terminal kinase;
MAPK, mitogen-activated protein kinase; MATRIX, material activated to render
inducible expression; MEKK3/6, Mitogen-activated protein/ERK kinase kinase 3/6;
MESA, modular extracellular sensor architecture; NFAT, nuclear factor of
activated T cells; NF-κB, nuclear factor κB; NIK, nuclear factor κB-inducing kinase;
PTGS2, prostaglandin-endoperoxide synthase 2; RIP, receptor-interacting protein;
scFv, single-chain variable fragment; sTNFR1, soluble TNF receptor 1; TRADD,
TNF-associated death domain; TRPV4, transient receptor potential vanilloid 4;
uPAR, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial
growth factor; VEGFR, VEGF receptor.
Nature Reviews Rheumatology
Review article
with a repressor domain, typically the KRAB domain from human
zinc finger KOX1 (dCas9–KRAB)
170–172
. However, the recently devel
-
oped CRISPRoff platform, which makes use of dCas9–KRAB fused
to DNMT3A and DNMT3L, results in long-term silencing of the target
gene even after transient delivery of the CRISPR reagent to the cell173.
Thus, strategies for delivering CRISPRoff RNPs or similar epigenetic
Table 2 | Cellular engineering strategies to mitigate OA-related pathological conditions
Cell-modifying approach Engineered
cell type OA-relevant
therapeutic function Result Refs.
Fusion of VP64 transactivator to
dCas9 for gRNA-speciic activation
of gene expression (CRISPRa)
HEK293T
cells Activation of IL1RN
expression Increased the expression of IL1RN and subsequent production of IL-1RA
by the engineered cells in vitro 224
Doxycycline-inducible gene circuit Human MSCs IL-1RA production In the presence of IL-1, ex vivo cartilage tissue containing the engineered
cells produced increased amounts of glycosaminoglycan and collagen
and showed partial recovery of tissue mechanical properties
225
Logic-gated gene circuit for NF-κB-
dependent cytokine-sensitive
activation of transgene expression
HEK293T
cells Expression of
IL-22RA, IL-4 and
IL-10
In a mouse model of imiquimod-induced psoriasis, cell implantation
increased the serum levels of IL-4 and IL-10; decreased the serum levels
of TNF and IL-22; and reduced the thickness and immune iniltration of
psoriasis lesions
157
dCas9–KRAB-mediated gene
knockdown hADSCs Knockdown of IL1R1
and TNFR1 The engineered cells had reduced levels of NF-κB activity and had an
improved capacity to differentiate into chondrocytes and produce
glycosaminoglycans in the presence of TNF and IL-1 in vitro
168
Autoregulated gene circuit using
synthetic NF-κB-responsive promoter
for transgene expression
iPSCs IL-1RA production In the presence of IL-1, ex vivo cartilage containing the engineered cells
produced increased amounts of glycosaminoglycans; had decreased
expression of pro-inlammatory genes; and had increased expression of
anabolic genes
161
Synthetic mechanogenetic NF-κB-
inducible and PTGS2-inducible gene
circuits
Primary
porcine
chondrocytes
IL-1RA production Ex vivo cartilage tissue containing the engineered cells had increased
expression of IL-1RA following mechanical and osmotic loading and
were protected against loss of glycosaminoglycan content following
treatment with IL-1
174
CRISPR–Cas9 gene insertion for
delivery of anti-inlammatory
molecules in vivo
iPSCs IL-1RA production In the K/BxN mouse model of inlammatory arthritis, injected hydrogel-
encapsulated iPSCs reduced clinical pain scores and structural damage
of articular cartilage, and increased serum levels of IL-1RA
166,167
Synthetic CAR that targets the
senescent marker uPAR Primary
mouse T Cells Apoptosis of
senescent cells In a patient-derived xenograft model of non-small-cell lung cancer
in mice and in mouse models of carbon tetrachloride-induced liver
ibrosis and NASH-induced liver ibrosis; CAR T cell treatment was
accompanied by T cell iniltration in the lungs and decreased expression
of senescence-associated markers
179
Engineered HLA-DR1 CAR linked to a
type II collagen peptide, for targeting
autoreactive T cells
Primary
mouse CD8+
T cells
Suppression of
collagen type
II-reactive T cells
In a mouse model of collagen-induced arthritis, CAR T cell treatment
reduced the levels of type II collagen-speciic autoantibodies and
Tcells and resulted in a lower incidence of arthritis.
