Optimizing gene transfection strategies for the targeted treatment of rheumatoid arthritis
SummaryRheumatoid arthritis affects an estimated 2.1 million Americans. The initial success of biologics that neutralize TNF alpha mean that these therapeutics dominate the US and European rheumatoid arthritis markets. Although the development of biologics has revolutionized treatment of rheumatoid arthritis therapies remain far from optimal. Since biologics are peptidic in nature they must be administered by injection. Moreover, multiple dosing is required and this can be disruptive to the patient.
Rheumatoid arthritis affects an estimated 2.1 million Americans and is characterized by chronic inflammation of the lining of the joints and the development of the pannus, a structure composed of fibroblast-like synovial cells that erode the joint and surrounding bone. The initial success of biologics that neutralize TNF alpha such as the decoy receptor Enbrel (etanercept) and the neutralizing anti-TNF alpha antibody Remicade (infliximab) mean that these therapeutics dominate the US and European rheumatoid arthritis markets (see Rheumatoid Arthritis - Novel Biologics Drive Innovation in the Global RA Market).
Although the development of biologics has revolutionized treatment of rheumatoid arthritis therapeutic options remain far from optimal. Since biologics are peptidic in nature they must be administered by injection. Moreover, multiple dosing is required and this can be disruptive to the patient. Furthermore, relatively high systemic doses are necessary to achieve constant therapeutic levels and this can be costly. Systemic exposure can also result in non-specific immune suppression and increase the susceptibility to infection. For example over the five month period following the approval of Enbrel thirty of the estimated 25,000 patients treated with the drug were reported to have developed serious infections including several with sepsis. Six of these patients died within two to sixteen weeks after initiation of treatment.
An alternative approach could be to use intra-articular gene therapy to deliver therapeutic genes directly to the synovium in order to have the protein synthesized at the site of inflammation. The DNA/RNA market which includes gene therapy is in its infancy with no significant products launched across the seven major markets. Despite the unproven nature of the sector in terms of market potential, it is commanding considerable interest across the biotechnology industry. Almost 100 companies are developing DNA/RNA therapies, with 229 products in development. The DNA/RNA market is forecast to generate $1.2bn by 2010 with antisense and gene therapies together generating 46% of these sales (see DNA/RNA Therapies).
Gene therapy technologies are advancing and technologies now include the use of viral vectors, non-viral vectors and cell therapy with genetically modified vectors. The various technologies as well as the markets and clinical applications are extensively covered in our recent feature Gene Therapy - technologies, markets and companies. Rheumatoid arthritis represents an excellent candidate for gene therapies since synoviocytes can be readily accessed by intra-articular injection and furthermore they have a low mitotic rate and as a result they are likely to express transduced genes for a considerable length of time.
Of the various technologies viral-mediated gene transfer is currently the most efficient system for delivering therapeutic proteins in vivo. Adeno-associated virus (AAV) is especially promising given its safety due in part to its low immunogenicity. Although at least 8 AAV serotypes exist, in the clinic recombinant AAV2 (rAAV2) has been the most validated for gene therapy. Studies have been conducted in a number of diseases and the use of this virus is particularly suited to situations where long-term gene expression is required. Barriers to the use of rAAV2 in the clinic include the sometimes inefficient gene transduction and also the prevalence of antibodies against this serotype in humans leading to the neutralization of target cell transfection. The capsids of the AAV serotypes differ considerably offering an opportunity to avoid the problem of antibody neutralization. In the Ann Rheum Dis study featured here, Janik Adriaansen from the and colleagues take the novel approach of cloning the capsid encoding domains of serotypes 1-5 into a common vector backbone containing AAV2 replication genes. The resultant hybrid serotype vectors (rAAV1-5) differ only in their capsids and the study compared the transduction efficiency of each of the hybrids using beta-galactosidase as a marker gene.
In their study Adriaansen et al found that the strongest synovial gene expression 12 days after rat intra-articular immunization was found with rAAV5, followed by a much lower expression using rAAV2. Transduction was highly limited or absent following inoculation with adenovirus or rAAV serotypes 1, 3 and 4 hybrids. Furthermore transduction was more rapid and more prolonged (at least 4 weeks) with rAAV5 compared to rAAV2. The improved transduction seen with the former corresponded to reduced neutralizing antibody titers suggesting that the greater efficacy was due, at least in part, to reduced neutralization. It should be noted that an estimated 80% of the human population carry antibodies to the capsid proteins of AAV2; very few patients carry AAV5 antibodies and therefore the advantage conferred to rAAV5 derived vectors should be maintained in the clinic. In addition to having differing immunogenicity rAAV2 and rAAV5 use distinct receptors to bind and infect target cells; moreover intra-cellular trafficking might be different for the two serotypes and these factors may also contribute to the improved transduction seen with rAAV5.
In a further study Adriaansen et al found that both rAAV2 and rAAV5 were able to effectively transduce human fibroblast-like synoviocytes in acute in vitro studies. This is an important finding since it suggests that the transduction observed when administered intra-articularly to rats will be replicated in humans.
Until now, no gene transfer studies employing non-serotype-2 vectors have been conducted in the inflamed joint. The present study makes the important advance of demonstrating that the use of rAAV5 vectors may considerably improve therapeutic efficacy. In particular rAAV5-derived vectors appear to be excellent candidate vectors for local gene therapy in patients with rheumatoid arthritis, allowing long-term expression of the transgene limited to the synovial compartment. The next challenge will be to select candidate genes for evaluation in rAAV5 vectors and there are a number of candidates (see our feature Rheumatoid arthritis: Emerging drug discovery targets and therapeutic candidates). Perhaps the most obvious starting point would be to focus on TNF and candidates could include those which code for anti-inflammatory mediators. Indeed the Dutch group are currently developing a rAAV5 vector able to transduce synoviocytes with a TNF-alpha inhibitor. The success of this approach will depend on the expressed protein being able to exit transfected cells. However very promising results have already been achieved with studies demonstrating that rAAV5 is able to transduce cells with the gene encoding this TNF blocking protein and that the expressed protein is secreted into the synovial tissue. Moreover evidence suggests that the secreted protein influences other cell types and in rodents inoculation with this rAAV5 vector has produced anti-inflammatory activity in animals with experimental arthritis validating the approach. According to Dr Adriaansen a clinical trail with this vector is planned during the next two years.