GENETICS 0889857X/02 $15.00
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GENE THERAPY FOR

RHEUMATOID ARTHRITIS
Lessons from Animal Models, Including
Studies on Interleukin-4, Interleukin-10,
and Interleukin-1 Receptor Antagonist
as Potential Disease Modulators

Fons A.J. van de Loo, PhD, and Wim B. van den Berg, PhD

IDENTIFYING THE GENE THERAPEUTIC TARGETS

IN ARTHRITIS

Rheumatoid arthritis (RA) and animal models of arthritis demon-

strate inammation of the synovial joints leading to the destruction of
articular cartilage and eventually loss of function. Although the cause
of RA is still not fully understood, it is long recognized that proinam-
matory cytokines, particularly tumor necrosis factor (TNF)- and in-
terleukin (IL)-1, have an important role in its pathogenesis. In vitro
culture studies using recombinant proteins and subsequent in vivo anal-
ysis in animals, including mice, rats, and rabbits, have shown that TNF
is a potent proinammatory mediator, whereas IL-1 is a potent cartilage
catabolic mediator. The in vivo relevance of both cytokines in experimen-
tal arthritis emerged from studies with neutralizing antibodies and solu-
ble receptors of TNF and IL-1 performed in mice and rabbits. The crucial

This work was supported by grant 902-27-218 from the Dutch Organization of Scien-
tic Research.

From the Department of Rheumatology, University Medical Center Nijmegen, Nijmegen,

The Netherlands

RHEUMATIC DISEASE CLINICS OF NORTH AMERICA

VOLUME 28 NUMBER 1 FEBRUARY 2002 127

128 LOO & BERG

role of these cytokines in arthritis was further substantiated in transgenic

and gene knock-out mice. Mice overexpressing human TNF developed
full signs of chronic destructive arthritis. Intriguingly, this TNF
transgenic arthritis could be completely blocked with antibodies against
the IL-1 receptor, implying that IL-1 is the pivotal downstream mediator
in this model.64 It also identies that TNF alone is not harmful to the
joint. More recent animal model studies have demonstrated that local
IL-1 production is independent of TNF and have identied IL-1 as the
pivotal mediator in arthritis.
Although the historic picture clearly underlines IL-1 as a major
therapeutic target, especially for cartilage protection, most clinical stud-
ies focused on blockade of TNF. The relative lack of interest in IL-1 was
further strengthened by the limited efcacy of IL-1 soluble receptor and
IL-1 receptor antagonist in the rst clinical trials in RA patients in sharp
contrast to the promising effects of anti-TNF antibodies and engineered
TNF receptors. It seemed that the initial choice of the soluble IL-1 type
I receptor for therapeutic studies was unfortunate. This receptor has
high afnity for IL-1 receptor antagonist (Ra); scavenging of endogenous
IL-1Ra seriously hampers the natural inhibitor of IL-1 activity and its
therapeutic application. Upcoming studies now focus on potential appli-
cation of the decoy type II receptor, which lacks high afnity for IL-1Ra
but still binds IL-1.
The other option, use of IL-1Ra itself as an inhibitor, remains a
moot point. Although the approach is straightforward, the efcacy is
hampered by the poor pharmacokinetics of the small molecule and the
need to fully occupy IL-1 receptors on the cell surface for prolonged
periods of time. IL-1 exerts full activation of responder cells on activation
of only 2% of the IL-1 receptors, and cells then remain activated for
days. In vitro studies revealed that IL-1 blocking could be obtained with
100- to 1000-fold excess of IL-1Ra over IL-1, making in vivo application
a difcult job. Our studies with IL-1Ra in experimental animal models
made it clear that arthritis could not be suppressed by repeated IL-1Ra
dosing with twice-daily injections but that arthritis was fully controlled
by sustained systemic application of IL-1Ra by miniosmotic pumps.32
This made IL-1Ra an obvious choice for rst attempts at local overex-
pression by local gene therapy.
Local gene therapy offers the potential advantage of conned and
prolonged overexpression of inhibitors at dened sites,1921, 67, 68 and this
avoids toxic systemic side effects, prevents blocking of the benecial
effect of the proinammatory cytokines in other tissue compartments,
and increases the local concentration of these anti-inammatory cyto-
kines. As such, it provides a challenging alternative to cytokine-directed
targeting in chronic arthritis, where systemic administration of neutraliz-
ing antibodies and engineered soluble receptors is nowadays accepted
as a promising therapeutic modality.
 GENE THERAPY OF RHEUMATOID ARTHRITIS 129

LOCAL GENE THERAPY IN COLLAGEN-INDUCED

ARTHRITIS

Interleukin-1directed Gene Therapy

Interleukin-1 Receptor Antagonist

To obtain local overexpression of IL-1Ra, we followed the ex vivo
approach introduced by Evans and Robbins,19 using retroviral gene
constructs and in vitro transfection of cells. A retroviral vector containing
human IL-1Ra cDNA was used to transfect the NIH-3T3 broblast cell
line, and clones, stable-transduced and producing high amounts of IL-
1Ra, were selected by limiting dilution. Two hundred thousand trans-
duced cells of an IL-1Raproducing clone were injected into the knee
joint cavity of mice shortly before the expected onset of collagen-induced
arthritis (CIA). This treatment markedly ameliorated the onset and sever-
ity of collagen arthritis in that joint, whereas control cells transduced
with an empty virus were ineffective.6 Detailed analysis revealed that
IL-1Raproducing cells lined up along the synovial lining cells on intra-
articular injection and remained at this site for at least 14 days. No graft-
versus-host reaction was seen with these cells, which are histocompatible
for the CIA-sensitive DBA1 mouse strain. Immunohistochemistry analy-
sis identied prolonged IL-1Ra production by these cells.
A safety preclinical study using transfer of retrovirally transduced
autologous synovial broblasts into metacarpophalangeal joints of post-
menopausal women with RA shortly before expected joint replacement
has provided evidence that the procedure is possible in patients and
yields consistent expression of IL-1Ra in the synovial tissue.21 A phase
II efcacy study is being planned.
A whole range of other virus-based vectors have now been evalu-
ated for the overexpression of IL-1Ra in experimental arthritis. Local
treatment of a rabbit knee joint with a replicationdecient herpes sim-
plex virus (T/0) carrying the IL1Ra gene signicantly inhibited leuko-
cytosis and synovitis in an experimental model of arthritis generated by
an overexpression of IL1. In contrast to retroviral vectors, which only
infect proliferating cells, adenoviral vectors can infect almost all cells,
and an intermediate step to enrich for transduced cells in vitro is not
needed. Interestingly, direct injection of adenoviral gene constructs in
the joint space results in almost selective infection of the synovial lining
cells, which form the rst cell layer encountered, with scant positively
in deeper layers. Topographically, this creates a situation much like the
condition after local injection in the knee joint of cells transduced in
vitro, because the transferred cells associate along the lining cells. Our
recent studies (Table 1) using replication-decient adenoviral vectors
with IL-1Ra showed clear efcacy in murine CIA.5, 7 Although the adeno-
viral vectors can be used to show the feasibility of different anti-in-
ammatory transgenes in experimental arthritis, their application in RA
is ambiguous because of transient gene expression. For this, adenoassoci-
130