184
Dimerized extracellular receptors
tethered to intracellular endogenous
signalling domains (GEMS platform)
WEN1.3
hybridoma
cells
IL-10 production Within the receptor-expressed cells, rerouted MAPK and JAK–STAT
signalling led to upregulated secretion of IL-10 in the presence of the
ligand of interest (RR120) in vitro
152
dCas9-transcription factor complex
containing synthetic gRNA that
targets a native transcription factor
(GEARs platform)
HEK293T
cells and
Jurkat T cells
IL-12 production In GEARN FAT-expressing Jurkat T cells, intracellular calcium signalling
could induce the expression of IL-12; in GEARSMAD2-expressing HEK293T
cell or GEARp65-expressing HEK293T cells, the detection of TGFβ or TNF,
respectively, could induce the expression of IL-12 in vitro
159
Dimerization of scFv-derived
modular receptors tethered to a
dCas9–transcription factor fusion
protein controlling IL-2 expression
(MESA platform)
Jurkat T cells IL-2 production VEGFR MESA receptor-expressing cells could secrete IL-2 in the
presence of soluble VEGF in vitro 226
Anti-GFP nanobody-based
synNotch receptor paired with
a GFP-immobilizing substratum
(MATRIX platform) for inducible
sTNFR1 expression
Mouse MSCs sTNFR1 production SynNotch-expressing cells selectively produced sTNFR1 in the
presence of GFP ligand; had reduced NF-κB transcriptional activity
(suggesting decreased TNF signalling) and reduced expression of
pro-inlammatory genes, including Il6, Ccl5 and Icam1, in vitro
197
CAR, chimeric antigen receptor; CRISPRa, clustered regularly interspaced short palindromic repeats activation; dCas9, catalytically dead Cas9; GEMS, generalized extracellular molecule
sensor; GEARs, generalized engineered activation regulators; GFP, green luorescent protein; hADSCs, human adipose-derived stem cells; HEK293T, human embryonic kidney 293T; IL-1RA,
interleukin-1 receptor antagonist; IL-22RA, interleukin-22 receptor subunit α; iPSC, induced pluripotent stem cell; JAK–STAT, Janus kinase–signal transducers and activators of transcription;
KRAB, kruppel associated box; MATRIX, material activated to render inducible expression; MESA, modular extracellular sensor architecture; MSCs, mesenchymal stem cells; NFAT, nuclear
factor of activated T cells; NF-κB, nuclear factor κB; p65, RelA subunit of nuclear factor κB; PTGS2, prostaglandin-endoperoxide synthase 2; RR120, Reactive Red 120 dye; SMAD2, mothers
against decapentaplegic homologue 2; sTNFR1, soluble tumour necrosis factor receptor 1; TGFβ, transforming growth factor β; uPAR, urokinase-type plasminogen activator receptor;
VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
Nature Reviews Rheumatology
Review article
editors to cells might enable sustained endogenous gene silencing to
mitigate OA.
Alternatively, transgene cassettes can be transduced into cells to
create synthetic gene circuits that are dynamically responsive to envi
-
ronmental cues and produce anti-inflammatory mediators. For exam-
ple, researchers have investigated whether native signalling through
the cartilage mechanosensitive channel TRPV4 could be comman-
deered to autoregulate mechano-dependent or osmo-dependent
transgene expression of IL-1RA in chondrocytes1 74 (Fig.4b). In the
engineered primary porcine chondrocytes, both mechanical and
osmotic loading of engineered cartilage could increase the expression
of IL-1RA when the transgene was inserted downstream of a synthetic
NF-κB-inducible promoter or a PTGS2 promoter and delivered to cells
via a lentivirus vector. Such artificial mechanogenetic circuits could
regulate the expression of IL-1RA and protect ex vivo engineered car-
tilage from IL-1-mediated degradation. These approaches, as well as
others, integrate artificial gene circuits with native signalling pathways
and highlight advancements that could be utilized for engineering
cells that can survey their microenvironments to fight OA-associated
pathological conditions.
