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Local delivery of a recombinant adenoassociated vector containing a tumour necrosis factor α antagonist gene in inflammatory arthritis: a phase 1 dose-escalation safety and tolerability study
  1. P J Mease1,
  2. K Hobbs2,
  3. A Chalmers3,
  4. H El-Gabalawy4,
  5. A Bookman5,
  6. E Keystone6,
  7. D E Furst7,
  8. P Anklesaria8,
  9. A E Heald8
  1. 1
    Seattle Rheumatology Associates/Swedish Medical Center Research, Seattle, Washington, USA
  2. 2
    Denver Arthritis Clinic and University of Colorado Health Sciences Center, Denver, Colorado, USA
  3. 3
    University of British Columbia, Vancouver, British Columbia, Canada
  4. 4
    University of Manitoba, Winnipeg, Manitoba, Canada
  5. 5
    University Health Network, Toronto, Ontario, Canada
  6. 6
    University of Toronto, Toronto, Ontario, Canada
  7. 7
    Geffen School of Medicine at UCLA, Los Angeles, California, USA
  8. 8
    Targeted Genetics Corporation, Seattle, Washington, USA
  1. A E Heald, Targeted Genetics Corporation, 1100 Olive Way, Suite 100, Seattle, Washington 98101, USA; alison.heald{at}


Objective: To examine the safety and tolerability of a single intra-articular injection of rAAV2-TNFR:Fc, an adenoassociated virus serotype 2 vector containing the cDNA for the human tumour necrosis factor–immunoglobulin Fc fusion gene (tgAAC94), in subjects with inflammatory arthritis.

Methods: In a double-blind, placebo-controlled, phase 1, dose-escalation study, 15 subjects with inflammatory arthritis (14 with rheumatoid arthritis and 1 with ankylosing spondylitis) not receiving tumour necrosis factor α (TNFα) inhibitors with persistent moderate (grade 2) or severe (grade 3) swelling in a target joint due to inflammatory arthritis received a single intra-articular injection of rAAV2-TNFR:Fc at 1×1010 (n = 5) or 1×1011 (n = 6) DNase resistant particles per ml joint volume or placebo (n = 4) into a knee (n = 14) or ankle (n = 1). Safety was assessed through adverse event monitoring. As a secondary objective, changes in injected joint tenderness and swelling scores, each measured on a four-point scale, were evaluated.

Results: Intra-articular injections of rAAV2-TNFR:Fc were well tolerated with no major safety issues. One event, mild knee pruritis, was considered probably related. Synovial fluid TNFR:Fc protein was not detected (nor expected) at the doses used. At 12 weeks after injection, a two-point decrease in swelling was noted in 2/11 and 2/4 subjects injected with rAAV2-TNFR:Fc and placebo, respectively.

Conclusion: A single dose of intra-articular rAAV2-TNFR:Fc appears to be safe and well tolerated in subjects without concurrent systemic TNFα antagonist use. It is thus feasible to proceed with larger trials to further test the safety and efficacy of local TNFR:Fc gene transfer as a therapeutic modality for patients with inflammatory arthritis.

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Tumour necrosis factor α (TNFα) has been implicated as a major participant in the inflammatory cascade that leads to the joint damage and destruction in diseases such as rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS). Although there is no cure, treatment has been revolutionised by the advent of anti-TNFα and other biological therapies. Clinical studies have shown that these agents improve the signs and symptoms, inhibit the structural damage and impact functional outcomes in patients with these inflammatory arthritides.13

Many patients with inflammatory arthritis have a few persistently symptomatic, swollen joints despite systemic therapy, including TNFα blockade.4 Since persistent joint swelling correlates with structural damage over time, these patients are ideal candidates for a localised, more concentrated delivery of anti-TNFα therapy directly to the joint. Other patients, who have monoarticular or oligoarticular arthritis, may also benefit from anti-TNFα therapy using localised gene transfer given the reduced risk of the adverse effects of systemic therapy.

