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Gene therapy for arthritis: progress towards a clinical trial

Aalbers, C.J.

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Citation for published version (APA):Aalbers, C. J. (2015). Gene therapy for arthritis: progress towards a clinical trial.

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Download date: 16 Oct 2020

E m p t y c a p s i d s a n d m a c r o p h a g e i n h i b i t i o n /d e p l e t i o n i n c r e a s e

r A A V - t r a n s g e n e e x p r e s s i o n i n j o i n t s o f b o t h h e a l t h y

a n d a r t h r i t i c m i c e6

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A B S T R A C TGene therapy has potential to treat rheumatic diseases, however, the presence of macrophages

in the joint might hamper AAV mediated gene delivery. We demonstrate that in arthritic,

but also in healthy mice, administration of agents that influence macrophage activity/

number and/or addition of empty decoy capsids substantially improve the efficacy of rAAV5-

transgene expression in the joint. Pre-treatment with triamcinolone or clodronate liposomes

improved luciferase expression over a period of 4 weeks. Both the expression per joint, and

the percentage of expressing knee joints improved. Similar results were seen when empty

decoy capsids were added to full genome containing capsids in a 5:1 ratio. In a study to assess

the duration of expression as well as to investigate the combination of these two approaches

we observed a synergistic enhancement of gene expression, sustained for at least 6 months.

The enhancement of gene expression was independent of the route of administration of

triamcinolone (intra-articular or intramuscular). In healthy mice it was demonstrated that the

combination improved expression of the transgene significantly, independently of the serotype

used. These data have implications for future applications of gene therapy to the joint and for

other tissues with an abundance of macrophages.

CJ Aalbers,1,2 N Broekstra,1 M van Geldorp,1 E Kramer,1 S Ramiro,2 PP Tak,1,2,#

MJ Vervoordeldonk,1,2 JD Finn1,2

1 Arthrogen B.V., Amsterdam, the Netherlands2 Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands

# Current address also: GlaxoSmithKline, Stevenage, U.K., University of Cambridge, Cambridge, U.K. and Ghent University, Ghent, Belgium

Manuscript in preparation

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I N T R O D U C T I O NRheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease affecting the synovial

tissue in multiple joints. The synovial joint is characterized by a capsule surrounding the joint

and the presence of lubricating synovial fluid within the synovial cavity. The intimal lining layer

is composed of two cell types, intimal macrophages and fibroblast-like synoviocytes (FLS). The

FLS are responsible for the lubrication of the joint (through production of hyaluronan), and the

macrophages are responsible for the removal of debris and undesirable substances from the

synovial fluid.1 During inflammation the presence of both cell types is increased by proliferation

of the FLS and migration of macrophages to the joint and both cell types produce inflammatory

mediators and bone- and cartilage-degrading enzymes.2

Several biological therapies are now available for RA, significantly improving the outcome of the

disease in many patients, but not all.3 Over 50% of patients will still have active disease and even the

majority of clinical responders to biological treatment will have one or more actively inflamed joints.

There remains an urgent need for the development of safe, efficacious, and cost-effective treatments

to specifically target actively inflamed joints. Intra-articular gene therapy has the potential to treat

arthritis, as the joint is an excellent target for the local delivery of gene therapy vectors.

There have been very few gene therapy clinical trials in RA, using either injection of cells that

were genetically modified with a retrovirus expressing IL-1 receptor antagonist into the joint (ex

vivo gene therapy), or injection of a recombinant adeno-associated viral vector (rAAV) encoding

a TNF antagonist (in vivo gene therapy).4,5 While intra-articular administration of an rAAV2 vector

encoding the TNF receptor IgG1 fusion gene (tgAAC94)6,7 was well tolerated, the treatment was

not effective. It is likely that lack of efficacy was due to insufficient levels of gene expression. This

notion is supported by the fact that local gene expression, as determined by a radioimmunoassay

for TNFR:Fc protein in synovial fluid and an RT-PCR assay for mRNA specific to rAAV2-TNFR:Fc in

synovial tissue, was not detected in the very limited number of samples that became available

during the study.7 Moreover, we have shown that intra-articular injection of etanercept into the

inflamed joint of RA patients does include clinical improvement, validating the therapeutic target

when administered to the site of inflammation.8 Importantly, this trial provided encouraging data

on safety and feasibility for further development of AAV-based intra-articular gene therapy in RA.