Repurposing native signal transmission via engineered receptors.
A separate synthetic biology strategy links desired cell behaviours to
targeted inputs through the design of CAR T cells175–178. The purpose of
the CAR is to provide a mimetic of native T cell receptor (TCR) signal-
ling to control cytokine production and T cell-mediated cytotoxicity
in response to a particular antigen of choice. For example, researchers
have engineered T cells that target senescent cells by CAR recogni-
tion of the urokinase-type plasminogen activator receptor (uPAR),
a surface marker that is broadly expressed on senescent cells179 (Fig.4c).
The researchers deployed these cells in several models of disease
associated with senescence, including lung adenocarcinoma and liver
fibrosis, achieving ablation of pathological senescent cells in vivo. In
the context of OA, dysregulated cells in the joint defined in part by the
expression of fibroblast-activating protein (FAP) are implicated in
disease180,181. Thus, the successful targeting of FAP by CAR T cells might
represent an opportunity to attenuate progressive loss of matrix in
OA
182
. Furthermore, proof-of-principle studies have shown the poten-
tial of engineered CAR T cells and regulatory T cells in ameliorating dis-
ease in models of autoimmune arthritis183,184. Hence, CAR T cell-based
strategies that eliminate cells that contribute to OA pathogenesis are a
promising future direction for cellular immunotherapy. Although the
current panel of clinically approved CAR T cell products is generated
through lentiviral transduction, the approach of RNA-guided gene
editing continues to gain traction as delivery and editing strategies
become more efficient185–187.
Customizing cell behaviour via orthogonal receptors and gene
circuits. In the context of cell engineering, orthogonality refers to the
capacity of an engineered signalling platform to function indepen-
dently of native pathways owing to having only minimal or no mutual,
overlapping components. Unlike approaches that repurpose native sig-
nalling, orthogonal receptors can link an individual, preselected output
to a single, specific input. For example, unlike the previously described
NF-κB-regulated or Ccl2-regulated gene circuits, an orthogonal circuit
could selectively discriminate between inputs from IL-1 and TNF. Impor-
tantly, many soluble factors such as cytokines and chemokines have
pleiotropic roles and are not exclusively pathogenic mediators. Thus,
incorporating disease-specific biomarkers to indicate whether cells
are in a deleterious setting would provide more sophisticated control
over the functionality of designer cells.
One approach to producing orthogonal input-to-output circuits
is inspired by a class of native receptors known as regulated intramem-
brane proteolysis (RIP) receptors. RIP receptors are typically juxtacrine
signalling channels, and, as such, generate cellular outputs onlywhen
in direct contact with immobilized ligand. This approach offers the
ability to spatially constrain transgene expression. The Notch1 recep-
tor is one such member of the RIP receptor family; Notch signalling is
activated following the generation of mechanical forces that exceed
~10 pN owing to receptor interactions with anchored ligands188,189.
This mechanical force induces a conformational change in the recep-
tor, exposing protease cleavage sites. Subsequent proteolysis of the
receptor enables translocation of the Notch intracellular domain
(NICD) to the nucleus, where the NICD modulates the expression of
target genes. A suite of artificial RIP receptors has been developed,
the most prominent of which is known as synNotch
190,191
. SynNotch
receptors are designed by replacing the Notch extracellular domain
with a chosen sensor motif (such as a single-chain fragment variable),
while also exchanging the NICD for an artificial transcription factor
(such as the tetracycline transactivatorsimilar to that used in sev-
eral doxycycline-inducible systems)192. Thus, synNotch serves as a
customizable, orthogonal signalling platform that tightly couples
receptor binding to a specific immobilized ligand and transgene
expression
193–195
. Cells that express synNotch gene circuits or other
orthogonal receptor platforms are commonly engineered using
viral transduction
193,196
, transposase insertion
197
or CRISPR-mediated
targeted integration198,199.