TNFα antagonists such as etanercept and infliximab have been administered directly into the joints of a limited number of patients with inflammatory arthritis with improvement in joint swelling, tenderness and range of motion.510 In general, intra-articular administration of etanercept and infliximab was well tolerated, but provided only temporary relief, because of the short half lives of the proteins.510

An alternative to intra-articular therapeutic proteins is the injection of DNA coding for therapeutic protein. This process, called gene transfer, offers the advantage of potentially providing sustained concentrations of the therapeutic protein within the joint, thus reducing the need for frequent intra-articular injections. Recombinant adenoassociated virus (rAAV) vectors are derived from adenoassociated virus (AAV), a naturally occurring, non-pathogenic, non-integrating and non-replicating virus that depends on a helper virus for replication.11 12 rAAV vectors do not contain any AAV coding sequences. They are efficient gene delivery vehicles and in non-dividing or slowly dividing cells the delivered gene can be expressed for the life of the transduced cell.11 13 rAAV vectors have been used to deliver genes to over 200 study subjects by various routes of administration for potential treatment of genetic disorders such as cystic fibrosis, haemophilia and hereditary emphysema (α1-antitrypsin deficiency) and neurological disorders such as Canavan disease, Batten disease, Parkinson disease and Alzheimer disease without any significant safety concerns.14 The favourable safety profile established thus far and long-term gene expression suggest that an AAV vector may be an efficient means to deliver a transgene that codes for a TNFα antagonist protein into the affected joint(s).

An rAAV serotype 2-based vector containing the human tumour necrosis factor–immunoglobulin Fc fusion gene (rAAV2-TNFR:Fc, or tgAAC94) was developed for clinical use. Preclinical studies were conducted in a rat model of experimental arthritis15 and in non-human primates to support clinical assessment of rAAV2-TNFR:Fc in humans. The potential efficacy of this modality of treatment was demonstrated in a study of AAV2-ratTNFR:Fc in Lewis rats with streptococcal cell wall-induced arthritis. A single dose of ∼1×1012 DNase-resistant particles (DRP)/ml of joint volume AAV2-ratTNFR:Fc administered via intra-articular injection resulted in suppression of arthritis in the injected joint as well as the contralateral joint.15 Additional pharmacological studies indicate that a single intra-articular administration of a high dose of rAAV2-TNFR:Fc (∼1×1013 DRP/ml joint volume) resulted in sustained and detectable expression as measured by TNFR:Fc RNA in joint tissues for ⩾365 and 90 days, the last timepoints tested, in healthy Lewis rats and healthy cynomolgus primates, respectively (unpublished results). Toxicology studies in Lewis rats have demonstrated that single and repeat intra-articular injections of rAAV2-TNFR:Fc were safe and well tolerated at doses up to 1×1013 DRP/ml joint volume (unpublished data).

This first phase 1 clinical study was undertaken to evaluate the safety and tolerability of a single intra-articular administration of rAAV2-TNFR:Fc in subjects not on systemic TNFα antagonists in order to assess the safety of rAAV2-TNFR:Fc without the confounding presence of other TNFα antagonists. To provide a wide safety margin, dose escalation began at 1×1010 DRP/ml joint volume. Therapeutic benefit was expected at a dose of 1×1012 DRP/ml joint volume, based on the study of Lewis rats with streptococcal cell wall-induced arthritis. Although the study was not powered for efficacy, clinical activity was assessed.


Study agent

The active study agent was rAAV2-TNFR:Fc (tgAAC94), a recombinant AAV serotype 2 vector genetically engineered to contain the cDNA for the TNFR:Fc fusion gene, which consists of the cDNA of the human tumour necrosis factor extracellular domain fused in-frame to the cDNA of the human IgG1 Fc domain (fig 1). The DNA sequence of TNFR:Fc in rAAV2-TNFR:Fc is identical to that used in the production of etanercept. rAAV2-TNFR:Fc was formulated in a sterile isotonic buffered salt solution. The placebo used was the same sterile isotonic buffered salt solution.