We are developing intra-articular gene therapy for RA using rAAV5.9-11 Comparing AAV

serotype 1 to 5 we have shown that AAV5 was the most efficient in transducing synovial tissue

after intra-articular injection in two different animal models of RA.12,13 The main target cells for

rAAV5 in the joint are FLS. AAV5 is effective in delivering genes to both human and rodent FLS,

in vitro14 as well as in in vivo in rat and mouse models of RA.12,13 AAV5 has the added benefit that

the majority of the human population is seronegative for AAV5.15

In an effort to improve transduction efficiency, we have investigated factors that can

influence in vivo transduction of rAAV vectors after intra-articular injection in animal models

of RA and in healthy animals. Here we present data that uncover an unexpected barrier to

efficient gene transfer to the joint, namely, that synovial macrophages significantly inhibit AAV-

mediated gene delivery, and we present strategies for overcoming this barrier. These data have

implications for future applications of local gene therapy to the joint, or to other tissues that

are abundantly infiltrated by macrophages.

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M E T H O D SVector and empty capsids production

An rAAV5 and rAAV2 vector was produced coding for Firefly Luciferase (Fluc) with a

cytomegalovirus (CMV) promoter (rAAV5.CMV.Fluc and rAAV2.CMV.Fluc; Children’s Hospital of

Philadelphia, Philadelphia, PA) as described previously.16 In brief, the plasmid encodes the Fluc

gene under the control of the CMV promoter and a human growth hormone polyadenylation

signal. The transgene cassette is flanked by AAV2 inverted terminal repeats and is packaged in

capsid from AAV5 or AAV2.17 The genome containing vector and empty AAV capsid particles were

purified by combined chromatography and cesium chloride density gradient centrifugation.18

Vector titers were determined by qPCR and expressed as viral genomes/ml (vg/ml).

Vector administration in mice

Intra-articular rAAV expression was investigated in male DBA mice (8-12 week old; Harlan Sprague

Dawley, Horst, the Netherlands). Groups consisted of 5 to 18 animals per experiment (indicated per

experiment in the legends of the figures). Mice were injected with rAAV5.CMV.Fluc or rAAV2.CMC.

FLuc in both knee joints and monitored periodically for luciferase expression (as indicated in the

legends of the figures, from 3 days up till a maximum of 6 months). For animals without arthritis the

vector was administered on day 1, in arthritic animals the vector was injected on day 17 or 24 after

immunization. Animals received 1.26e10 to 1.65e10 vg of rAAV per knee joint (in a volume of 5 ul) in

healthy or CIA mice as indicated in the legends of the figures. Empty capsids were co-administered

with the genome containing particles in several groups in a 1:5 or 1:20 full:empty ratio, as indicated.

Collagen-induced arthritis

Arthritis was induced and evaluated as previously described.19,20 Bovine type II collagen (2 mg/

ml in 0.05 M acetic acid; Chondrex, Redmond, WA) was mixed in an equal volume of Freund’s

complete adjuvant (2 mg/ml of Mycobacterium tuberculosis; Chondrex). The mice were

immunized intradermally at the base of the tail with 100 µl of emulsion (100 µg collagen) on

day 0. On day 20, mice received an intraperitoneal booster injection of 100 µg type II collagen

in NaCl. The severity of arthritis was assessed using an established semiquantitative scoring

system (0-4; 0 = normal, 1 = swelling in 1 joint, 2 = swelling in >1 joint, 3 = swelling in the entire

paw, and 4 = deformity and/or ankylosis. The cumulative score for all 4 paws of each mouse was

used to represent overall disease severity and progression. In the long-term follow-up study

presented in figure 3, due to technical problems of the IVIS, data are not available for time point

8 weeks. In addition, a total of 15 animals (2-4 per group) were sacrificed prior to the end of the

experiment due to reaching a humane endpoint (clinical score >12) before the end of the study.