Table 1. LOCAL (KNEE) AND IPSILATERAL (PAW) EFFECTS ON COLLAGEN-INDUCED ARTHRITIS OF INTERLEUKIN (IL) -1 RECEPTOR
ANTAGONIST, IL-4, AND IL-10 VIA ADENOVIRAL TRANSDUCTION OF THE MURINE SYNOVIUM
Intra-Articular Incidence of Collagen-induced Arthritis
Injection in Knee
(1  107 plaque Number of Number of Bone Erosion
forming unit [PFU]) Knee Joints Evaluated Ipsilateral Paw Joints Evaluated in Knee
Ad5.CMV-Luc* 90% (22)5 100% (22) 100%
Ad5del70-3 90% (22) 100% (22) 100%
Ad5.CMV-IL-1Ra 73% (22) 80% (22) 53%
Ad5.C3-Tat/HIV-IL-1Ra 50% (24) 42% (24) 10%
Ad5.RGD.CMV-IL-1Ra 36% (22) 54% (22) 39%
Ad5.CMV-IL-4 100% (41) 60% (41) 25%
Ad5.CMV-IL-10 75% (7) 40% (10) ND

*Adenovirus expressing luciferase (LUC) used as a control for Ad5.CMV-IL-1Ra, Ad5.C3-Tat/HIV-IL-1Ra, and Ad5.RGD.CMV-IL-1Ra
Adenovirus (empty/no transgene) used as a control for Ad5.CMV-IL-4 and Ad5.CMV-IL-10
Adenoviral vector containing IL-1Ra gene under the control of the disease-inducible promoter (C3-Tat/HIV)
Tropism modied adenoviral vector with RGD motif in the HI loop of the ber knob
Bone erosion was evaluated by radiographic analysis with an arbitrary score ranging from 0 to 5; mean values of Ad5.CMV-Luc (3,8) and Ad5del703 (2,8) were set
to 100%
CMV  cytomegalovirus, IL  interleukin, RA  receptor antagonist, TAT  transactivator of transcription, RGD  adenoviral vector with Arg-Gly-Asp, HIV 
human immundeciency virus, ND  not done.
 GENE THERAPY OF RHEUMATOID ARTHRITIS 131

ated virus (AAV) vectors were used as a highly efcient gene-delivery

system, which can facilitate long-term transduction. AAV has the advan-
tage that it can infect dividing and nondividing cells, and the AAV
genome integrates at a site-specic manner in a locus on human chromo-
some 19. The AAV-mediated expression of IL-1Ra showed a pronounced
inhibition of lipopolysaccharide (LPS)-induced joint inammation in
rats.62 Moreover, transgene expression could be reactivated by a second
LPS injection 80 to 100 days after recombinant AAV injection showing
the stable transduction of synoviocytes necessary for long-term protec-
tion against arthritis.62, 63 Final studies to be mentioned here regard
the systemic treatment by engraftment of human (hIL)-1RII transfected
human keratinocytes in the back of mice with CIA.11
Apart from suppression of joint inammation, local overexpression
of IL-1Ra also normalized the synthetic function of the chondrocytes in
articular cartilage. Joint inammation has a profound suppressive effect
on chondrocyte proteoglycan synthesis, and this lack of matrix formation
contributes to net cartilage damage during joint inammation. Using the
technique of the combination of RA synovial broblasts with coim-
planted cartilage in severe combined immunodeciency (SCID) mice, it
was shown that retroviral IL-1Ra transduction of the broblasts pre-
vented progressive chondrocyte-mediated cartilage degradation.58 In ear-
lier studies with neutralizing antibodies, we identied IL-1 as the crucial
mediator of this inhibition, and the studies with IL-1Ra gene transfer
further substantiated this crucial role of IL-1 in cartilage destruction in
arthritis. Although it is beyond the scope of this article, we would like
to mention that liposomal-mediated IL-1Ra gene transfer to rabbit knee
joints and the adenoviral transfer of IL-1Ra to joints in horses also
showed clear protection against cartilage erosion in an osteoarthritis
model.22, 23

Novel Interleukin-1 Inhibitors

Recently, novel IL-1 homologues were found, which indicates that
the IL-1 family is signicantly larger than IL-1, IL-1Ra, and IL-18. Most
of them (IL-1H1, IL-1H2, IL-1H3, FIL1, and IL-1HY2) have great similar-
ities to IL-1Ra and may show improved antagonistic properties.42, 45, 67
The association of increased intracellular IL-1Ra type 1 in inamed
joints during the resolution of CIA suggests an inhibition of IL-1.24
The discovery of novel IL-1 homologs and new information about the
intracellular IL-1 signaling pathway (Myd88, IL1 receptorassociated
kinases [IRAK], nuclear factor kappa B [NFB]) should trigger new
strategies to block IL-1 with gene therapy in arthritis.