Figure 1

The rAAV serotype 2-based vector containing the human tumour necrosis factor–immunoglobulin Fc fusion gene (rAAV2-TNFR:Fc) genome consists of human cytomegalovirus immediate–early promoter (P CMV IE), a splice donor/splice acceptor site (SD/SA), the cDNA for the human TNFR:Fc gene (huTNFR:Fc) and a bovine growth hormone poly A sequence (BGH pA), flanked by the AAV2 inverted terminal repeat sequences (ITR).

Study design

In this double-blind, placebo-controlled, dose-escalation study, subjects were enrolled in two cohorts of up to eight subjects with six subjects in each cohort receiving rAAV2-TNFR:Fc and two receiving placebo. The two cohorts received escalating dose concentrations of rAAV2-TNFR:Fc, ranging from 1×1010 to 1×1011 DRP/ml. The volume injected depended on the joint. Knees were injected with 5 ml and ankles with 2 ml. The placebo group was included to distinguish adverse events related to rAAV2-TNFR:Fc from intra-articular injections. A third cohort was to receive a dose concentration of 1×1012 DRP/ml, but the study was stopped prior to this cohort because of slow enrolment.

Study population

Study subjects were recruited from three rheumatology practices in the USA and four in Canada. The study was approved by the institutional review boards at each site, and by institutional biosafety committees at US sites. Written informed consent was obtained prior to conduct of any study procedures. The study was overseen by an independent Data Monitoring Committee (DMC).

Entry criteria included a confirmed diagnosis of RA, PsA or AS according to published criteria1618 and persistent moderate (grade 2) or severe (grade 3) joint swelling in at least one peripheral joint eligible for injection. Other entry criteria included age greater than 18 years, and for subjects with rheumatoid arthritis, an adequate trial of at least one disease-modifying antirheumatic drug (DMARD) prior to screening. Subjects on DMARD(s) were required to be on the same regimen for the previous 3 months, with no changes in dose in the 4 weeks prior to screening. Key exclusion criteria included current use of a TNFα antagonist, disease severe enough to warrant use of systemic TNFα antagonist therapy in the next 3 months and discontinuation of TNFα antagonists in the past because of safety concerns.

Study procedures

After completion of screening procedures, subjects in each cohort were randomised to receive a single intra-articular injection of rAAV2-TNFR:Fc at the dose level for the cohort or placebo. Subjects were evaluated at screening, the day of dosing, day 3 and weeks 1, 2, 4, 8, 12 and 24 after dosing.

Safety was evaluated by serial medical history and physical examination, complete blood count (CBC) with white blood cell differential, blood chemistries and urinalysis. Pre-existing and induced anti-AAV2 capsid neutralising titres (NT) in serum and synovial fluid were measured prior to dosing and 4, 12 and 24 weeks after dosing using a microtitre assay. Presence of AAV2-TNFR:Fc genomic DNA in systemic circulation was evaluated by DNA PCR analysis using TNFR:Fc-specific primers and probes on DNA samples isolated from blood collected prior to, 3 days, and 2, 4, 8 and 12 weeks after dosing (limit of detection 15 copies per μg DNA). To assess gene expression in synovial fluid, the amount of functional TNFα antagonist activity was quantified by quantitative radioimmunoassay with an etanercept (TNFR:Fc) standard curve (Biomonitor A/S, Copenhagen, Denmark; limit of detection 7 ng/ml).

