AMSTERDAM, The Netherlands—The possibility of achieving TNF inhibition without the need for (or supplemental to) systemic TNF-inhibitor therapy was brought tantalizingly closer this month with publication by lead researcher Janik Adriaansen, MD, of data showing that a gene for a TNF-blocking agent carried by an adeno-associated virus (AAV) construct injected into the joints could reduce joint inflammation in an animal model of rheumatoid arthritis (RA). The report, published early online in Annals of the Rheumatic Diseases,¹ also shows that expression of the TNF-blocker could be regulated by a disease-inducible nuclear factor (NF)-κB-responsive promoter.

"[T]his work supports the feasibility of local gene therapy using an rAAV5 vector to deliver a therapeutic gene directly to the inflamed joint. Additionally, disease-regulated transgene expression for physiologically responsive gene therapy might be feasible and could contribute to the safety and effectiveness of the gene therapeutic approach. Thus, this study provides the rationale for further research towards clinical development of rAAV5-NF-κB-TNFRI-Ig intra-articular gene therapy," writes Dr. Adriaansen, with the division of clinical immunology and rheumatology at the University of Amsterdam, The Netherlands.

Researchers aim for local gene therapy in individual joints

Recombinant AAV is a popular vector for gene therapy in chronic diseases because these viruses are not pathogenic in humans and can establish long-term expression of the transgenic protein. The researchers combined an rAAV serotype 5 (rAAV5) viral vector with a chimeric human soluble p55 TNF receptor-immunoglobulin (TNFR-Ig) coupled to the Fc part of murine IgG1 (TNFRI-Ig). This therapeutic gene was then placed under control of a disease-inducible NF-κB promoter.

"As we intend to develop intra-articular gene therapy for RA, we evaluated the effects of these gene constructs on arthritis activity and cytokine levels as well as the biodistribution after intra-articular injection in the adjuvant arthritis model of RA," Dr. Adriaansen writes.

This approach might be appropriate for RA patients who have only a few symptomatic joints and who are not considered candidates for systemic TNF-inhibitor treatment, or for patients who are ACR20 responders to systemic TNF-inhibitors but still have a few symptomatic joints. "Local gene transfer might allow persistent production of a TNF inhibitor directly in the inflamed joint and enhance the bioavailability at the site of inflammation when the right vector is used," Dr. Adriaansen writes.

The therapeutic gene was made by cloning a chimeric hTNFRI/mIgG gene into a pVAX2 plasmid. In this study, the researchers compared a construct with the TNF-inhibitor gene under control of a disease-inducible NF-κB promoter to the same gene under control of a cytomegalovirus immediate early (CMV) promoter and to a control vector that used the CMV-shuttle plasmid without a gene transcription cassette. In vitro effects were tested in fibroblast-like synoviocytes (FLS) isolated from synovial biopsies from RA patients and cultured. Local gene transfer was done in rats immunized with Mycobacterium tuberculosis in mineral oil to produce paw swelling.

The animals' right ankle joints were injected on day 12 with 2 x 1010 viral molecules of NF-κB-controlled TNFRI-Ig vector, CMV-controlled vector, or control empty vector in 50 μL saline (10 animals per group). Two weeks later the animals were sacrificed, and hind paws and organs examined for biodistribution.

The findings showed that:

•  Human FLS transfected with rAAV5 could produce TNFRI-Ig in vitro, and this production was responsive to activation of NF-κB by inflammatory stimuli (TNF-α, IL-1β, or lipopolysaccharide, LPS).
  
•  Both rat and human TNF were neutralized by the TNFR-Ig produced by the transduced cells, but the antibody had a much higher affinity for human TNF.
  
•  TNFRI-Ig was found in joints treated with one of the TNFR-Ig vectors but not in those treated with the control vector.
  
•  TNFRI-Ig protein was produced to a much greater degree in the synovial cells of rats injected with the NF-κB-controlled vector than in those injected with the CMV-controlled vector.
  
• less paw swelling and a reduction in synovial inflammation than those treated with either the CMV-TNFRIIg or the control vector.
  
•  These animals also had significantly lower levels of the inflammatory mediators TNF-α, IL-1β, and interleukin 6 (IL-6), and higher levels of the anti-inflammatory cytokine interleukin-10 (IL-10).

The biodistribution analysis showed that the transfected gene stayed where it had been injected. Slightly elevated levels of TNFRI-Ig-mRNA were found only in the spleen and draining lymph nodes.

The investigators conclude, "The present study shows that intra-articular injection of a human TNFRI-Ig expressed by an rAAV5 vector with an NF-κB inducible promoter in rats with adjuvant arthritis results in secreted and bioactive TNFRI-Ig, able to block TNF-α in the injected joint providing a therapeutic effect."

Daniel E. Furst, MD, the Carl M. Pearson professor of rheumatology at the University of California, in Los Angeles, told CIAOMed, "This is a way to deliver a drug to a single locale. It may be very good for oligoarthritis or monoarthritis, if data show that it leads to fewer adverse events. This would serve a restricted but important segment of the market." Dr. Furst pointed out a number of questions that need further study, including duration of effect, whether there are systemic effects, whether the construct remains in the joint, and whether the gene therapy leads to decreased adverse events relative to systemically delivered TNF-inhibitors.

Senior author Paul P. Tak, MD, PhD, told CIAOMed that many of these points are currently under investigation and he expects to see gene constructs used in clinical trials in patients within 4 years. Dr. Tak is professor of medicine and director of the division of clinical immunology and rheumatology at the University of Amsterdam, The Netherlands.

"This study provides proof of concept that TNF inhibition by intra-articular gene therapy might be possible," Dr. Tak said. "This is what we had hoped for, and it worked."

Compared with the lenercept (Ro 45-2081) intravenous human recombinant TNF-receptor p55 IgG1 fusion protein Dr. Furst and colleagues studied in RA patients, Dr. Tak said that the localized gene therapy construct may have some advantages. "It is probably less immunogenic. It stays localized at the site of inflammation. And the production of the therapeutic protein in the joint is regulated by disease activity: there is more production when it is needed, and when the disease activity is low there is little expression of the therapeutic protein," he said.

Reference

1. Adriaansen J, Khoury M, de Cortie CJ, et al. Reduction of arthritis following intra-articular administration of an adeno-associated virus serotype 5 expressing a disease-inducible TNF-blocking agent. Ann Rheum Dis. 2007 March 15; [Epub ahead of print].