524. A Novel Retroviral Envelope as a Candidate for Pseudotyping Retroviral and Lentiviral Vectors

RNA VIRUS VECTORS into the bloodstream. Female Il2rg-/- mice were injected with G-CSF on four consecutive days and subsequently transplanted with lineage negative bone marrow cells derived from male donor mice (Il2rg-/- or wild type). Il2rg-/- donor cells were cultured overnight with codon optimized IL2RG lentiviral vectors driven by either the strong spleen focus forming virus (SF) promoter or a 1.1 kb section of the native IL2RG promoter (cPr). Mice were monitored monthly via flow cytometry for reconstitution of blood lymphocyte populations. After 6 months, mice were sacrificed and spleen lymphocyte populations and levels of donor cell chimerism were compared to mice given wild type or gene therapy treated cells after 2 Gy irradiation conditioning. Reconstitution of T and B cells in mice given LV.SF.coIL2RG vectortreated cells was equally effective in the G-CSF and 2 Gy groups and similar to lymphocyte populations in wild type mice. Levels of bone marrow donor cell chimerism were 2 to 5-fold lower in the G-CSF group compared to the 2 Gy group. Lymphocyte reconstitution when using a vector driven by the weaker, but safer cPr promoter had more variation between individual mice, with the majority of mice having measurable levels levels of T and B cells. The cPr group had lower vector marking and lower donor cell chimerism than mice treated with the SF vector but comparable to mice given LV.cPr.coIL2RG transduced cells after 2 Gy irradiation. The successful reconsitution of the lentiviral coIL2RG vector transduced mice after G-CSF treatment demonstrates the feasibility of mobilization factors as a way to improve engraftment of vector-treated donor HSCs in gene therapy of SCIDs. Administration of these vectors in conjunction with G-CSF may result in better reconstitution and a more effective treatment in future gene therapy clinical trials for SCID-X1.

522. Development of Human Mesenchymal Stem Cell-Based Tumor-Homing Producer Cells for Efficient Delivery of Retroviral Replicating Vectors to Brain Tumors

Shuichi Kamijima,1 Akihito Inagaki,1 Kei Hiraoka,1 Masamichi Takahashi,1 Quincy Tam,1 Emmanuelle Faure-Kumar,1 Janet Treger,1 Brooke Bogan,1 Christopher R. Logg,1 Noriyuki Kasahara.1 1 Medicine, University of California, Los Angeles, CA.

We have previously demonstrated highly efficient tumor-selective replication and therapeutic efficacy of prodrug activator (‘suicide’) gene therapy delivered by retroviral replicating vectors (RRVs). In the United States, an improved RRV expressing the yeast cytosine deaminase (yCD) prodrug activator gene is now being evaluated in multicenter Phase I/II clinical trials for patients with recurrent glioblastoma. To further improve the effiency of intratumoral vector dissemination and extend this approach to multi-focal malignancies, we are pursuing studies to employ human mesenchymal stem cells (hMSC) as tumor-homing cellular carriers that will produce and deliver RRV to multiple tumor sites. Commercially available hMSC isolates derived from bone marrow, adipose, or cord blood were obtained. In addition to these isolates, fetal liver derived hMSC (FL-hMSC) were isolated and characterized by flow cytometry using MSC-specific cell surface markers (CD29, CD44, CD73, CD90, CD105 and CD166 for positive fraction and CD14, CD34 and CD45 for negative fraction). Migratory activity of MSC was determined by an in vitro Boyden chamber assay in the presence of conditioned medium from target U87 glioma cells. Each MSC isolate exhibited different degrees of tumor tropism and isolates which showed high migratory activity were chosen for further in vivo experiments. To examine in vivo migration activity, MSC were labeled with firefly luciferase or DiI and injected into left brain contralateral to pre-established U87 xenograft in the right brain. MSC migrating toward tumor sites were observed by in vivo imaging system and confirmed by histology. RRV producer MSC were generated by transducing MSC with RRV. We found that spinoculation method on recombinant human fibronectin coated plate S202

can achieve efficient initial transduction level . Vector production titers from 1x105 producer MSC were more than 1x105 transducing units/ml. Cytotoxicity assays confirmed efficient prodrug activator function in target U87 glioma cells with vectors released from RRV producer MSC. Soft agar colony formation assay showed no colony formation with both naïve MSC or RRV producer MSC after 2 weeks incubation period, which indicates no malignant transformation of MSC by integrated RRV sequences. In conclusion, we have confirmed migration activity of MSC toward target glioma cells in vitro and in vivo, and developed methods to generate MSC to serve as RRV producer cells for improved delivery of RRV to cancer cells.

