1055 Infusion of Thymidine Kinase (TK)+ Donor Lymphocytes Promotes a Rapid and Wide Immune Reconstitution and Permits Long Term Survival after Hematopoietic Transplantation from a Haploidentical Donor[.]
cDNA for the human tumor necrosis factor receptor-immunoglobulin (lgG I) Fe fusion (TNFR:Fc) gene (tgAAC94) may be a means to provide local high concentrations ofsoluble TNFR:Fc for an extended period of time In the first phase I study oftgAAC94, single IA injections of Ixl 10 and Ix I011 DNase resistant particles (DRP)/mL joint volume were shown to be safe and well-tolerated in II subjects with inflammatory arthritis not on TNF-a antagonists Based on the safety profile, this phase 1/2 study was initiated to evaluate the safety ofsingle and repeat IA administration oftgAAC94 in inflammatory arthritis patients with and without systemic TNF-a antagonist use Adults with persistent moderate or severe inflammation in a target joint (n=120) are to be enrolled and receive a single IA injection of either tgAAC94 (IxIOII, IxlO 12 or Ixl0 13 DRP/mL joint volume) or placebo, followed by open-Iabcl tgAAC94 after 12 to 30 weeks, depending on when swelling in the target joint meets criteria for re-injection Eighty-five subjects with either rheumatoid arthritis (n=64), psoriatic arthritis (n=17) or ankylosing spondylitis (n=4), have been enrolled to date Preliminary analysis indicates reduction of tenderness and swelling of the injected joint after study agent administration The subjects, 46 of whom were on systemic TNF-a antagonists, received an injection of blinded study agent into thc knee (n=38), ankle (n=15), wrist (n=15), MCP (n=9) or elbow (n=8) Thirty-nine subjects have received a second injection of open-label tgAAC94 Among the first 41 subjects randomized to the two lower doses of tgAAC94 or placebo, of 10 subjects (70%) who received placebo qualified for open label tgAAC94 prior to 30 weeks, in contrast to 16 of 31 subjects (52%) who received tgAAC94 The median time to second injection was 120, 129 and 145 days for subjects who received placebo, tgAAC94 Ix I0" DRP/mL and tgAAC94 IxlO 12 DRP/mL, respectively Administration site reactions, consisting ofmild to moderate increase in tenderness and swelling ofthe injected joint, sometimes accompanied by erythema or pruritis, have been reported in -10% of subjects One subject developed a serious adverse event, culture negative septic arthritis, 15 weeks after injection oftgAAC94 IxlO 12 DRP/mL or placebo that the investigator considered probably related to study agent Single and repeat IA injections oftgAAC94 at doses up to IxlO 13 DRP/mL appear to be safe and well-tolerated in subjects with and without systemic TNF-a antagonists Enrollment is ongoing and treatment effect and duration are being evaluated 1054 Phase I Clinical Trials of Intramuscular Injection of rAAV2 and rAAV1-Pseudotyped Versions of an Alpha-1 Antitrypsin (AAT) Vector in AAT-Deficient Adults Terence R Flotte , I Mark L Brantly, Lindo T Spencer, I Margaret Humphries; Thomas J Conlon,' Carolyn T Spencer; Amy Poirier, I Wendy Garlington; Dawn Baker,' Sihong Song,' Ken I Berns,' Nick Muzyczka,' Richard O Snyder, Sue Washer,' Barry Byrne.' 'Powell Gene Therapy Center; Universit ofFlorida, Gainesville, FL A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype (rAAV2) alpha-I antitrypsin (AAT) vector (utilizing a CMV enhancer/beta actin promoter/hybrid intron [CBA] expression cassette) was performed in 12 AAT-defieient adults, 10 of whom were male All subjects were either homozygous for the most common AAT mutation (a missense designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease There were four dosage cohorts, ranging from 2.lx10 12 vector genomes (vg) to 6.9x10 13 vg, with subjects per cohort Subjects were injected sequentially in a dose escalating fashion with a minimum of 14 days between patients Subjects who had been on AAT protein replacement discontinued that therapy 28 days prior to vector administration There were no vector-related S402 serious adverse events in any of the 12 participants Vector DNA sequences were detected in the blood between I and days after injection in nearly all patients receiving doses of 6.9x I0 12 vg or higher Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected One subject who had not been on protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82nM), which was detectable 30 days after receiving an injection of2.1x I0 13 vg Unfortunately, residual but declining M-AA'r levels from the wash -out of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts Based on a desire to inerease the potency for M-AAT expression per vector genome, we further pursued pseudotyping ofthis same vector genome into AAV serotype I capsids (rAAV I-AA'r) Preclinical data indicated that the potency per vector genome was approximately 500-fold higher in mice with this cassette Toxicology and biodistribution studies in mice and rabbits indicated that 1M injection ofrAAVl-AAT posed some risk of injection-site