Tumor Necrosis Factordirected Gene Therapy

Compared with the overwhelming interest in TNF as a therapeutic

target in RA, surprisingly little has been done on TNF-directed gene
therapy. This might be linked to paradoxic effects noted. When adenovi-
132 LOO & BERG

ral gene delivery of a human p55 TNF receptor, rendered dimeric by

fusion to an IgG backbone. A rebound to greater inammation was
observed at later time points, however, despite high levels of the TNFR
fusion protein, which is probably related to an increase in anticollagen
type II antibodies.65 The lack of effect of anti-TNF treatment in estab-
lished CIA is in line with observations about neutralizing antibodies in
this model,32 but the increase in autoimmune antibodies identies a risk
of prolonged and effective TNF neutralization.
Local treatment with adenoviral p55 TNFR-IgG fusion protein in
the knee joint of rats with CIA failed to show a benecial effect, although
systemic treatment was effective.25, 43 It can also be argued that anti-TNF
treatment exerts its effect mainly at other sites such as the lymphoid
system. Mageed et al53 followed an elegant approach showing the great
efcacy of TNF blocking in T-cell activation. Ex vivo infection of spleno-
cytes from arthritic DBA/1 mice with a retroviral vector encoding a p75
TNFR prevents the transfer of arthritis to recipient SCID mice. It identi-
es that gene transfer manipulation of the immune system with TNF
inhibitors can ameliorate arthritis.
The lack of a local effect might be linked to viral inammation or the
immunogenicity of soluble TNFRI, which hampers the administration of
higher virus particles. Transduction using recombinant AAV (no expres-
sion of viral genes) results in a very mild inammatory response. Intraar-
ticular injection of 1.5  10 particles of recombinant AAV to express a
lowimmunogenic form of human soluble TNFRI (STNFRI2.6D) effec-
tively reduced the severity of the arthritic disease in humanTNF
transgenic mice.81 The approach of injecting multiple joints (wrist, ankle,
and knee), although no soluble TNFRI could be found in sera, probably
mimics the effect of systemic adenovirus delivery.

Interleukin-1 and Tumor Necrosis Factor

Combination Therapy

Recently, high and steady levels in the circulation were achieved by

using a slow-release hyaluronic acid vehicle for IL-1Ra and binding of
polyethylene glycol (PEGylation) to TNFRI. A combination therapy of
both modied inhibitors showed greater than additive ameliorative
effects in CIA and antigeninduced arthritis (AIA).9 An adenoviral gene
construct encoding the IL-1 type I receptorIgG fusion protein was more
efcacious in the treatment of antigen-induced arthritis in the rabbit
knee when treatment was combined with a gene encoding a TNF-soluble
type I receptor protein.25 This showed the strength of combining several
cytokine inhibitors on arthritis, a strategy that could be easily transferred
to the local levels (inamed joint) by a multiple gene expression cassette
in a viral vector.
 GENE THERAPY OF RHEUMATOID ARTHRITIS 133

Interleukin-10 Gene Therapy in Arthritis

Apart from control by overexpression of cytokine inhibitors, addi-

tional control of arthritic and destructive processes by local overexpres-
sion of modulatory cytokines is an obvious alternative.34, 50 IL-10 and IL-
4 are known as inhibitors of TNF and IL-1 production by synovial cells.
Moreover, they suppress T helper (Th) 1driven processes. Additional
relief may be expected from upregulation of inhibitors such as IL-
1Ra, IL-1soluble and TNF-soluble receptors, and tissue inhibitor of
metalloproteinases by synovial cells or articular chondrocytes.
Adenoviral-mediated overexpression of murine IL-10 in the knee
joint of mice shortly before the expected onset of CIA had a moderate
suppressive effect on local arthritis (see Table 1). This is probably a
result of the fact that IL-10 has proinammatory potential as well as
upregulation of adhesion molecules and Fc receptor expression, thereby
enhancing leukocyte inltration at the local site.49, 51
Moreover, IL-10 is a potent suppressor of TNF but has limited effect
on IL-1 production in vivo. Remarkably, the most pronounced effect of
IL-10 overexpression in the knee joint is the complete suppression of
arthritis in the ipsilateral hind paw (see Table 1), without a consistent
effect on other paws. This ts with the concept that TNF is an important
mediator in spreading arthritis to ipsilateral sites and that the local
suppression of TNF and IL-1 is sufcient to prevent signicant diffusion
of these mediators to the nearby paw.
Additional studies by a number of investigators showed efcacy of
systemic (intravenous) injection of an adenoviral construct of viral IL-10
in CIA. The effect was convincing before the onset of arthritis but
virtually absent in established arthritis.2, 37 Viral IL-10 lacks immunostim-
ulatory properties and is predominantly immunosuppressive, making it
more suitable for therapy. A recent study also examined local efcacy
after periarticular injection in mouse paws.78 Intra-articular injection in
the ankle is technically hardly possible, and periarticular localization
might explain the common nding of suppression of arthritis expression
in injected paws by this approach. It probably generates more of a local
depot of IL-10, with sufcient leakage to the joint spaces and limited
direct activation of the synovial tissue by viral antigens. The common
observation of efcacy in noninjected paws is probably linked to the use
of high dosages, causing a systemic suppressive effect on CIA, including
manipulation of collagen type II immune responses in lymphoid organs.
With respect to cartilage damage, transfer studies in SCID mice
identied that retrovirally expressed IL-10 reduces invasion of RA syno-
vial broblasts into the human articular cartilage, a therapeutic activity
not found with TNFR gene transfer.36, 57 Perichondrocytic cartilage degra-
dation was still ongoing after IL-10 gene transfer by either murine or
viral origin, and effective suppression of this characteristic element of
cartilage destruction is only found with IL-1Ra. These observations
further underscore separate activities of IL-10, TNFR, and IL-1Ra and
argue for combination therapies. A triple-gene transfer of IL-1Ra, IL-10,
134 LOO & BERG

and soluble TNFR resulted in deep invasion of synovial broblasts and

partly antagonized the benecial effects observed in double-gene trans-
fer in the SCID mouse model, however.56 This showed that we still do
not fully understand the interplay of multiple genes in the treatment of
arthritis.