In 2023, researchers developed a platform known as MATRIX
(material activated to render inducible expression) that combines
synNotch-based cell engineering and biomaterial design. In this sys-
tem, synNotch-expressing cells are designed to interact with bioma-
terial surfaces that have been engineered to capture soluble ligands
of interest
197
(Fig.4d). The MATRIX platform enables localized cell
responses to bulk, soluble inputs and can potentiate the expression
of anti-inflammatory factors, CRISPR-based transcriptome modi-
fiers and stem cell differentiation factors. Future implementations of
MATRIX might involve the transplantation of synNotch cells embed-
ded in scaffolds or hydrogels to the joint to locally mitigate cytokine
signals that promote OA. Another opportunity for leveraging syn
-
thetic RIP receptors involves programming synNotch with affinity
motifs so that the receptors can directly recognize damaged cartilage.
This approach might enable cells to recognize disease-relevant fea-
tures of the matrix for localized production of transgene products
in a pathology-dependent manner. Designing engineered cell tech-
nologies to detect OA-specific biomarkers, such as degraded matrix
constituents, should provide opportunities to improve the delivery
of genetically encoded cargos to the OA joint.
Clinical perspectives and challenges
The standard treatments for OA have remained stagnant for decades;
challenges contributing to this stagnation include poor and/or expen-
sive animal models, difficulties with patient recruitment for clinical
trials, and the diversity in idiopathic, post-traumatic and multi-joint
OA subtypes. The majority of studies discussed in this Review showed
promise in reducing joint degeneration in rodent models of surgically
induced OA, a scenario that approximates prophylactic treatment
following a joint injury to block the development of PTOA in humans.
Although these models are valuable for therapeutic screening owing
Nature Reviews Rheumatology
Review article
to their relatively low cost and high throughput, they have proven to
incompletely predict efficacy in larger animal models or humans.
Despite idiopathic OA being the most common clinical presentation
of disease, fewer models are available that reliably achieve develop-
ment of advanced OA in the absence of injury. Furthermore, spon-
taneous OA models tend to be more time-consuming and costly to
implement. Another limitation to many preclinical model studies is that
they rely on histological outcomes to assess treatment success, and
such measures do not necessarily predict improvements in pain
and function. More translationally relevant metrics related to joint
function are typically associated with higher cost and technical bur-
den. Therefore, the efficacy of novel interventions in animal models
of PTOA cannot be assumed to reliably predict the results of human
clinical trials, where patients often present with late-stage, idiopathic
disease that might most benefit from an intervention that can reverse
the effects of pre-existing cartilage loss and other severe joint structural
changes. Additionally, because most existing treatment strategies
target articular cartilage and the synovium, there tends to be a lack of
consideration of other aspects of the total joint. OA-relevant crosstalk
between joint tissues, including the cartilage and synovium
200
, but also
the meniscus
201
, infrapatellar fat pad
202,203
and subchondral bone
204
,
must be considered with regard to determining their role in influencing
the efficacy of treatment and their targetability with therapeutic agents.
These challenges motivate the need for new, durable interventions that
can selectively deliver therapeutic factors in a regulated fashion to
mitigate total joint disease.
Both RNA medicines and ‘smart’, engineered cells have the poten-
tial to address the pleiotropy of mediators involved in OA progres-
sion, ranging from pro-inflammatory cytokines to matrix-degrading
enzymes. The application of these technologies in other disease areas
has set the stage for more in-depth exploration in OA. In the case of
RNA medicines, advances in RNA chemistry and delivery technologies
have enabled proof-of-concept studies that indicate that the historical
limitations of RNA-based therapies in OA are surmountable. How-
ever, major hurdles remain, particularly in the context of chronic and
late-stage disease. RNAi strategies such as siRNA, miRNA or anti-miRNA
might be most appropriate for early intervention in PTOA, whereas
transient or permanent expression of growth factors enabled by mRNA
or CRISPR–Cas9 technologies might hold more promise in advanced
stages of disease. Furthermore, translating transient, siRNA, miRNA
or anti-miRNA therapies will require studies determining their persis-
tence after intra-articular injection in the human synovial joint, with or
without carriers. Notably, encouraging evidence is available showing
sustained target knockdown with siRNA in humans that extends for up
to 6 months after a single treatment
205
, indicating the potential of this
approach for clinically relevant, durable management of chronic dis-
eases. Another delivery route that should be explored in OA is systemic
administration (especially subcutaneous), which has the potential
to reduce the requirement for clinical visits for intra-articular injec
-
tion and to expand treatment modalities to patients with multi-joint
OA. Self-administered, subcutaneous injections are already being
utilized, for example, in patients with rheumatoid arthritis receiving
anti-TNF antibody therapy206,207.