Macrophage inhibition/depletion

Macrophage inhibition was initiated by two different compounds, clodronate containing

liposomes (Ordered from www.clodronateliposomes.com; Dr N van Rooijen, Amsterdam,

the Netherlands) and triamcinolone (B.-M.S., Utrecht, the Netherlands). Clodronate

liposomes were injected intravenously at a dosage of 5ul/g bodyweight with a concentration

of 5 mg liposomes per 1 ml suspension 2 days prior to intra-articulr vector administration.23

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Triamcinolone (5 mg/kg) was administered intramuscularly in the femoral biceps muscle, in a

volume of 50 ul also two days prior to vector injection. Taking into account the faster metabolic

rate in mice (factor 12.5), a dose of 5 mg/kg bodyweight was used and injected intramuscular,

similar to the route of systemic administration in RA patients. Control groups received an

intramuscular injection with 50 ul NaCl. In follow-up experiments, intramuscular injection of

triamcinolone was compared to intra-articular administration of the glucocorticoid (same

dose in a volume of 5 ul). A control group received an intra-articular injection with 5 ul NaCl.

Imaging of luciferase expression

Luciferase expression was measured at different time points after vector administration, from

day 3 up till 6 months depending on the experiments. Luciferase expression was detected as

described previously.14 In brief, D-luciferin potassium-salt substrate (Caliper Life Sciences,

Hopkinton, MA, USA) was injected intraperitoneally (150 mg/kg of body weight, in a volume of

approximately 200 µl). Photon counts were acquired 10 minutes after substrate administration

for 5 minutes using a cooled charge-coupled device (CCD) camera system (Photon Imager,

Biospace Lab, Paris, France) and image processing and signal intensity quantification and

analysis were performed using M3 Vision (Biospace Lab). The number of photons emitted per

second per square centimetre per steradian was calculated as a measure of luciferase activity.

General animal conditions and ethics statement

Immunization, intra-articular injections and in vivo imaging were performed under isoflurane

anaesthesia (3% isoflurane and oxygen). At the end of the experiments, animals were sacrificed by

cardiac puncture under isoflurane anaesthesia, followed by cervical dislocation. The studies were

reviewed and approved by the animal care and use committee of the University of Amsterdam

(Amsterdam, the Netherlands; Permit Numbers: ART 102881, ART 102656, ART 102793, ART 102948,

ART 103021 and ART 111AB) and carried out in strict accordance with the recommendations in the

Dutch Law on Animal Welfare (Dutch: “Wet op Dierproeven”). Animals were maintained under

pathogen-free conditions in the animal facility of the University of Amsterdam.

Statistical analysis

Luminescence over time was investigated using generalized estimating equations (GEE) to allow

for longitudinal analysis (including all available longitudinal data and allowing unequal numbers

of repeated measurements).21 All other statistics were analyzed using where appropriate the

T-test or Mann Whitney U in Graphpad Prism (Ja Jolla, CA, USA). For all tests, differences with a

p-value of <0.05 were considered significant.

R E S U LT SInflammation affects intra-articular rAAV5 gene expression

A hallmark of synovial inflammation is the proliferation of FLS in the synovial tissue of RA

patients.2 In a healthy joint the intimal lining layer is 1-2 cell layers thick, whereas in the inflamed

joint this is increased to 8- to 10 cells. This is also true in mouse models of RA, including in

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the collagen-induced arthritis (CIA) model. As FLS are the primary target cells for rAAV5 in

the joint, we hypothesized that administration of rAAV5 after the onset of inflammation in the

CIA model would lead to higher expression, due to a higher number FLS present in the joint at

the time of inflammation. To test this hypothesis, we administered an rAAV5 vector encoding

the Firefly luciferase gene (rAAV5.CMV.Fluc) intra-articularly in mice with CIA, before (day (d)

17) or after (d24) the onset of arthritis. Surprisingly, we found that administration of an rAAV5

vector after the onset of inflammation (d24) resulted in lower expression compared to vector

administration before the onset of inflammation (d17) (Figure 1).