523. Reducing In Vivo Clearance of Lentiviral Vectors by Immune Cells in order To Improve Efficacy and Safety of Gene Therapy

Nisha G. Sosale,1 Irena I. Ivanovska,1 Richard K. Tsai,1 Philip K. Zoltick,2 Yale Goldman,1 Dennis E. Discher.1 1 University of Pennsylvania, Philadelphia, PA; 2Harvard, Cambridge. Macrophages and dendritic cells take up foreign microbes from the circulation and other tissues and present these foreign components to the adaptive immune system. Uptake of viruses, including lentiviral vectors, by macrophages not only makes viral delivery inefficient, but also contributes to an unwanted immune response to vector components as well as delivered gene products. Macrophage uptake of micron-size particles and red blood cells is inhibited by CD47 display on the surface of target particles, through an interaction between CD47 and macrophage receptor SIRPa. To elucidate the impact of CD47-SIRPa interactions on uptake of lentiviral vectors by macrophage, a novel lentiviral vector was engineered to present an oriented human CD47-GFP fusion protein on the vector envelope. Limiting uptake of the vector by innate immune cells should reduce clearance of the vector, including antigen presentation and limits to re-administration. Using TIRF coupled AFM and western blotting, we have demonstrated that engineered LVs display CD47-GFP. Cell culture studies show LV display of CD47 reduces transgene expression in human THP-1 macrophages, while it enhances transgene expression in other cell types. In vivo results likewise show that lentiviral vector display of CD47 reduces clearance of the vector from the bloodstream, and transgene expression in splenic macrophages. Use of this CD47-lentiviral vector, thus has the potential to increase both delivery to target tissues, and the safety of in vivo gene therapy.

524. A Novel Retroviral Envelope as a Candidate for Pseudotyping Retroviral and Lentiviral Vectors

Wenqin Xu,1 Jill Russ,1 Kristen Gorman,1 Kyle Delaney,1 Maribeth V. Eiden.1 1 Section on Directed Gene Transfer, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, National Institutes of Health, Bethesda, MD.

Gibbon ape leukemia virus (GALV) pseudotyped vectors have been widely used in retroviral gene delivery. Wild type GALV envelope fails to pseudotype lentiviral vectors. However, replacement of the GALV transmembrane domain (TM) with that of a murine retrovirus (MuLV) renders this chimeric envelope capable of pseudotyping lentiviral vectors. More recently it was determined that three amino acid residues important for the cleavage of R peptide residues by HIV protease restrict the GALV TM from pseudotyping lentiviral vectors. We have isolated a novel retrovirus from koalas which, although highly related to GALV (70% nucleotide identity), uses a different receptor to efficiently infect human cells in culture. In contrast to GALV that uses the phosphate transporter PiT1 (SLC20A1) as a receptor, this isolate termed KoRVB employs the thiamine transporter THTR1 (SLC19A2) to infect human cells. Epitope tagged GALV or Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

RNA VIRUS VECTORS KoRVB envelope receptor binding subunits can be used as biomarkers to determine the distribution and expression levels of these receptors/ transporters on appropriate target cells. KoRVB can package MuLVbased retroviral vector with high efficiency, suggesting the potential use in specific retroviral gene delivery into THTR1 expressing cells. Like GALV, wild type KoRVB envelope cannot package lentiviral vectors. Therefore we constructed KoRVB mutants with residue modifications similar to those that confer lentiviral pseudotyping capabilities to GALV and assessed the ability of various mutations to allow KoRVB to package lentiviral vectors. These findings will be presented.

