246. Characterization of Peripheral Cardiac Progenitors

Cell Therapies I HSV-TK phosphorylates the prodrug ganciclovir (GCV), an analog of guanosine nucleoside. The phosphorylated GCV is incorporated into host DNA and terminates the elongation of DNA strands, resulting in cell death. The therapeutic range of GCV is narrow and the administration of GCV often induces adverse reactions. Aiming at a more potent and thus less toxic TK, mutations in the HSV-TK had been screened in previous studies. However, no studies have been conducted on the direct comparison of the efficacy of mutant TKs for iPS cells. In the current study, we examined some TK mutants with substitution at the nucleoside binding site that had been reported before (Black, et al., 1996; Black, et al., 2001; Mercer, et al., 2002; Balzarini, et al., 2006). We made iPS cell clones that constitutively express individual mutant TK. Treatment of the TK-iPS clones with GCV revealed that cells with mutant SR11 (IF160 LL+A168Y), SR39 (LIF159IFL+AL168FM), Q7530 (IF160LL+AL168YF), and A168H were 10-fold more sensitive to GCV than wild type TK. Some other mutants that had been shown to have an enhanced killing activity in their original reports did not show an advantage over the wild type. These results suggested that TK mutants were more potent than the wild type and have to be screened and selected for a specific purpose.

244. Development and Qualification of Assays for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Use

Jennil Patel, Wei Dang, Aparna Krishnan, Derek Kong, David Hsu, Larry Couture Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, Duarte, CA Stem cell therapy holds the promise for numerous degenerative diseases and injuries. Human embryonic stem cells (hESCs) are the starting cell population for many of these potential therapies since they are capable of seemingly indefinite proliferation in the pluripotent state and have the ability to differentiate into all cell types found in the adult. It is vital that the cellular therapeutics derived from these highly proliferative cells are well characterized prior to clinical use and the assays used to characterize these cellular products are well developed. At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and early phase clinical studies. We have developed a systematic approach to characterize hESC-derived cell products using well developed technologies such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. We have standardized the development and qualification of each assay which involves the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, generation of assay qualification report and standard operating procedure (SOP). In addition, we have established a general guideline to assist the selection of appropriate markers for different products. Here we describe the application of this systematic approach to develop and qualify identity and purity assays for hESC-derived cardiomyocytes produced through hESC differentiation procedure in suspension culture. Selection of markers for the characterization of cardiomyocyte products will be discussed, and development and qualification of the assays will be reported.

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245. MicroRNA Expression in Bone MarrowDerived Human MSCs

Ian H. Bellayr1, Jennifer G. Catalano2, Raj K. Puri1 Division of Cellular and Gene Therapies, US Food and Drug Administration, Silver Spring, MD, 2Office of Industries, US International Trade Commission, Washington, DC 1

Multipotent stromal cells (MSCs) are being studied in the field of regenerative medicine for their capacity for multi-differentiation. These cells can be isolated from multiple tissue types. The current literature indicates that MSCs have an immunoregulatory capacity, which can suppress the immune system. MicroRNAs (miRNAs) are short non-coding RNAs that are responsible for regulating gene expression. Through targeting binding to gene transcripts, miRNAs have been observed to impact MSC function such as proliferation, differentiation, migration and apoptosis. Studies have shown that various miRNAs are expressed in MSCs; however, the impact of cellular expansion and donor variability on the miRNA expression is not well understood. Six commercially available MSC lines were expanded from passage 3 to 7 and their miRNA expression was evaluated using microarray technology. Statistical analyses of our data revealed that 71 miRNAs out of 939 examined were expressed by this set of MSC lines at all passages and the expression of 13 miRNAs were significantly different between passage 3 and 7. The expression of six miRNAs with the largest fold changes was further evaluated using RT-qPCR for both the original MSC lines and a second set of seven MSC lines expanded from passage 4 to 8. By RT-qPCR only 2 miRNAs, miR-638 and miR-572 were upregulated at passage 7 compared to passage 3 for the original MSC lines by 1.71 and 1.54 fold, respectively; and upregulated at passage 8 compared to passage 4 for the second set of MSC lines, 1.34 and 1.59 fold, respectively. These 2 miRNAs distinguish aging MSCs expanded in culture. These novel results may be useful in establishing critical quality attributes for limiting clinical applications of MSCs beyond specific cellular expansion protocols.