Clinical response was evaluated by examination of the target joint for tenderness on a scale ranging from 0 (none) to 3 (severe) and swelling on a scale ranging from 0 (none) to 3 (severe), based on guidelines published in the Dictionary of Rheumatic Diseases.19

To determine whether a systemic response occurred after local administration, systemic efficacy measures were collected. These included a tender joint count (68 joints) and a swollen joint count (66 joints), patient self-assessment of pain (10 cm visual analogue scale (VAS)), patient and physician global assessment of disease activity (10 cm VAS), patient assessment of disability using the Health Assessment Questionnaire (HAQ),20 and determination of the erythrocyte sedimentation rate and C-reactive protein. In addition, subjects with AS completed the Bath Ankylosing Spondylitis Functional Index (BASFI) and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI).21 22

For subjects with rheumatoid arthritis, clinical response was determined using the American College of Rheumatology (ACR) 20 response, according to previously published criteria.23 Disease activity was also assessed using the modified 28-joint Disease Activity Score (DAS), developed by the European League Against Rheumatism (EULAR).24 25


Study enrolment and disposition

A total of 18 subjects were screened for the study. Two subjects did not qualify and a third withdrew prior to dosing. The remaining 15 subjects included 12 women and 2 men with RA and 1 man with AS (table 1). In all, 10 subjects were taking methotrexate and 5 were taking prednisone. As per study entry criteria, none were taking TNFα antagonists, although one subject randomised to rAAV2-TNFR:Fc at a dose of 1×1011 DRP/ml (P13) had received infliximab in the past, which had been discontinued because of treatment failure.

Table 1 Demographics and baseline characteristics

In the first cohort, seven subjects were randomised to receive either a single intra-articular injection of rAAV2-TNFR:Fc at a dose of 1×1010 DRP/ml joint volume (n = 5) or placebo (n = 2). In the second cohort, eight subjects were randomised to receive a single intra-articular injection of either rAAV2-TNFR:Fc at a dose of 1×1011 (n = 6) DRP/ml joint volume or placebo (n = 2).

Of the 15 subjects, 14 completed the study. One subject who received rAAV2-TNFR:Fc at a dose of 5×1011 DRP (total) into the knee (P11) withdrew from the study 8 weeks after dosing because of a flare of RA.


Intra-articular injections, 14 into knees and 1 into an ankle, of either rAAV2-TNFR:Fc or placebo, appeared to be safe and well tolerated. A total of 50 adverse events were reported (table 2). Although no infections were reported among placebo recipients, a similar number of subjects experienced infections in the two active treatment groups. The incidence of other adverse events was similar across treatment groups.

Table 2 Incidence of adverse events by dose group

Three related adverse events were reported. Two subjects, both of whom received rAAV2-TNFR:Fc at a dose of 1×1010 DRP/ml joint volume, experienced one adverse event each that the investigator considered to be possibly related to study agent. These included nasopharyngitis and mild sinus congestion, 17 and 49 days after study agent injection, respectively. One subject, who received the rAAV2-TNFR:Fc at a dose of 1×1011 DRP/ml joint volume, developed mild pruritis of the injected knee, 14 days after study agent injection, which the investigator considered probably related to study agent.

Four serious adverse events were reported, all in the same 88-year-old woman with RA who received a single injection of rAAV2-TNFR:Fc at a dose of 1×1011 DRP/ml joint volume into the knee. She was hospitalised with labyrinthitis, myocardial infarction and two episodes of congestive heart failure 25, 32, 47 and 75 days after study agent administration, respectively. The investigator considered all four events unlikely to be related to study agent.

Baseline serum anti-AAV2 capsid NT ranged from <1/4 to 1/1024 (table 1). None of the subjects treated with placebo or the lower dose of rAAV2-TNFR:Fc experienced a fourfold or greater rise in NT, which would be considered an induction of NT due to study agent administration. Among the six subjects who received rAAV2-TNFR:Fc at a dose of 1×1011 DRP/ml joint volume, five (83%) experienced a fourfold or greater rise in titres to a maximum ranging from 1/128 to 1/8192 (geometric mean 1/1176). By 24 weeks after injection, NT in these five subjects had decreased to 1/64 to 1/1024 (geometric mean 1/152). No adverse effects were associated with development of anti-AAV2 capsid NT.