Immunosuppressive agents improve rAAV5 transgene expression

An explanation for decreased expression in animals with inflamed joints could be degradation or

neutralization of the vector before it is able to transduce the target cells. During inflammation,

there is not only an increase in the number of FLS, but there is also an increase in the number

and activation of macrophages. We hypothesized that the decreased expression could be due

to vector neutralization by macrophages (for example through phagocytosis or opsonization by

soluble factors [complement]). To investigate this possibility we examined whether administration

of agents that influence macrophage-activity and/or -number had an effect on rAAV5 expression.

Triamcinolone, a glucocorticosteroid, acts by inhibiting the activation and proliferation

of immune cells, including macrophages.22 Alternatively, clodronate containing liposomes

were used to deplete macrophages.23 The two agents were administered 48 hours before

vector administration. Both triamcinolone and clodronate liposomes improved rAA5.CMV.Fluc

luciferase expression over a period of 4 weeks, showing that either depletion or inhibition of

macrophages leads to an increase in gene expression (Figure 2a).

Figure 1. Luciferase expression is influenced by vector administration before or after the onset of arthritis. (a) After induction of arthritis, mice (n = 5 per group) were injected with 1.65e10 vg/joint of a rAAV5-transgene vector encoding the gene for luciferase (rAAV.CMV.Fluc5 ) intra-articularly in the knee joint either on day 17 before onset of arthritis or day 24 after onset of arthritis. Imaging was performed 3 days after vector injection and thereafter weekly up to 4 weeks (group day 24) or 5 weeks (group day 17). (b) The right panel shows a representative picture of the imaging results. Luminescence is shown per group as average; error bars, SEM.

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Fig

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We hypothesized that an alternative approach to avoid macrophage vector neutralization

was to add empty capsid particles upon vector administration. These empty capsids could act

as a decoy and therefore increasing the chances that full virus particles will be able to reach the

target cells. This strategy is similar that previously used to avoid neutralizing anti-AAV capsid

antibodies.24 When empty capsids were added to full genome containing capsids in a 5:1 ratio,

expression improved significantly (Figure 2b). These data supported our hypothesis that the

vector is likely being neutralized by macrophages. In support of this hypothesis, we observed

that all three treatment groups (triamcinolone, clodronate liposomes, empty capsid) also

showed an increased percentage of positive joints (Figure 2c). In figure 2d the luminescence

28 days after vector administration is shown, demonstrating a significant difference between

administration of the vector alone after onset of disease compared to the groups that received

treatment. As triamcinolone is an anti-inflammatory agent, arthritis activity was closely

monitored. As expected, mice treated with triamcinolone or clodronate liposomes showed a

delayed onset of arthritis (results not shown).

Triamcinolone and decoy capsids have a synergistic effect on rAAV5 expression

As we had found that pharmacological inhibition of macrophage activity or addition of empty

decoy capsids resulted in increased gene expression, we performed a long term follow up study

to assess the duration of improved gene expression, as well as to investigate the combination

of these two approaches. As triamcinolone is a therapeutic compound that is already approved

for the treatment of RA, we selected this compound for the follow-up studies. The effect of

addition of triamcinolone and/or empty capsids was analysed longitudinally using generalized

estimating equations (GEE), allowing us to include all available longitudinal data and allowing

unequal numbers of repeated measurements. Similar to the previous study, we observed

increased expression using triamcinolone or empty capsid alone, however it was clear that the

combination of macrophage inhibition and decoy capsids resulted in a synergistic enhancement

of gene expression (Figure 3a and Table 1, 5.85 fold enhancement, p<0.001) and that this

enhancement was sustained for up to 6 months post vector administration. As expected, due

to its anti-inflammatory effect, arthritis activity was lower in groups treated with triamcinolone

up till week 4 (Figure 3b), however long term arthritis activity was comparable between groups.

Table 1. Generalized estimating equations analysis showed a significant improvement in luminescence when both triamcinolone and empty capsids were added.