Interleukin-4 Gene Therapy in Arthritis

As a follow-up to the IL-10 studies, murine IL-4 was overexpressed

in the knee joint, again using the murine CIA model. In contrast to IL-
10, endogenous IL-4 levels are low in inamed joints, making IL-4 an
obvious therapeutic modality. When evaluating the levels of IL-4 pro-
duced in the knee joint after local gene transfer, it was evident that the
kinetics of IL-4 expression are curiouslow expression in the rst days
and higher expression after a week. So far, this is the only adenoviral
gene construct where this kinetic pattern was seen; all other gene con-
structs analyzed, including IL-1Ra, IL-12, IL-15, IL-17, and transforming
growth factor (TGF)-, showed the highest expression in the rst days.
The reason for these peculiar kinetics is unclear. The high levels at later
stages were not caused by substantial elicitation of endogenous IL-
4 production, because similar kinetics was observed in IL-4 knock-
out mice.
Early observations in the CIA model were disappointing. No sig-
nicant reduction of inammation in the IL-4injected knee joint was
seen (see Table 1). In line with the observations about IL-10, moderate
suppression of spreading to ipsilateral hind paws was seen. On more
careful analysis of joint histologic ndings of the treated knee, however,
it was clear that IL-4 had markedly reduced the erosive changes. Al-
though proteoglycan loss from the articular cartilage matrix as identied
by loss of Safranin-O staining was not reduced, the degree of chondro-
cyte death was highly diminished at day 7 after injection of IL-4. More-
over, excessive proteoglycan breakdown neoepitopes indicative of irre-
versible breakdown and occurrence of collagenase-mediated loss of
collagen type II were seen in the control arthritic group but hardly in
the IL-4treated mice. Follow-up to day 14 revealed that the control
group proceeded to major erosive cartilage damage, including marked
surface erosions and tissue loss, whereas the IL-4 mice were highly
protected.47 Several recent publications conrmed the IL-4 protective
effect on cartilage and bone; however, they all mentioned reduction of
joint inammation based on joint swelling. Histologically, the preventive
administration of adenoviral vector containing the IL4 gene (AdIL4)
was less pronounced on the cellular inltrate (mono/lymphocytes),
whereas suppression of cartilage and bone destruction was more impres-
sive.16, 39, 76, 79 It was found that low levels of IL-4 (as a result of small
amounts of injected viruses) are proinammatory, whereas high levels
of IL-4 (high number of injected virions) are anti-inammatory.39, 47, 48, 79
We want to emphasize the peculiar kinetics of IL-4 production in our
experiments and those of others,13 which are low early in disease and
 GENE THERAPY OF RHEUMATOID ARTHRITIS 135

increase thereafter. We postulate that the initial Th1-mediated inamma-

tory response was not prevented but that with increasing levels of IL-4,
the subsequent secondary effects on cartilage and bone were inhibited.
Furthermore, the increasing IL-4 levels could contribute to the transition
of inammation into a Th2 phenotype. Prevention of the initial Th1
reaction by high IL-4 levels (high number of virions) may also prevent
the subsequent local development of Th2-related inammation. A similar
observation was made in IL-2R/IL-4R chimeric cytokine receptor
transgenic mice. Redirecting the immune response to a Th2 phenotype
without blocking the initial local Th1 inammatory response did aggra-
vate the inammatory response in CIA.14 The presence of eosinophils
in the synovial inltrate may conrm a Th2-related secondary joint
inammation in our studies. It is also possible that cartilage and bone
destruction has a lower threshold for the protective effect of IL-4 than
does inammation.
In addition to prevention of major cartilage erosion, IL-4 also abol-
ished excessive bone erosion in murine CIA (see Table 1). Pronounced
osteoclast activation was found in the control arthritic group with im-
pressive ingrowth of granulation tissue from the synovial membrane,
whereas this aggressive phenotype was hardly seen in the IL-4 mice.
Reverse transcriptase polymerase chain reaction analysis and immunolo-
calization on the synovial tissue suggest that IL-4 exerted its suppressive
activity at multiple levels. TNF and IL-1 were suppressed, stromelysin
was inhibited, and IL-12 and IL-17 levels were markedly reduced, im-
plying that a nonimmune inammatory and destructive pathway as well
as Th1-driven activation were inhibited. With respect to characteristic
elements of bone erosion, it became evident that osteoprotegerin (OPG)
ligand [RANKL]) is reduced, with similar levels of the endogenous
inhibitor OPG. OPG ligand has recently identied as the crucial mediator
of osteoclast differentiation and activation. Because RANKL upregula-
tion can be induced by the cytokines TNF and IL-1 as well as by the
Th1 T-cell pathway (IL-17), a dual hit of IL-4mediated suppression is
conceivable.48 As yet, it is unclear whether IL-4 can directly reduce
RANKL.
A recent study in rat adjuvant arthritis demonstrated the efcacy of
retroviral IL-4 injected in the ankle. As previously discussed for IL-10
injections in the paw, it is unlikely that paw injections target the joint
spaces, and most of the expression probably occurs at periarticular sites,
which is compatible with signicant serum levels of IL-4 and enhanced
systemic Th2 function.13 In line with the murine studies, IL-4 reduced
bone destruction in adjuvant arthritis.79 Comparative studies with Chi-
nese hamster ovary (CHO) cells engineered to produce IL-4, IL-10, or
IL-13 and engrafted at weekly intervals showed efcacy in TNF
transgenic mice only for IL-4.10
In conclusion, local IL-4 treatment provides impressive control of
cartilage and bone destruction. The limited effect on cell inltration
implies that IL-4 should be combined with an anti-inammatory treat-
ment to provide acceptable symptomatic relief at the site, however.
Recent studies with systemic IL-4 combined with low-dose steroids
136 LOO & BERG

demonstrated marked synergy of this mixture.33, 35 It remains to be seen

whether low steroid dose can be combined with local IL-4 gene therapy.
A combination of the anti-inammatory properties of IL-4 with en-
hanced Fas ligandmediated apoptosis of granulocytes by gene therapy
showed a benecial effect in CIA.28