A major barrier to the development of RNA-based therapeutics is
the identification of effective targets that will mitigate OA, with many
candidates highlighted in this Review. A single genetic target might
not be sufficient to holistically remedy a total joint disease such as
OA, especially as patients often seek intervention after their joints
are already severely deteriorated. A related challenge is specifically
targeting the relevant intra-articular cells or tissues. One strength of
RNA-based strategies is the ability to quickly synthesize sequences
that target the expression or repression of, in theory, any gene in the
genome. The complexity of OA, combined with this technological fea-
ture, suggests a future opportunity to deliver cocktails (for example,
siRNAs that target multiple genes or RNP formulations that incorporate
multiplexed gRNAs to target multiple genomic sites) that can more
comprehensively address the pathological features of OA. For exam-
ple, blocking proteases, combined with promoting the expression of
pro-chondrogenic genes, might be an effective strategy for tipping
the balance in favour of cartilage anabolism over cartilage catabolism
in osteoarthritic joints. Drug combination approaches are commonly
used in cancer therapies, indicating a promising avenue for tackling
the complexity of OA.
The cell design strategies described in this Review introduce
the power of synthetic biology for commandeering cellsignalling
networks to elicit pro-regenerative functions. By engineering cells to
produce therapeutic products in a regulated manner, outputs can be
calibrated to the demands of the pathological condition, potentially
reducing deleterious adverse effects. Concurrent breakthroughs in
our understanding of OA pathophysiology highlight which cellular
decision-making processes to manipulate, via either RNA medicines
or cell design, and which desirable cellular functions to elicit in the
context of OA. The expansion of the CRISPR toolkit and related delivery
modalities will facilitate the manipulation of genetic and/or epigenetic
features that contribute to OA. Already, the field has made dramatic
strides that circumvent concerns associated with double-strand
breaks, including the use ofbase editors208–210, prime editors211–213,
and epigenetic regulators through the use of the aforementioned
CRISPRi and CRISPRoff systems. Future efforts might combine
nucleic acid delivery strategies to engineer native joint cells in situ
with artificial gene circuits. The growing body of preclinical evidence
validating these approaches in vivo should lead to their future clinical
translation in OA.
Effective deployment of cell-based therapies for OA will also
require considerable innovation to achieve safe and long-term per-
sistence of therapeutic cells. Identifying the best cell source for delivery
to the joint requires further investigation. For example, autologous
cells are the safest choice, with the lowest risk of rejection; however,
the use of an allogeneic ‘off-the-shelf’, universal cell product would
greatly simplify the manufacturing process in terms of meeting the
considerable quality control demands. Notably, genome engineer-
ing strategies with CRISPR–Cas9 have been implemented to mitigate
therapeutic cell-sourcing problems in the context of CAR T cell design
for cancer immunotherapies, and such strategies have potential in the
treatment of the chronic inflammation and cartilage degradation asso-
ciated with OA. Other considerations include developing strategies to
promote the survival and retention of engineered cells within the joint
after administration; the use of biomaterials are one such promising
strategy that warrants further exploration
167,214
. Overcoming these
challenges in the design and deployment of engineered cell therapies
is an area of ongoing and future investigation.
Gene and cell therapy products are intrinsically more complex and
less well-established than traditional small molecule drugs or recombi-
nant biologic drugs. Thus, the field is slowed by bespoke manufacturing
programmes that lack industry-wide standardization. Advances in
technology, such as the potential for the aforementioned off-the-shelf,
universal products, offer promise towards alleviating this burden.
Furthermore, in the past few years, notable progress has been made in
Nature Reviews Rheumatology
Review article
the translation of RNA therapies, CRISPR-based products and ex vivo
engineered cells to the clinic and/or various approaches have entered
the regulatory space through sponsor initiatives aimed at tackling
life-threatening or severely disabling diseases. Lessons learned in these
domains can be applied to employ these technologies in OA, despite
the challenges associated with navigating an only partially charted
regulatory terrain.