Group Luminescence coefficient SE P

rAAV.CMV.Fluc - - -

+ Triamcinolone 1.29 1.81 0.477

+ Empty capsid 0.86 1.81 0.635

+ Triamcinolone and empty capsid 5.85 1.80 0.001

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Figure 3. Improvement of intra-articular rAAV5-luciferase expression by addition of empty capsids and/or triamcinolone. Arthritic mice (n = 13 per group) were injected intra-articularly with 1.65e10 vg/joint of rAAV5.CMV.Fluc vector with or without the addition of empty capsid (1:5 full to empty) or triamcinolone (5 mg/kg i.m.). Mice were followed weekly for 1 month and thereafter monthly up till 6 months. (a) Luminescence increased up till 4 months for all groups, and then remained stable. (b) Arthritis activity was scored at each time point and a clinical score was calculated. Initially arthritis activity was lower in groups treated with triamcinolone. The clinical score and luminescence are shown per group as averages; error bars, SEM. P<0.001 Fluc plus triamcinolone/empty capsids versus Fluc alone.

Local triamcinolone administration is as effective as systemic administration

on increased gene expression of rAAV5

In the clinic, triamcinolone is usually administered locally by intra-articular injection into the

inflamed joint. Therefore we compared the efficacy of local versus systemic administration

of triamcinolone on enhancement of gene expression. To test this, triamcinolone was

administered systemically (intra-muscular) or locally (intra-articular) two days prior to intra-

articular administration of rAAV5.CMV.Fluc + empty capsid (empty:ful ratio of 5:1). As control,

saline was injected intra-articularly. Luciferase expression was monitored over time by IVIS

imaging up till 30 days. We observed similar increases in gene expression when triamcinolone

was administered locally or systemically, indicating that local administration of triamcinolone

was as effective as systemic administration (Figure 4).

AAV empty capsids improve transgene expression of rAAV5 in the absence

of inflammation

All previous experiments were performed in CIA models, in which animals experienced significant

inflammation in the joint at the time of vector administration. We then decided to investigate

whether the enhancement in luciferase expression by adding empty capsids could also be seen

in healthy joints. While the number of synovial macrophages increases during inflammation, even

healthy synovium contains a significant number of macrophages. When empty capsids were added

to genome containing particles in 2 different ratios we observed a dose dependent increase in

luminescence (Figure 5). The increase was 4.8 fold on average in animals injected with empty capsids

in a 5:1 ratio to full capsids (p < 0.01), and 20 fold when a 20:1 empty to full ratio was used (P<0.05).

Next, we investigated the effect of the combination of the two approaches on the expression

of the transgene in healthy joints. As can be seen in Figure 6, both empty decoy capsid and

triamcinolone treated animals showed increased gene expression compared with vector alone

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Figure 4. Improvement of intra-articular rAAV5-luciferase expression by addition of empty capsids and/or triamcinolone. Mice with CIA (n = 18 per group) were injected with triamcinolone either systemically (i.m.) or locally (i.a.) two days prior to i.a. administration of rAAV5.CMV.Fluc (1.65e10 vg/joint) + empty capsid (full:empty ratio of 1:5). Luciferase expression was monitored over time by IVIS imaging for a follow-up of 30 days after vector administration. Luminescence is shown per group as average; error bars, SEM.

Figure 5. Addition of empty capsids in 2 different ratios (1:5 and 1:20 full:empty) improves intra-articular rAAV5-luciferase expression in healthy mice. Healthy mice (n=7 per group) were injected i.a. with 1.65e10 vg/joint rAAV5-CMV-FLUC. In 2 groups empty capsids were added in different ratios. Luciferase expression was measured weekly until mice were sacrificed after 4 weeks. Luminescence at week 4 is shown per group as averages; error bars, SEM. * P < 0.05 and ** P < 0.01 in groups with empty capsid addition versus the control group that only received genome containing vector (one-tailed unpaired t test).

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Figure 6. Effect of empty capsid and triamcinolone on intra-articular rAAV5 gene expression in healthy mice. Healthy mice (n=17 per group, 34 total injected joints) were injected with 1.26e10 vg/joint rAAV5-CMV-Fluc intra-articularly +/- empty capsid (5:1 empty to full ratio) preceded 2 days prior with i.m. administration of either saline (NaCl) or triamcinolone. Luciferase expression was measured weekly by IVIS up to week 8. (a) Luciferase measurement over time for all groups. (b) Luciferase expression in all groups at week 8. Data shown are average per group + SEM. * p<0.05, ** p<0.01, *** p<0.001 as determined by one-tailed Mann Whitney test.

animals, but the combination of empty capsid and triamcinolone gave rise to the highest increase

in gene expression levels, similar to what was observed in inflamed (CIA) joints. These results

support the hypothesis that macrophages in the intimal lining layer inhibit AAV-mediated

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expression, even in healthy joints, and that either adding decoy capsid particles and/or inhibiting

macrophage activity can overcome this inhibition, leading to increased gene expression.