246. Characterization of Peripheral Cardiac Progenitors

José R. Navarro-Betancourt1, Salomón Hernández1, Felipe Masso2, Luis A. Lara-Martínez1, Alejandra Contreras-Ramos3, Andrés R. Martinez-Calderón1, Victor M. Arenas1, José L. Aceves-Chimal4 1 Molecular Biology, Universidad Panamericana, Mexico City, Mexico, 2Cellular Biology, National Institute of Cardiology “Ignacio Chávez”, Mexico City, Mexico, 3Cellular Biology, Mexico Children’s Hospital “Federico Gómez”, Mexico City, Mexico, 4 Cardiothoracic Surgery, “20 de Noviembre” National Medical Centre, Mexico City, Mexico Introduction We propose that cells expressing cardiomyocytespecific structural proteins have a specific cardiac differentiation commitment and that an immunophenotype distinguished by the presence of cardiac stem cell (CSC) related surface markers, namely CD117, CD34 and Sca-1, identifies peripheral cardiac progenitors, therefore we explored the presence of these attributes in bone marrowderived cells, aiming to establish an association between cardiac differentiation potential and a particular cellular profile. Methods Whole bone marrow was extracted from Wistar rat femur, cells were isolated through enzymatic desegregation and plated for incubation. Twenty-four hours later non-adherent supernatant cells were recovered and re-seeded, seventy-two hours later adherent cells were harvested and underwent dilution cloning. Flow cytometry was used to characterize each clone searching particularly for the surface markers CD117, CD34 and Sca-1. The subpopulation expressing the highest levels of these markers was isolated and labeled bone marrow supernatant (BMSN). Subsequently, a complete immunophenotypic profile of BMSN was performed, their capacity to differentiate into Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

Clinical Trials Spotlight Symposium typical mesenchymal tissues was assessed using a commercial kit. BMSN were evaluated through rt-PCR and q-PCR for the expression of cardiac-related genes. The presence of proteins coded by these genes was evaluated through western blot and immunofluorescence. Co-cultures of green fluorescent protein (GFP) labeled BMSN with mature cardiomyocytes were performed, the expression of prototypal cardiac proteins was verified through immunofluorescence. In a rat model of myocardial infarction, 5x10 6 GFP-labeled BMSN were injected directly into the infarcted tissue. Cardiac function was tracked with left-ventricle ejection fraction (LVEF), measured though transthoracic echocardiography, and myocardial perfusion measured through single-photon emission computed tomography (SPE-CT). Myocardium histological analysis was carried out to evaluate engraftment. Results In addition to CD117, CD34 and Sca-1, BMSN express CD29, CD44, CD90, CD105 and CD106. BMSN are able to differentiate into osteocytes, but not into adipocytes or chondrocytes. BMSN express the following genes: GATA4, MEF2, Nkx2.5, a-MHC, connexin-43 and troponin-T, connexin-43 reached a 60% higher expression than mature cardiomyocytes. Through westernblot analysis the presence of GATA4, MEF2, Nkx2.5, a-MHC, connexin-43 and troponin-T in CMSC-like is unequivocal. By means of immunofluorescence protein detection only a meager proportion was positive for GATA4, a-MHC and troponin-T, compared to the quantity presumed by western-blot. BMSN phenotypically differentiate into cardiomyocytes when co-cultured with mature cardiomyocytes, cells that expressed Troponin-T before co-culture acquired spontaneous rhythmic contractile movement at the seventh day of follow-up. Sixty days after BMSN injection LVEF increased from an average value of 29% to a mean of 56% and myocardial perfusion increased significantly. BMSN co-expressing GFP and Troponin T were detected inside and around the infarcted area of treated rats. Conclusions The available data allows to infer an association between CSC-related surface marker expression, cardiomyocytespecific gene transcription, prototypal cardiac protein presence and a differentiation potential restricted to cardiac tissue.

Clinical Trials Spotlight Symposium 247. Phase I Study of Second Generation Chimeric Antigen Receptor-Engineered T Cells Targeting IL13Rα2 for the Treatment of Glioblastoma