inflammation and biodistribution to distant sites, including gonads and semen, both ofwhich were dose -related, but neither of which were significant within the dose range (per kg) planned for the rAAVI clinical trial Based on this a phase I trial has now been initiated with the pseudotyped vector Four subjects have now been injected with up to X 1013 vg ofrAAVI-AAT One of the four showed a level of total AAT peaking at II microM 30 days after injection, but it is unclear whether this was due to vector expression or to an intercurrent bacterial epididymitis, with its associated fever and acute phase reaction, which could have induced endogenous Z-AAT to be transiently elevated To date , there are no vector-related adverse events noted Nine additional subjects are anticipated to receive doses ranging up to Ix I0 14 vg Supported by grants from NHLBI, NCRR, and Applied Genetic Technologies Corporation 1055 Infusion of Thymidine Kinase (TK)+ Donor Lymphocytes Promotes a Rapid and Wide Immune Reconstitution and Permits Long-Term Survival after Hematopoietic Transplantation from a Haploidentical Donor C Bonini; F Ciceri,' M T Lupo Stanghellini,' A Bondanza,' Z Magnani,' S Perna , M Bernardi,' J Pcccatori,' P Scrvida,' F Crippa,' S Kaneko; V Valtolina ,' M Ferrari; E Provasi ,' M Salornoni,' L Turchetto,' S Torna.' K Traversari,' P Bruzzi,' L Castagna,' A Santoro,' J Apperley," S Slavin ,' S Colombi,' C Gallo Stampino,' M Bregni, I C Bordignon.' 'Hematology H.S Raffaele Milan, Italy; 2lnjec/iol/s Disease H.S Raffaele, Milan I/aly; 3MolMed, SpA, Milan, Italy; 4£pidemiologia Clinica, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; sHematology, Ie Humanitas, Razzano, Italy; "Hematology, Hammersmith H., London, United Kingdom; 7BMT Unit, Hadassah, Jerusalem, Israel Haploidentical family donors represent the ideal solution to offer every patient with high risk leukemia the potential cure ofhematopoietie stem cell transplantation (HSCT) However, T-cell depletion required to prevent graft-versus-host disease (GvI-lD) results in prolonged immunosuppression ultimately leading to opportunistic infections and disease relapse We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK-DLI) after haploidentieal I-ISCT (haplo-I-ISCT) could provide early immune reconstitution and selective control of GvI-lD In a phase II clinical trial (protocol MMTK007), 29 patients (median age 52) were transplanted for high risk leukemia No immune reconstitution and no Gvl-lD were observed in absence ofTK-DLI Seventeen patients received TK-DLI starting at d+42 TK cells cngraftrncnt, observed in 14 patients, was necessary and sufficient for a rapid and effective Molecular Therapy Volume15.Supplement I ~b y 2007 Copyright © The American Society o f Gene Therapy immune reconstitution (lR) , with a median of 144 (10 I-336) CD3+ , 59 (28-149) CD4+ and 86 (52-279) CD8+ cells/mel at day +100 after I-ISCT.Allhough a massive increase ofTK cells was observed in all 14 patients, transduced cells represented only a median of 48% (10-90) of circulating CD3+ cells, suggesting that TK cells played not only a direct effect, but also a critical homeostatic role in promoting T cell immune reconstitution, As shown in Table I, the oligoclonal T cell repertoire enriched in activated lymphocytes observed months after HSCT was progressively replaced by a polyclonal, resting immune repertoire and normalized in the following 12 months High numbers 01'1' cell precursors specific for CMV and EBV were detected at immune reconstitution (average of 61 and 96 g[FN specific spots/lO"5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0 002) Accordingly, the cumulative infectious mortality beyond d+ 100 post transplant was 12.5%, with last event at d+166 Six patients developed acute Gvl-ID(grade [ to IV) that was effectively controlled by the suicide gene/prodrug machinery These results indicate that TK-DLI abrogate late mortality associated to haplo-HSCT in adults of all ages from week I post-vector delivery in subjects with detectable T cell responses to AAV capsid , None of the subjects developed an antibody to the LPL transgene product Together, these results confirm previous findings (Blood 2006; I08( I I): I38a) that cellular immunity to AAV capsid in humans is not restricted solely to AAV serotype vectors or vectors with heparin binding domains Moreover, these findings show that this response arises not only after systemic administration of the vector, but after direct injection into the skeletal muscle as well The kinetics of the immune response were slower after intramuscular injection compared with hepatic artery delivery, although this may also reflect a dosing effect Thus CD8+ T cell responses to capsid occur following vector injection with multiple serotypes and in at least two distinct tissue types Table I- 1058 In Vivo Lentiviral SIN-LTR Vector Integration Is Nearly Random and Shows a Preponderance of Satellite DNA in Postmitotic Rodent Eyes - - - - - - - - - - r - . - - r - L IN.