Combination of empty capsid and triamcinolone is not specific for rAAV5,

but enhances intra-articular gene expression using other serotypes as well

Our studies thus far focused on AAV5 as this serotype is very effective in transducing FLS, however

we hypothesized that the effect of macrophage neutralization on gene transfer using AAV is not

serotype specific. AAV uptake by macrophages is a general phenomenon utilizing scavenger

receptors, and thus this should not be limited to one specific serotype, or any specific virus as

macrophages are known to take up a wide range of viruses and bacteria. To test this hypothesis,

we performed an experiment where we evaluated if triamcinolone and empty capsid could

enhance gene expression from a serotype that is very different from AAV5, being AAV2. AAV5

and AAV2 share only 57% homology at the amino acid level, making them two of the most diverse

serotypes of AAV known. To test this, we performed an experiment identical to that described

above (Figure 6), but now with AAV2. Similar to results seen with rAAV5, both empty decoy capsid

and triamcinolone treated animals showed increased gene expression compared with vector

alone animals following rAAV2 administration (Figure 7). The combination of empty capsid and

triamcinolone gave rise to the highest increase in gene expression levels, similar to what was

observed in rAAV5 treated animals, providing further validation of this approach.

D I S C U S S I O NWhile there have been many advances in the treatment of RA, there remains an urgent need

for safe and effective therapies targeting inflamed joints that are unresponsive to systemic

therapies. To address this need, we and others have proposed local delivery of rAAV as a

potential platform for the long term expression of therapeutic genes.14,25-27 rAAV vectors have

been extensively used for in vivo gene therapy and have been shown to be safe and effective in

pre-clinical models as well as in clinical trials.4,28

In the course of developing an rAAV-based gene therapy product for the treatment of

RA, we have used rAAV5 expressing firefly luciferase combined with in vivo imaging of mice

to investigate factors affecting intra-articular gene expression. Of importance, we found that,

following intra-articular administration of rAAV5 in arthritic animals, not all joints are effectively

transduced (usually <50%), and expression in injected joints was quite variable. To ensure

sustained local production of effective doses of therapeutic proteins in the joint, in particular

in the rheumatoid synovium, an optimized gene delivery system is clearly needed.

Here we present data showing that synovial macrophages represent a previously

unrecognized barrier for efficient intra-articular gene transfer. Our data suggests that

agents that can deplete and/or inhibit activation of macrophages (clodronate liposomes or

triamcinolone), as well as the addition of empty decoy capsids to the vector preparation, can

improve luciferase expression after local injection in the joint. Using a combination of both

approaches resulted in a synergetic effect. Interestingly, also in healthy joints the expression of

luciferase was improved using a combination of triamcinolone and empty capsids, and this was

not limited to the use of an rAAV5 vector but also applies to other serotypes.

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Triamcinolone, a glucocorticosteroid which is frequently used in the treatment of RA, had

a positive effect on transgene expression in a similar order of magnitude as seen for clodronate

liposomes. Systemic administration of glucocorticosteroids is known to exert a local effect in

the joint by decreasing the number of macrophages in synovial tissue of RA patients29, indicating

that the effect of triamcinolone is at least partly related to the depletion of macrophages.30

Both systemic and intra-articular administration of glucocorticosteroids can be accompanied

by side-effects.31 In case of mono- or oligoarthritis, the preferred route is intra-articular.. We

demonstrated that intra-articular injection of triamcinolone was equally effective in increasing

gene expression compared with systemic administration, which is an important finding in view

of the possible application of this approach for intra-articular gene therapy in RA.