Conclusions
Despite many promising preclinical leads, widely adopted, disease-
modifying therapies for OA have yet to emerge. The pleiotropy of
molecular drivers of OAincreases the demand for selectivity and
well-regulated delivery of therapeutic factors to mitigate joint disease.
Breakthroughs in the fields of drug delivery, RNA chemistry and cell
engineering have positioned these technologies as candidate strategies
to achieve these therapeutic goals. Notably, RNA-based technologies
such as siRNA and CRISPR-based systems are benefitting both from
the engineering of carrier systems and from the chemical optimiza-
tion of the RNA components, which, in the case of siRNA, enables
carrier-free delivery. In addition, cell-design strategies enable robust,
disease-relevant input-to-output programmability. Such approaches
demonstrate the power of synthetic biology to commandeer cellsignal-
ling networks to prescribe pro-regenerative functions via gene circuit
design and CRISPR-based gene editing. Engineered cells, such as CAR
T cell therapy, have already proven revolutionary in demonstrating
disease-modifying behaviours in the context of cancer immunotherapy
and, more recently, in mitigating inflammation. Such advances fore-
shadow the utility of applying synthetic biology tools to engineer cells
for OA disease modification.
As the population ages and risk factors associated with OA
onset and progression become more prevalent, interest in durable,
disease-modifying osteoarthritic agents will only continue to rise.
Advances in chemical and delivery technologies for RNA medicines are
elevating this class of drugs towards the mainstream, motivating the
development of such technologies for new applications such as for
the treatment of OA. Furthermore, emerging, cutting-edge cell manipu-
lation and synthetic biology techniques are evolving that will yield a
next generation of ‘intelligent’ cell therapies capable of overcoming
the weaknesses of previous cell-based tissue engineering and related
approaches. The progression of genetically regulated treatments
for OA might facilitate safer and longer lasting treatment options to
improve patient outcomes.
Published online: xx xx xxxx
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Acknowledgements
The work of the authors is supported by the National Institutes of Health National Institute of
Arthritis and Musculoskeletal and Skin Diseases (NIH NIAMS) R21 AR079245 (C.L.D., J.M.B.,
R.D., B.L.W.), R21 AR078636 (C.L.D., J.M.C., N.F.), R21 AR079683 (C.L.D., J.M.B., B.L.W.), and R01
AR078666 (C.L.D., R.D., C.R.D.), as well as the NIH National Institute of Biomedical Imaging
and Bioengineering (T32-EB021937, C.R.D.). Additional support was provided by the Arthritis
National Research Foundation Judy E. Green Valiant Women’s Fellowship.J.M.C is supported
by NIGMS of the National Institutes of Healthunder award number T32GM007347, theNatural
Sciences and Engineering Research Council of Canada (NSERC), and the Rheumatology
Research Foundation (RRF).
Author contributions
All authors researched data for the article, wrote the article and reviewed and/or edited the
manuscript before submission. C.R.D., C.L.D., B.L.W., J.M.C., R.D., N.F. and J.M.B. contributed
substantially to discussion of the content.
Competing interests
C.L.D., C.R.D., and R.D. hold patents related to drug delivery (US20170096517A1 (C.L.D.);
WO2018213361A1 (C.L.D.); WO2019068098A1 (C.L.D., C.R.D.); US20180064749A1 (C.L.D.);
WO2023059833A1 (C.L.D. and R.D.); WO2023034561A2 (C.L.D.); US2023/018982 (C.L.D.,
C.R.D. and R.D.; iled)). J.M.B., B.L.W., and C.L.D. hold patents related to cell engineering (US-
11319555-B2 (J.M.B.); US-10954513-B2 (J.M.B.); US-20230295262-A1 (J.M.B., B.L.W. and C.L.D.;
iled)). These patents relect long-standing interest and innovation in the ields of RNA delivery
and cell design.
Additional information
Peer review information Nature Reviews Rheumatology thanks Farshid Guilak and Christopher
H. Evans and the other, anonymous, reviewer(s) for their contribution to the peer review of this
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