RAC protocol PI: Philip Mease, MD Sponsor: Targeted Genetics Ad hoc Reviewer: Robert H. Carter, M.D.

Summary statement This protocol addresses a major clinical need - localized therapy for RA patients with partial responses to systemic therapy. The study profits from the expression of a protein that has been well studied in this population. The rAAV-delivered gene therapy has advantages over repeated administration of proteins while using a vector currently thought to be relatively safe.

Potential Concerns: 1.Transgene-associated 2.Immunological 3.Disease-specific

Potential Concerns: 1.Transgene-associated A. Systemic exposure Circulating vector Circulating cells B. Integration Rare Difficult to document

Potential Concerns: 2. Immunological A. Pre-existing antibodies to AAV B. Induced antibodies to TNF-R-Fc

Potential Concerns: 3. Disease-specific A. Participant selection criteria B. Withholding therapy

Initial Critique and Responses (taken in order of written comments)

Initial Critique and Responses I. The trial is designed to recruit participants with RA with one or two active joints despite therapy with disease-modifying drugs. This leads to three problems: A.This criteria increases the chance of recruiting subjects whose disease in some form of arthritis other than RA B.This criteria will make this trial not comparable to other trials in RA, which require six active joints C.This criteria will decrease the ability to detect responses in other joints (“contralateral effect”).

Initial Critique and Responses A.This criteria increases the chance of recruiting subjects whose disease in some form of arthritis other than RA It is highly unlikely, but possible, that patients with persistent swelling in one or two joints could have another cause of arthritis, such as crystalline arthritis or infection, superimposed on their RA. The distinction will need to be based on good clinical judgment. If the Investigator deems it necessary, joint aspiration will be performed, and the synovial fluid will be sent for crystal examination, Gram stain and culture to rule out those possibilities prior to injection of tgAAC94. [Response to Dr. Simari] If a patient has significant polyarticular disease, then it is anticipated that systemic anti-TNF therapy would be suggested and not involved in this study.

Initial Critique and Responses B. This criteria will make this trial not comparable to other trials in RA, which require six active joints. The primary purpose of this study is to evaluate the safety of intra- articular injections of tgAAC94. It is not necessary to change the entry criteria to match those used in other clinical trials, because there are no plans to compare the results to other trials.

Initial Critique and Responses C. This criteria will decrease the ability to detect responses in other joints (“contralateral effect”). [Other TNF-inhibitor] trials were conducted in patients with active RA involving multiple joints, hence the requirement for involvement of at least six joints. In contrast, this trial is aimed at patients who have reasonably well-controlled disease in most joints, but one or two persistently inflamed and swollen joints which are at risk for cumulative damage.

II. Systemic effects A.Studies did show DNA in other organs, suggesting systemic spread of the virus. B.The trials in rodents demonstrated an effect of inoculation into one joint on disease in the contralateral joint. The inoculation into a single joint could have reduced inflammation in the non-injected joint either directly by systemic protein production (or release) or indirectly by secondary effects of the reduction of the inflammation in the injected joint (decreased local production and release of inflammatory mediators in the injected joint, or decrease systemic acute phase response). C.An important question to be addressed is whether the effect of intra- articular inoculation is local or systemic. … Ultimately, if this therapy appears successful, one question will be whether there is any benefit to intra-articular therapy over IM. Initial Critique and Responses

In the non-human primate model, after a single intra-articular injection, biodistribution of vector DNA was detected in the spleen, left (non-injected) femoral tibial joint tissues and right iliac lymph node by the limit of detection PCR assay (1 in 104 cells to 1 in 103 cells). At the intra-articular injection site (bone, cartilage, synovium, tendon and ligament) gene transfer was documented in a quantitative DNA PCR assay and the amount of vector DNA was dose dependent, with the highest amounts being … 6000 copies/cell. In contrast, while vector DNA in the contralateral joint was quantitated in this assay, the amount of vector was not dose dependent and the highest amount of vector DNA (1.2 copies/cell) was three orders of magnitude lower than at the injection site. Human TNFR:Fc transgene mRNA was present at the injection site using a qualitative RNA-specific PCR assay. In samples from non-injection sites there was no consistent detectable expression, particularly in the spleen and lymph node. II A. Studies did show DNA in other organs, suggesting systemic spread of the virus.