Christine E. Brown1, Renate Starr1, Lihong Weng1, Darya Alizadeh1, Jamie R. Wagner1, Julie Kilpatrick1, Todd L. Harshbarger1, Massimo D’Apuzzo1, Julie A. Ressler1, Michael C. Jensen2, Jana Portnow1, Michael E. Barish1, Stephen J. Forman1, Behnam Badie1 1 City of Hope, Duarte, CA, 2Seattle Children’s Research Institute, Seattle, WA T cell immunotherapy is emerging as a powerful strategy to treat cancer, and may offer new opportunities to improve outcomes for patients with glioblastoma (GBM), the most aggressive of primary brain tumors and among the most lethal of human cancers. We have optimized a chimeric antigen receptor (CAR) T cell therapy targeting the tumor associated antigen IL13Rα2 for the treatment of GBM. This T cell product utilizes CD62L-enriched central memory T cells (Tcm), engineered by lentiviral transduction to express a second-generation 4-1BB-containing CAR (IL13BBζ). We report here initial findings from our first-in-human clinical trial [NCT02208362], evaluating the safety, feasibility and bioactivity of weekly intracranial infusions of autologous IL13BBζ Tcm in patients with recurrent IL13Rα2+ GBM. Enrolled patients undergo leukapheresis to collect autologous Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

PBMC and, concurrent with IL13BBζ Tcm manufacturing, tumor biopsy or resection is performed, with placement of a reservoir/ catheter device. Following baseline MR and PET imaging and recovery from surgery, patients are treated on a 4-week therapeutic regimen consisting of 3-weekly intracranial infusions of IL13BBζ Tcm followed by one rest week for toxicity and disease assessment. To date, we have completed treatment of the first low dose cohort of three resection patients, and find that local delivery of IL13BBζ Tcm post surgical resection is safe and well-tolerated with no grade 3 or higher toxicities attributed to the therapy observed. Importantly, we have also obtained early evidence for antitumor activity following CAR T cell administration. For all patients in which sample was available, CAR T cells were detected in the tumor cyst fluid or cerebral spinal fluid (CSF) by flow cytometry for a minimum of 7 days post treatment. One patient of particular interest presented with a recurrent multifocal GBM, including one metastatic site in the spine and extensive leptomeningeal disease. This patient was initially treated per protocol with six local infusions of IL13BBζ Tcm into the resection cavity of the largest recurrent tumor focus in the posterior temporal-occipital region. Encouragingly, this CAR T cell injection site remained stable without evidence of disease recurrence for over 7-weeks, while other disease foci distant from the CAR T cell injection site continued to progress. Based on our preclinical laboratory experiments showing the efficacy of intraventricular delivery of CAR T cells, this patient was then treated on a compassionate use protocol with five weekly intraventricular infusions of IL13BBζ Tcm without any other therapeutic interventions. One week following the final intraventricular CAR T cell infusion, all intracranial and spinal tumors had regressed with most decreasing more than 75% by volume, and this patient remains clinically stable four months following the start of CAR T cell treatment. These early clinical findings suggest that intracranial delivery of second-generation IL13Rα2-targeted CAR T cells is safe and well-tolerated, and that after adoptive transfer, CAR T cells survive and maintain activity, capable of eliciting potent antitumor responses against recurrent multifocal glioblastoma.

248. Results of a Phase 1 and 2a Trial on rAAV. sFlt-1 in Treatment of Wet Age-Related Macular Degeneration

Chooi-May Lai1, Aaron L. Magno2, Martyn A. French3, Samuel B. Barone4, Steven D. Schwartz5, Mark S. Blumenkranz6, Mariapia A. Degli-Esposti1, Ian J. Constable1, Elizabeth P. Rakoczy1 1 Centre for Ophthalmology and Visual Science, The University of Western Austrialia, Nedlands, Australia, 2Molecular Ophthalmology, Lions Eye Institute, Nedlands, Australia, 3School of Pathology and Laboratory Medicine, The University of Western Austrialia, Nedlands, Australia, 4Avalanche Biotechnologies, Menlo Park, CA, 5Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA, 6The Byers Eye Institute, University of Stanford, Palo Alto, CA Purpose: To assess safety following subretinal rAAV.sFlt-1 injection at one year. Secondary endpoints were assessed for evidence of biologic activity. Methods: Under the protocol, Phase 1 followed a dose escalation design where eligible subjects received subretinal injection of low dose (LD, N=3, 10E10 vg) and high dose (HD, N=3, 10E11 vg) rAAV.sFlt-1, or control regimen (N=2). In Phase 2a eligible subjects were randomized to receive HD (N=21) rAAV. sFlt-1 or control regimen (N=11). All 40 subjects were assessed using ophthalmic exam and laboratory testing, and received intravitreal ranibizumab retreatment according to protocol-driven criteria for worsening wet Age-related Macular Degeneration (AMD). Results: During the 1-year follow-up to the primary endpoint, no serious adverse events related to rAAV.sFlt-1 were observed. However, transient adverse events such as subretinal hemorrhage, inflammation, S97