REMOTE EFFECTS BY LOCAL GENE THERAPY

CIA is an autoimmune polyarthritis model, which rst shows mac-

roscopic signs of arthritis in the paws. On analysis of the knee joints,
expression of arthritis was also often noted at those sites. The onset of
disease in knees and ankles is generally linked, and the level of expres-
sion between knees and ankles showed a good correlation in this arthritis
model. It is an intriguing nding that nonspecic inammation elicited
in the knee is a sufcient trigger to generate expression of smoldering
autoimmune arthritis in such a joint and the ipsilateral ankle, whereas
systemic inammation enhances overall expression in multiple joints.73
Spreading of arthritis seemed to be cytokine dependent, because anti-
IL-1 antibodies and anti-TNF antibodies were particularly efcient in
reduction of spreading.
Injection of NIH-3T3 broblasts into a knee joint of collagen type II
immunized DBA/1 mice provoked sufcient inammation to trigger
enhanced expression of CIA in that joint and in the ipsilateral hind paw.
When these cells were retrovirally transduced to produce high amounts
of IL-1Ra, it not only suppressed joint inammation in the knee joint
but also in the nearby paw,6 and this suppression was of the same order
of magnitude. Arthritis in joints of the contralateral knee or ankle was
unaffected.
In theory, there are a number of possible mechanisms for this effect
as listed in Table 2. The most obvious one is the local generation of TNF
and IL-1 in the control arthritic joint, where spreading of these mediators
to the ipsilateral hind paw provokes arthritis. Local suppression or
scavenging of IL-1 in the knee joint by overproduced IL-1Ra prevents
this ux and arrests expression at the ipsilateral site. In addition, IL-1Ra
produced in the knee and diffusing to the nearby ankle may contribute
to this phenomenon. Trafcking of the injected broblasts to the nearby
paw is less likely. Local IL-1Ra gene therapy by injection of recombinant
adenoviral vectors into the knee joint of collagen-induced type II immu-
nized mice conrmed the protection in the knee and in the noninjected
ipsilateral ankle joint.5, 7
A distal contralateral effect was seen with a combination of adenovi-
ral overexpression of IL-1 type I receptorIgG fusion protein and TNF
soluble type I receptor protein in antigen-induced arthritis in the rabbit.25
In studies with IL-10 and IL-4 overexpression, the remote protection of
local gene therapy in the knee was noted not only in the nearby ipsilat-
eral paw49 but also in the contralateral knee13, 44, 51, 54, 76 and even in the
untreated front paws.39 This implies that local expression of the anti-
 GENE THERAPY OF RHEUMATOID ARTHRITIS 137

Table 2. POSSIBLE OR HYPOTHESIZED MECHANISMS OF PROTECTION AT

REMOTE SITE OF TRANSGENE EXPRESSION
Transgene protein
Leakage of the transgene proteins to the blood circulation at other sites of
inammation or the immune system
Overow of transgene protein containing exosomes to the untreated joints
Draining of the transgene protein (in exosomes) to the lymphatic system, causing
suppression of the immune response in the lymph nodes
Saturation and diffusion of the interstitial uid by transgene proteins (distal effects to
the ipsilateral joints)
Viral transgene DNA
Transduction of macrophages or dendritic cells, which migrate to either the inamed
joints or the immune organs (homing of cells)
Leakage of adenoviral vectors to remote joints or immune organs (trafcking of
viruses)
Spreading of disease
Interference with disease spreading to other joints by reducing the spillover of
mediators or (auto) antigens to the system
Modulation of dendritic cells or antigen-presenting cells or T-cell function and
trafcking of these cells to other sites, where they suppress the disease
Interference with the peripheral or central nervous nociceptive system could be of
importance if the old dogma that the nervous system is implicated in the symmetric
onset of rheumatoid arthritis

inammatory cytokines can alter the systemic development of the in-

ammatory response. In our studies with adenoviral overexpression of
IL-10, the protection of the ipsilateral hind paw was seen even in the
absence of a local anti-inammatory response in the knee.49 It is argued
that IL-4 and IL-10 exert proinammatory activities at the local level but
that spreading of the disease could still be prevented by the inhibition
of IL-1 and TNF. Another plausible explanation could be that IL-4 and
IL-10 exert their distal protective effects by affecting the immune system.
The clearance and distribution of the adenoviral vector and viral IL-10
transgene was studied in CIA by Watanabe et al.77 They showed that the
mechanism of protection of distal joints was caused by leakage of viral
IL-10 to the circulation and that although large amounts of adenoviruses
were detected in the liver, the number of transduced cells in the distal
joints was too low to account for the observed protection. Furthermore,
they also found protection of the injected joints. In contrast to mamma-
lian IL-10, viral IL-10 lacks the proinammatory properties. We used
much smaller viral dosages (.001% of their dosage) and found a re-
stricted ipsilateral protection. In our experiments, we could not measure
IL-10 or IL-1Ra transgene overow into the blood circulation, and this
may explain our failure to obtain a contralateral effect. Others also found
no evidence for systemic leakage of the locally synthesized transgene
proteins (IL-4, soluble IL-1RI, soluble TNFR, and IL-1Ra).6, 25, 40, 4749 The
leakage of inhibitors to the draining lymphatic system was not studied,
however. Recently, it became known that the draining lymph nodes of
inamed foot joints had a higher (2) ow rate and higher concentra-
tions of proteins and immunoglobulins. More importantly, the lymph
138 LOO & BERG