The findings reported in this manuscript may also be relevant for the interpretation of the

lack of efficacy in the phase I/II clinical trial with tgAAC94. In this study, adults with persistent

moderate or severe inflammation in a clinically inflamed joint received a single intra-articular

injection of rAAV2 expressing the human TNFR:Fc gene.7 Arthritis activity is strongly correlated

with macrophage infiltration of the synovial tissue.32 Based on the findings presented here, we

postulate that the presence of macrophages at the site of injection may have diminished the

transduction efficacy of the vector, thereby preventing efficient transgene expression to induce a

therapeutic effect. Other factors, including neutralizing antibodies, may also have played a role.

For transduction efficacy of rAAV vectors, the presence of macrophages is not only important

in inflamed tissue, but as presented here, also in healthy tissues. Thus, the combination of

triamcinolone and empty capsid is not only effective for increasing expression in diseased joints,

but also in healthy joints. These results may be relevant for a wider range of applications using rAAV

vectors delivered to tissue containing macrophages. This notion is supported by a previous study

showing that blocking rAAV8 uptake by macrophages via scavenger receptors (using polynosinic

acid (poly[i]) can reduce AAV endocytosis in vitro, and that poly(i) enhances liver targeted gene

expression in vivo in a rat model of hyperbilirubinemia.33 As expected, we found that enhancement

of gene expression by macrophage inhibition is applicable to AAV serotypes other than AAV5 as well.

Recently, it was reported that addition of a significant amount of empty capsids to the AAV

transgene composition after intravenous administration is able to overcome the inhibitory

effect of (pre-existing) neutralizing antibodies and has an ameliorating effect on transgene

expression in the liver.24 It was shown that addition of empty decoy capsid to the final vector

formulation can adsorb these antibodies and thus overcoming their inhibitory effect. Based on

these findings, we hypothesized that empty capsids could act in a similar manner as decoy for

macrophages, thereby preventing uptake and degradation of the transgene-containing rAAV

vector. Indeed, we found that addition of empty decoy vectors to the full vector preparation in

a ratio of 1:5 or 1:20 (empty to full) has a positive effect on transgene expression in the joint. The

exact mechanism of action of these decoys is currently not known, and it is unclear if empty

capsids have a higher affinity for macrophages compared to full capsids, or if the abundance of

total capsid particles is overwhelming the macrophage scavenger pathway, resulting in more

rAAV particles able to transduce the synovial FLS.

Empty capsids can be part of the vector preparation or be produced independent of

the rAAV vector batch, and if desired, added to AAV vector preparations, or administered

separately to an individual. In future studies, the ratio of empty to full particles needs to be

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Figure 7. Effect of empty capsid and triamcinolone on intra-articular rAAV2 gene expression in healthy mice. Healthy mice (n=17 per group, 34 total injected joints) were injected with 1.26e10 vg/joint rAAV2-CMV-Fluc intra-articularly +/- empty capsid (5:1 empty to full ratio) preceded 2 days prior with i.m. adminstration of either saline (NaCl) or triamcinolone. Luciferase expression was measured weekly by IVIS up to week 4. a) Luciferase measurement over time for all groups. b) Luciferase expression in all groups at week 4. Data show is average per group + SEM. ** p<0.01, *** p<0.001 as determined by one-tailed Mann Whitney test.

investigated in more detail, also in the context of the level of inflammation and number of

macrophages present. Reducing the number of capsids present in the preparation may also

reduce immunotoxicity. An interesting approach to further enhance the safety of the use of

empty capsids to bind neutralizing antibodies, is the mutation of the receptor binding site

of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a

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target cell 24, thereby also reducing the potential for targeted immunotoxicity. Whether such

an approach is also feasible in reducing the detrimental effects of macrophages on transgene

expression needs to be evaluated in future studies

In conclusion, we provide evidence that intra-articular macrophages represent a barrier

to efficient gene transfer, and that triamcinolone or empty decoy capsids alone result in

improved gene expression in inflamed and healthy joints. Importantly, the combination of a

glucocorticosteroid and empty capsids led to a synergistic increase in gene expression. These

data have implications for future applications of gene therapy to the joint, but also to other

tissues with an abundance of macrophages.

A C K N O W L E D G E M E N T SThe authors want to thank L Bevaart and FA Koopman for their contribution in writing the

application for the animal ethical committee and their assistance in getting the studies initiated.

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