Vector DNA was detected transiently in the systemic circulation, 3 days after intra-articular injection in one of five (20%) and two of six (33%) subjects who received rAAV2-TNFR:Fc at doses of 1×1010 and 1×1011 DRP/ml joint volume, respectively. All other peripheral blood specimens were negative.

Synovial fluid analyses

Anti-AAV2 capsid NT in synovial fluid ranged from <1/4 to 1/64 at baseline (table 1). All four subjects with follow-up synovial fluid samples who received rAAV2:TNFR:Fc at a dose of 1×1011 DRP/ml experienced a fourfold or greater rise in NT to a maximum of 1/256 to 1/2048 (geometric mean 1/512). TNFR:Fc protein was not detected in any of 12 post injection synovial fluid samples from 8 subjects who received rAAV2-TNFR:Fc. Median baseline synovial fluid white cell count was 500 cells/mm3 (range 72 to 31 800 cells/mm3) and did not change appreciably after rAAV2-TNFR:Fc administration. Median baseline synovial fluid protein level was 5.6 g/dl (range 1.6 to 62 g/dl) and also did not change appreciably after rAAV2-TNFR:Fc administration.

Clinical activity

Individual changes in swelling and tenderness are depicted graphically in fig 2. Decreases in the tenderness and swelling scores were noted among subjects in both cohorts. Subjects on methotrexate did not appear to have a better or worse outcome than those who were not on methotrexate. The subject who had previously failed infliximab (P13) had complete resolution of tenderness and swelling of the target joint 4 weeks after administration of rAAV2-TNFR:Fc 1×1011 DRP, but the tenderness and swelling recurred 12 weeks after injection, and was back to baseline 24 weeks after injection.

Figure 2

Individual changes in the tenderness score (scale 0 to 3) and swelling score (scale 0 to 3) for each subject in cohort 1 (top panel) and cohort 2 (bottom panel). Subjects in cohort 1 received either recombinant adenoassociated virus serotype 2-based vector containing the human tumour necrosis factor–immunoglobulin Fc fusion gene (rAAV2-TNFR:Fc) at a dose of 1×1010 DNase-resistant particles (DRP)/ml joint volume or placebo. Subjects in cohort 2 received either rAAV2-TNFR:Fc at a dose of 1×1011 DRP/ml joint volume or placebo. Each individual graph is labelled with the subject’s publication number (P01–P15) and treatment assignment (placebo or rAAV2-TNFR:Fc). Subjects currently taking methotrexate (MTX) are denoted “on MTX”. One subject (P13) had failed infliximab (INF) in the past.

A two-point decrease in swelling (ie, from 3 (severe) to 1 (mild), or 2 (moderate) to 0 (none)) was considered to be clinically significant and readily distinguishable on physical examination. At 12 weeks after injection, a two-point decrease in swelling was noted in 2/11 and 2/4 subjects injected with rAAV2-TNFR:Fc and placebo, respectively. Both active subjects with two-point decreases in swelling had baseline serum anti-AAV2 NT<1/4.

Baseline systemic efficacy measures for each subject are shown in table 1. There were no significant changes in these parameters 12 weeks after injection between dose groups (table 3).

Table 3 Changes in systemic efficacy measures 12 weeks after injection

At 12 weeks after intra-articular injection, one subject who received placebo (P15) and one subject who received rAAV2-TNFR:Fc at 1×1011 DRP/ml joint volume (P12) achieved an ACR20 response.

Baseline DAS, listed for individual subjects in table 1, ranged from 1.7 to 6.5. Using the EULAR response criteria, one subject who received placebo (P15) achieved a good response, one subject who received placebo (P14) achieved a moderate response and one subject who received rAAV2-TNFR:Fc at 1×1011 DRP/ml joint volume (P12) achieved a moderate response at week 12.

The one subject with ankylosing spondylitis (P08) received rAAV2-TNFR:Fc at 1×1011 DRP/ml joint volume into the knee. This subject had an ASAS50 response, defined as greater than 50% reduction in all four domains of the Assessments in Ankylosing Spondylitis (ASAS; now Assessment in SpondyloArthritis international Society),26 at week 12.