concentrations of IL-1 (10), IL-6 (6), TNF (2), IL-1Ra (21), IL-
10 (13 or greater), IL-8 (4), granulocyte macrophage colony-stimulat-
ing factor (GM-CSF)(6), vascular endothelial growth factor (VEGF)
(2), and macrophage inammatory protein-1 (50) with a lymph:se-
rum ratio of greater than 1 in all RA patients indicated local production
of cytokines.60 The fact that lymph cytokine concentrations reected the
intensity of local joint inammation was demonstrated by the normaliza-
tion of these cytokine levels in lymph uid after intravenous predniso-
lone injection, whereas concentrations in serum showed little or no
change. The cytokines in the lymph could have been drained from the
inamed joint, but increased cytokine expression in draining lymph
nodes has been demonstrated during adjuvant arthritis.4 A boost of the
immune system often follows the onset of arthritis, and a spillover of
cytokine inhibitors to the draining lymphatic system could suppress this
amplifying effect, thereby inhibiting spreading of the disease.
Trafcking of genes or cells carrying the gene, although less likely,
cannot be excluded in all these studies. When the viral vector is applied
intra-articularly in a joint already heavily inamed and carrying a orid
exudate in the joint space, the adenoviral vector may infect a number of
different cell types. With adenoviral (Ad) vectors carrying green uores-
cent protein (GFP), Lechman et al44 demonstrated that a similar subset of
cells infected in the injected knee was able to migrate to the noninjected
contralateral knee joint. Infection of T cells is not likely, as these cells
tend to lack the Coxsackie and adenovirus receptor necessary for adeno-
virus recognition. Dendritic cells (DCs) were the obvious candidates for
traveling to other sites, and they may inuence arthritis at remote sites
depending on local priming with transduced genes and soluble cyto-
kines. DCs are antigen-presenting cells (APCs), and intravenous admin-
istration of genetically modied DCs expressing IL-4, Fas ligand, IL-1Ra,
or CTLA4-Ig into established CIA resulted in almost complete remission
of disease via suppression of the Th1 response.38, 69 It is unlikely that
DCs are targeted by the intra-articular adenovirus injection, however, as
only the rst one or two cell layers of the lining are effectively targeted.
Furthermore, DCs are difcult to infect with Ad5 vectors, which were
used in these studies; a multiplicities of infection (MOI) of more than
100 was necessary to transduce these cells in vitro. It is possible that the
migrating cells are not DCs but macrophages (genetically or phenotypi-
cally altered). It was demonstrated that macrophages transfected with
adenovirus to express IL-4 and injected into a single kidney markedly
reduced inammation in the contralateral noninjected kidney, whereas
systemically injected IL-4transfected macrophages have no effect.40
Macrophages in the inamed area do not die locally but emigrate to the
draining regional lymph nodes,8 where they may exert an immunoregu-
latory effect.
Periarticular injection of AdvIL-10 or retroviral vectors carrying rat
IL-4 protected only the injected ankle and failed to induce trafcking of
immunoregulatory cells capable of suppressing distal disease.13, 76 In this
case, the transfected cells are probably broblasts or myoblasts, which
are not known to be able to migrate. Intramuscular injection of recombi-
 GENE THERAPY OF RHEUMATOID ARTHRITIS 139

nant AAV-IL-4 into the tarsus area of mice had a profound therapeutic
effect on CIA, with spreading of the virus to all major organs.16 The
probable explanation is leakage of AAV to the blood circulation, as
muscles are well-vascularized tissues.
Recently, a new concept was added to the possible mechanisms
behind the distal protective effects of the local gene delivery treatment.
Robbins et al69 proposed that the local expression of anti-inammatory
factors modied the function of APCs, which are then able to modulate
the immune response in the contralateral joint. Furthermore, they dem-
onstrated that exosomes, probably derived from APCs, were present in
the synovial uid of adenovirally transfected joints and contained the
transgene protein. An intra-articular injection of exosomes derived from
AdvIL-10infected APCs reduced the delayed type hypersensitivity
(DTH) reaction in the treated and untreated paws. Taken together, these
results suggest that modied APCs as well as transgene protein con-
taining exosomes can transfer the protection from the treated to the
untreated contralateral joints.
There is probably not one mechanism to account for the distal
effects of local gene therapy (see Table 2), and it may depend on the
amount of viruses, type (tropism/size) of viral vector, site and time of
application, and cell types targeted. The observation of contralateral
protection may argue that local events in inamed joints not only destroy
joints but drive the disease at remote sites. These studies proved the
feasibility of therapeutic gene therapy in small joints, with a promising
protective effect on nearby joints. Given the existence of some interrela-
tion between arthritis in nearby joints in RA patients, this should ease
application in human patients, where it might prove sufcient to treat
only the larger joints. On the other hand, one might argue that clinical
application should be performed under controlled conditions, which
would imply that joints should be washed out before adenoviral gene
transfer so as to deliberately target local synovial lining cells.

IMPROVEMENT OF FOCAL THERAPY

Successful gene therapy depends on the efciency of gene delivery.

Viruses are the obvious vehicles for this, but none has a tropism for
infecting synovial joints more specically. Apart from retention of the
gene (a persistent synovial transgene expression up to 7 months with
recombinant AAV was reported),76 targeting of cells or selection of cells
as a carrier to deliver the gene product at particular sites is a crucial
element of successful therapy.

Synovium

When adenoviral gene constructs are injected into the knee joint
cavity of the mouse, the usual way to infect cells is through the Cox-
sackie and adenovirus receptor (CAR) recognized by the ber knob of
140 LOO & BERG

the virus. It was noted that infection in normal mouse joints was high
but that expression dropped dramatically (vefold) in chronically in-
amed joints, which is more close to the clinical situation in RA patients.
One way to enhance infection of cells under inamed conditions is
to make use of upregulated integrin expression of the synovial cells.
Adenoviral vectors with an Arg-Gly-Asp (RGD) motif introduced in the
HI loop of the ber knob were constructed.41, 66 This allowed this virus
to enter cells through integrin interaction independently of the CAR. On
injection of these viruses carrying rey luciferase/GFP as a marker
gene, it was found that infection was greatly enhanced (up to 70-fold)
in chronically inamed joints. This correlated with the relative higher
mRNA expression of the integrin V than that of the CAR in synovial
tissue. To get an impression of applicability in RA patients, ex vivo
incubations of synovial tissue specimens with RGD viruses were per-
formed. Infection levels with normal viruses are highly variable in tissue
specimens of various RA patients, which is probably linked to large
differences in cellular inltrates. Nevertheless, a dramatic increase in
transduction levels was consistently noted with the RGD viruses, rang-
ing from 10- to 50-fold increments. This is in line with the upregulated
v3 integrin in inamed synovium.31 In murine CIA, we showed that
using an RGD-modied adenoviral vector carrying IL-1Ra signicantly
improved the efciency of gene therapy for arthritis (Fig. 1, see Table 1).7
Another major advantage of the RGD modication is that these
adenoviruses are not recognized by the neutralizing antibodies against
Ad5 that are present in almost all individuals.12 For the same reason,
others changed the Ad5 ber knob for the Ad35 ber knob, resulting in
a chimeric vector (Ad5-b35 vector). Only 4% of the RA patients had
neutralizing activity in their synovial uid against Ad35, which is far
below the 72% of RA patients who had neutralizing activity inhibiting
Ad5 transfection. The Ad5-b35 vector also had an altered tropism and
transfected synoviocytes about 10 times more efciently than Ad5 vec-
tors with their wild-type ber knobs.27
Although RA patients lack neutralizing antibody (Ab) against
Ad5RGD and Ad15 chimeric vectors, it is unclear whether these rst-
generation (E1a- and E1b-deleted) viruses do raise no or fewer cytotoxic
T lymphocytes, which may curtail gene transfer after a second dose
of virus. Modication of adenoviruses chemically with monomethoxy-
polyethylene glycols can escape recognition of the adaptive immune
system and can be used for readministration of adenoviral vectors.17