Initial Critique and Responses In summary, the exact mechanism of the observed systemic effect after local intraarticular delivery (in the experimentally induced rat model) is not completely understood. We do not know if we will observe a similar systemic effect after intra-articular delivery in the proposed clinical trial. As mentioned above, recent clinical trials of intra-articular administration of etanercept at doses of up to 8 mg per injection were considered safe and did not result in any systemic effect (Bliddal et al., 2002). However, we plan to measure the circulating levels of TNFR:Fc protein. II B. The trials in rodents demonstrated an effect of inoculation into one joint on disease in the contralateral joint.

Initial Critique and Responses Our current pre-clinical data supports equivalent efficacy with both approaches in the rat SCW model, despite very different levels of circulating, systemic TNFR:Fc (approximately 2 log difference in serum TNFR:Fc). The benefit of intra-articular route of delivery is the potentially reduced exposure to systemic TNFR:Fc protein compared to either etanercept or intramuscular delivery of a rAAV- TNFR:Fc vector. II C. Ultimately, if this therapy appears successful, one question will be whether there is any benefit to intra-articular therapy over IM.

III. [The] protein that will be expressed, is immunogenic, despite having a human TNF-R fragment and the Fc portion of human IgG1. The hinge region, the junction between the two components, is thought to be the target. Perhaps the inflammation at the site of expression of the fusion construct will have an adjuvant-like effect. There is a risk that treatment with the active agent could induce antibodies that would reduce the efficacy of etanercept. Initial Critique and Responses

If we can develop and validate a reliable assay to detect antibodies to the human TNFR:Fc protein we will test the serum or joint fluid collected during the trial. III. [The] protein that will be expressed, is immunogenic…

IV. For some chimeric proteins, efficacy has been linked to particular allotypes of the FcR, which alter affinity. Initial Critique and Responses

This is a small study focused on safety. We will take this suggestion into consideration for future trials. IV. For some chimeric proteins, efficacy has been linked to particular allotypes of the FcR, which alter affinity.

V. There is theoretical possibility that adenovirus could reach the joint and rescue the AAV. There is no evidence that this is a problem from previous studies with AAV. Should there be restrictions on participation in this trial in subjects with RA and upper respiratory symptoms? Initial Critique and Responses

Subjects who are acutely ill should be excluded from the protocol under the last exclusion criterion, which includes “other concurrent medical condition that, in the opinion of the Investigator, would make the subject unsuitable for the study”. We do not believe that it is necessary to exclude subjects with upper respiratory symptoms, per se, because the tgAAC94 particles are non-replicating as they lack the wild-type AAV viral genes (rep and cap) required for viral replication and encapsidation of DNA. Thus, even in the presence of a helper virus infection (adenovirus 5) the particles are not capable of replication. Simultaneous joint infection with both wild-type AAV (containing rep and cap) and adenovirus 5 is required to rescue the vector. V. … theoretical possibility that adenovirus could reach the joint and rescue the AAV.

VI. The selection criteria suggest that participants should have active disease despite therapy with a DMARD, and that the DMARD will be continued for the study. A.Consideration should be given to making this more specific, perhaps including a trial of methotrexate at a full dose. This option would include candidates with persistent disease while on stable methotrexate. This is a relatively common scenario. B.The protocol states that, “all changes in concomitant medication during the study period will be recorded” - how will they be considered? C.There is also significant variability of immunosuppressive properties of different DMARDs. Should these be considered? Initial Critique and Responses

Although most patients will likely be receiving methotrexate, it is not necessary to standardize baseline treatment, because this is primarily a safety study. … However, baseline medications and changes in medications will be recorded, so it will be possible to perform exploratory retrospective analyses about at the conclusion of the trial to see if the “strength” of the DMARD at baseline, or addition of other medications has an impact on the outcome of the study. VI. DMARD selection criteria.

Potential Concerns: 1.Transgene-associated 2.Immunological 3.Disease-specific

Summary: 1.Transgene-associated A.Systemic exposure Risk from protein itself is low There is circulating vector but little evidence of problem in preclinical studies. Theoretical risk from transfected circulating cells (dendritic) but little evidence of clinical problem at this time.

Summary: 1.Transgene-associated B. Integration of transgene Difficult to define No evidence of risk from preclinical studies but these might not define long-term risk

Summary: 2. Immunological There is a risk of immunizing to the expressed protein, and antibodies should be measured Agree that understanding “contralateral effect” not the goal here. FcR allotypes might be linked to side effects. Consider genotyping.

Summary: 3. Disease-specific Accept rationale for oligo-articular activity, with added safety of not denying those with polyarticular disease systemic TNF-inhibitor Later study should compare intra- articular to intra-muscular. Accept diverse DMARD for phase I but would limit to those with disease on methotrexate in Phase II.