Although there have been vast improvements in the treatment of inflammatory arthritis, the problem of synovitis persisting in a few joints remains a therapeutic challenge.4 Historically, individual problematic joints have been treated with intra-articular steroid injections, radioactive synovectomy or surgical intervention.2729 These therapies have not been shown to have long-term benefit in refractory oligoarticular or monoarticular arthritis.

Local expression of proteins with antiarthritic properties following direct gene transfer to the inflamed joint may provide long-term benefit for persistent synovitis. Numerous preclinical studies of local and systemic gene therapy for inflammatory arthritis using an assortment of vectors and transgenes have been promising.30 31 However, few clinical studies have been reported to date.31 32 For local in vivo delivery of therapeutic genes, rAAV vectors may have the greatest potential due to their established safety and sustained transgene expression with the requirement for infrequent administration.

In this study, intra-articular injections of rAAV2-TNFR:Fc appeared to be safe and well tolerated at doses up to 1×1011 DRP/ml joint volume in inflammatory arthritis subjects not on TNFα antagonists. The pattern of adverse events with either dose of rAAV2-TNFR:Fc was similar to placebo. None of the four serious adverse events reported were considered related to rAAV2-TNFR:Fc vector.

At the doses administered, rAAV2-TNFR:Fc DNA was transiently detected by PCR analysis in the systemic circulation 3 days after intra-articular injection, indicating limited systemic biodistribution. In the non-human primate model TNFR:Fc protein at ∼3.7 ng/ml was detected in joint lavage obtained from animals injected with ∼1×1012 DRP/ml joint volume (unpublished results).

TNFR:Fc protein was not detected in synovial fluid, but the doses administered in this study did not reach the levels administered in the proof of concept study (1×1012 DRP/ml). Pre-existing immunity to AAV capsid may limit the transduction potential of rAAV vectors. In this small study, the impact of elevated anti-AAV2 capsid NT at the time of rAAV2-TNFR:Fc vector injection on transduction efficiency could not be evaluated since TNFR:Fc protein was not detected in synovial fluid. However, no adverse effects related to development of NT after the highest dose were noted.

Clinical evaluation of injected joints revealed decreases in joint swelling and tenderness in subjects who received doses of rAAV2-TNFR:Fc as well as placebo. This small, phase 1 study was not powered to show efficacy and it is not clear how reliable simple clinical measures of joint swelling and tenderness are in discriminating change in a single joint. Current composite response indices such as the ACR and DAS are unlikely to detect changes referable to a single joint and underscore the need for development of validated measures to assess the response of a single joint to new local therapies for inflammatory arthritis. Although a variety of outcome measures have been used in small clinical studies to measure improvement after intra-articular or systemic TNFα antagonist protein administration,7 9 10 33 34 or radiation synovectomy,35 none have been validated. Validated measures to assess the response of a single joint to local treatment for inflammatory arthritis are currently under development.36

In summary, intra-articular injections of rAAV2-TNFR:Fc at doses up to 1×1011 DRP/ml joint volume appear to be safe and well tolerated in subjects not receiving TNFα antagonists. This study supports the feasibility of direct AAV2 vector-mediated intra-articular gene delivery to the arthritic joint. A phase 1/2 study is currently underway to evaluate the safety and further explore efficacy of higher and repeat doses of rAAV2-TNFR:Fc.37


The authors would like to thank the subjects for participating in this study, Tara Allen, Linda Wilson and Glenna Peterson at Targeted Genetics Corporation for performing the anti-AAV2 capsid neutralising titre and vector in peripheral blood assays and Alice Henning at EMMES Corporation for preparing the final statistical report.



  • Competing interests: None declared.

  • Funding: This study was supported by Targeted Genetics Corporation (Seattle, Washington, USA).

  • Ethics approval: Obtained.