T Cells

Another way to express inhibitors or modulatory proteins at the

site is to make use of the migratory capacity of T cells. It was found that
pathogenic lymphoid cells engineered to express TGF ameliorate CIA.15
It is known that Th1 cells display good migratory characteristics (chemo-
kine receptors) to enter joint tissues, whereas the protective Th2 cells
 GENE THERAPY OF RHEUMATOID ARTHRITIS 141

Figure 1. Increased efcacy of IL-1Ra gene therapy for collagen-induced arthritis using
either tropism modied adenoviruses or conventional viruses with a disease-inducible
promoter instead of the constitutive ieCMV promoter. Collagen-type II immunized mice
were injected with 10 PFU of adenovisuses into knee joint cavity at the time of arthritis
onset. In the tropism-modied viruses was the RGD (Arg-Gly-Asp) motif introduced in the
HI loop of the ber knob (Ad5.CMVIL-1Ra). The disease-inducible two component expres-
sion system used the complement 3 promoter to regulate the expression of HIV transactiva-
tor of transcription (Tat) which in turn activates the HIV-LTR promoter to transcribe the IL-
1Ra gene (Ad5.C3-Tat/HIV-IL-1Ra). Paws were macroscopically scored on a scale of 02
from day 1 after i.a. injection of the viruses. *P.05 compared to the Ad5.CMV.Luc group.
n  10 mice per group. IL1Ra  interleukin1 receptor antagonist; ieCMV  immediate
early cytomegalovirus; PFU  plaque forming unit; and HIVLTR  human immunode-
ciency viruslong terminal repeat.

have difculty in reaching this site. T cells are, however, relatively

resistant to adenoviral infection because of their lack of CAR receptors
and low levels of V3 and V5 integrins. Upregulation of the integrin
expression by cell activation or exposure to macrophage-CSF and GM-
CSF permits adenoviral transfection. It should be technically possible to
engineer T cells with the desired chemokine receptor makeup of Th1
cells but additionally engineered to produce modulatory cytokines such
as TGF and IL-4.
The principle of an active tolerance based on IL-4-transduced immu-
nospecic T cells to inhibit transfer of disease has recently been proven
in a diabetes model in mice.80 By manipulation of the T-cell receptor on
these cells, it is also possible to direct the antigen specicity, for instance,
to the joint specic antigen collagen type II,1 and to use such cells as
gene carriers in anti-inammatory gene therapy in autoimmune arthritis.

Chondrocytes

Protection against degradation and enhancing tissue repair are the

two objectives for transduction of articular chondrocytes in arthritis.
142 LOO & BERG

Chondrocytes reside in cartilage and do not move freely. A chondrocyte-

specic transfection offers the chance of site- or joint-restricted expres-
sion without the chance of migration of the transduced cells to other
tissues. Chondrocyte-specic transgene expression can be easily obtained
using the cell-specic collagen type II promoter for the regulation of
transgene. Achieving infection of chondrocytes in the presence of their
surrounding extracellular matrix is a more difcult task, however. From
studies on cartilage accessibility, we showed that penetration into carti-
lage was impossible for protein of more than 150 kd in size and with
decreased retention of anionic proteins.74 During arthritis, when the
cartilage matrix has a reduced glycosaminoglycan content (reduced an-
ionic charge) or breakdown of the collagen network, the penetration of
larger or anionic molecules could theoretically be possible. Goater et al.26
demonstrated the in vivo transduction of chondrocytes with recombi-
nant AAV-cytomegalovirus-LacZ in cartilage from arthritic knee joints
in TNF transgenic mice. In human cartilage organ culture, it was shown
that GFP gene delivery by AAV was not restricted to the supercial
layers but extended to the deep cartilage layers.3 AAV viruses are around
20 nm in size and adenoviruses are ve to six times that size, too large
to be able to penetrate into the cartilage matrix. As it was shown
that even with adenoviruses (Adv), the chondrocytes on the surface of
degenerated cartilage could be transduced,29 we hypothesize that this
was caused by depletion of the articular cartilage of proteoglycans,
which may open the matrix for penetration of larger molecules. This
showed that chondrocyte-specic transduction is within our reach, and
this may offer new opportunities to protect and repair cartilage and to
achieve site-specic expression after intra-articular injection of viral vec-
tors.

Muscles

Gene delivery to skeletal muscles is an easy way to achieve systemic

secretion of therapeutic proteins. Direct injection of plasmid DNA is
possible but results in low expression and high variability. Transduction
of muscle bers can be greatly enhanced using electrotransfer71 and
resulted in sustained expression for more than 12 months. Muscle cells
do not proliferate and have a long life span; although the plasmid DNA
remains extrachromosomal, a prolonged transgene expression can be
obtained.
Recent studies showed that AAV vectors could give a sustained
transgene expression of several months in the joint. Jennings et al30
showed that human and murine type B synoviocytes are poorly trans-
ducible by AAV2 and AAV5. Only after coinfection with an adenovirus
could this cell express the transgene, indicating the necessity of second-
strand DNA synthesis. Intra-articular injection of AAV, however, resulted
in periarticular transgene expression of the suprapatellar muscle tissue.
Also, human RA synovial tissue engrafted on the backs of SCID mice
had low AAV transduction efciency in vivo. This showed that AAV
 GENE THERAPY OF RHEUMATOID ARTHRITIS 143

could not be used for transfection of the synovium. AAV has the poten-
tial to transduce muscle cells with high efcacy, although a lag time in
transgene expression of several weeks must be anticipated.

CONTROLLED EXPRESSION OF GENES

As mentioned previously, future therapies are likely to make use of

cassettes of genes, combining the targeting of multiple mediators as well
as including growth factors to optimize recovery of the damaged joint.
Apart from improved targeting to synovial tissue, a major challenge for
the future is the proper targeting of chondrocytes in the articular carti-
lage. Present adenoviral gene constructs are too large to allow for ef-
cient penetration of the dense cartilage matrix, and further research is
needed to design applicable carriers for this tissue. Lipid carriers might
be an alternative.52
Apart from targeting, the controlled expression of the gene is an
obvious goal. So far, most studies have been done with the constitutive
cytomegalovirus promotor. It makes more sense to create an intelligent
promoter system, which can sense the need for expression of a particular
inhibitor and only activates the gene when needed. Varley et al75a engi-
neered a two-hybrid system, with a complement C3 promoter activating
the transactivator of transcription (Tat) gene and Tat activating the
human immunodeciency virus promoter in front of the gene of interest.
The complement promoter is turned on under conditions of acute in-
ammation and is silenced when inammation is suppressed. Testing of
such a hybrid system with IL-1Ra as the gene of interest in the model
of CIA has been successful (see Table 1 and Fig. 1) and has resulted in
a more efcient inhibition of arthritis.5
The recent observation of long-term retention of AAV-IL-1Ra in the
knee joint of rats and the reactivation of IL-1Ra production on rechal-
lenge with an inammation-inducing agent such as LPS63 provides a
way to protect against exacerbations. This is important in arthritic dis-
eases that undergo spontaneous ares and remissions. Goater et al26
demonstrated that recombinant AAV transduction of primary broblast-
like synoviocytes could be enhanced by pretreatment with TNF. Several
recent reports from Robbins et al70 (AAV-IL-1Ra) and Jennings et al30
(AAV-vIL-10) could not conrm that a second inammatory response
could restore transgene expression using AAV in the joint, however.

SIDE EFFECTS AND PRECAUTIONS

Risks involved with gene therapy surround the use of the various
vector systems and the trafcking of genes to remote sites. Nonviral
vectors hold the least concern but show low efcacy of gene transfer.
Viral vectors have been genetically disabled to minimize their ability to
replicate and cause pathologic changes. Engineering of viral vectors
144 LOO & BERG

continues to improve, including removal of immunogenic components

and further reduction of the risk of recombination events toward the
impossible. An advantage of the ex vivo gene therapy approach with
infection and selection of cells in vitro is the opportunity to do safety
screenings before transplantation of the cells. The rst clinical trials in
RA patients have been done with the retroviral ex vivo approach for
safety reasons, but further trials are likely to focus on gutless adenovi-
rus or AAV virus as a carrier. AAV is an attractive candidate as it is a
nonpathogenic virus, can infect dividing and nondividing cells, and can
integrate site specically at the AAVS1 locus on chromosome 19, which
results in stable transfection and long-lasting expression of the
transgene.78 Recombinant AAV lacks the Rep protein, and this protein is
an absolute requirement for the site specicity of AAV DNA integra-
tion.46 The limited packaging size of 5 kb is a disadvantage, although
the strategy of transsplicing vectors (spontaneous intermolecular recom-
bination of two AAV vectors into circular concatamers) expands the
information to be transferred to cells up to 10 kb.18 As a consequence of
the absence of Rep proteins, which are essential for targeted integration
into the genome, the rAAV randomly integrates, with the theoretic risk
of causing insertional mutations (oncogen activation). If this is true,
rAAV must be applied with the same precaution as was done with
retroviral vectors using the ex vivo method as described by Evans and
his colleagues,1921 and this may hamper its application in human beings.
If the focus of future therapy is on local treatment in joints, the total
amount of viral elements introduced is low and the risk is negligible.
Thousands of patients have participated in gene therapy studies over
the last decade, and it is fair to say that the safety record is impressive
so far.

SUMMARY

Evidence from animal models convincingly supports the fact that

gene therapy can be an advantageous strategy in the treatment of chronic
destructive RA. In this article, we review the state of the art in anticytok-
ine gene transfer into the synovial arthritic joint with the emphasis on
IL-1Ra, IL-4, and IL-10 effects on CIA in mice.
In CIA, only high and continuous release of IL-1Ra protein systemi-
cally by miniosmotic pumps could prevent disease onset and was
curative in mice. Local gene transfer seemed to be the obvious way to
reach the high local levels that are demanded for protection. It was
shown that local IL-1Ra overexpression reduced arthritis incidence and
severity as well as tissue destruction. In line with observations about
neutralizing antibodies and soluble receptors, gene therapy with TNF
soluble receptors provided anti-inammatory activity in early arthritis
but not in advanced arthritis. The limited efcacy at later stages and
poor protection against destruction imply that the combination of gene
 GENE THERAPY OF RHEUMATOID ARTHRITIS 145

constructs for TNF and IL-1 inhibitors is the obvious direction for fu-
ture therapy.
Apart from targeting of proinammatory cytokines, adenoviral
overexpression of IL-10 and IL-4 may have therapeutic applicability.
Local injection of AdIL-10 in the knee joint was effective at the site, but
also highly reduced spreading to ipsilateral sites. High local dosages
caused suppression in contralateral sites as well. The reports on the anti-
inammatory effect of AdIL-4 are conicting; however, all present data
showed that IL-4 overexpression provides impressive protection against
cartilage and bone erosion.
Apart from the local effects in the injected joint, it is becoming more
and more clear that local treatment also affects arthritis in nearby joints.
This is an intriguing general nding, which may enlarge the therapeutic
applicability of gene transfer in human arthritis.
Proving the feasibility of gene therapy in experimental arthritis,
most research efforts are now focused on improving local gene delivery
by enhanced viral infection of synovial cells, using RGD-modied ade-
novirus, or achieving prolonged persistence and regulated expression
with AAV. Elegant future alternatives are the application of in vitro
engineered T cells as a vehicle capable of specic homing to joint tissues.
The feasibility of viral transduction of chondrocytes to obtain a tissue-
specic approach to treat articular cartilage damage in arthritis needs
further attention.

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Address reprint requests to

Fons A.J. van de Loo, PhD
Department of Rheumatology
University Medical Center Nijmegen
Geert Grooteplein 26-28
6500 HB Nijmegen
The Netherlands

e-mail: a.vandeloo@reuma.azn.nl