525. A Novel Shuttle Vector Approach To Identify Genes Involved in Hematopoietic Stem Cell Disorders Using Vector-Mediated Genotoxicity

Dustin T. Rae,1 Ryan Maynard,1 H. Joachim Deeg,2 Grant D. Trobridge.1 1 Pharmaceutical Sciences, Washington State University, Pullman, WA; 2Fred Hutchinson Cancer Research Center, Washington State University, Seattle, WA.

Here we report a novel retroviral shuttle vector approach to perform mutagenesis screens to identify genes and gene networks involved in the development of hematopoietic stem cell disorders (HSCs). Myelodysplastic syndromes (MDS) are a group of HSC disorders that are characterized by dysplasia that often progress to acute myeloid leukemia (AML). However, little is known about the mechanisms of disease progression. Several mutations in the transcription factor AML1 (RUNX1) have been identified, and account for 15%-40% of MDS-refractory anemia. Our goal is to identify genes activated by vector-mediated genotoxicity that collaborate with AML1. In our approach, a lentiviral shuttle vector that expresses a patient-derived AML1 mutant gene (AML1-D171N) is used to transduce mouse bone marrow prior to transplantation. Following infusion, expression of AML1-D171N causes MDS/AML formation which has been previously established in a mouse model. Our shuttle vector contains a bacterial origin of replication and a kanamycin resistance gene to allow rescue of integrated vector proviruses as bacterial plasmids, and rapid identification of integration sites. In addition, our vector contains a library of short hairpin RNAs (shRNAs), allowing for knockdown of distant genes, with the potential to identify collaborating gene networks. Using our approach, we expect to identify dysregulated genes near integrated vector proviruses, and gene networks that may collaborate with AML1-D171N to promote MDS and AML. We have established efficient high-throughput recovery of shuttle vector plasmids in bacteria from vector-transduced cells. Here we also report engraftment data and the development of MDS in transplanted mice. We also describe our bioinformatics pipeline and vector integration sites identified using our shuttle vector approach. The genes and gene networks identified in our screen may be used as therapeutic targets or biomarkers for detection and treatment of MDS/AML. In addition, our approach of using a shuttle vector expressing a shRNA library has broad potential to identify genes and gene networks involved in other stem cell disorders.

526. A Systematic Approach to the Establishment and Characterisation of Endothelial Progenitor Cells for Gene Therapy Yuan Zhao,1 Natalie Werling,1 Robin Thorpe.1 Biotherapeutics, National Institute for Biological Standards and Control, London, United Kingdom.

1

It has been recently demonstrated that endothelial progenitor cells (EPCs) have increasing potential for gene therapy or regenerative cell therapy for cardiovascular diseases and cancer. However, current therapies involving EPCs are inefficient due to the very low level Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

of EPCs in the available sources e.g. in blood. One solution is to derive in vitro an expanded population of EPCs from circulation. In addition, EPCs like other progenitor cells have an intrinsic predisposition of differentiating into mature cell types, e.g. mature endothelial cells; therefore, establishing a sufficient amount of EPCs alongside maintaining the EPC characteristic phenotype during genetic modification and long-term culture presents a significant challenge to the field of gene and cell therapies. In this study, we have systematically investigated EPCs from different sources and used multiple parameters including cell surface markers and a tubule formation assay to identify factors that influence the establishment, characteristics and vector transduction capability of EPCs. Our results show the considerable promise, as well as certain limitations in the establishment and manipulation of genetically modified EPCs for gene therapy. Whilst obtaining high transduction efficiency and robust in vitro tubule formation of EPCs using lentiviral vectors, we also observed that lentiviral vector-transduction significantly altered EPC phenotype as demonstrated by an increased percentage of CD34+ progenitor cells and an increased expression of adhesion molecule CD144 (VE-cadherin). Taking account of the increased expression of CD144 reported in cancer patients, the altered expression of ECP related markers, e.g. VE-cadherin and the enrichment of CD34+cells after vector transduction indicates the importance of extensive characterisation and vigorous safety control of genetically modified EPCs before they are accepted for clinical use.

527. Lentiviral Vector Transduction of Human CD34+ Cells Requires a Wash/Rest Phase between Vector Exposures To Produce Additional Benefit but Does Not Require High Dose Cytokines Michael R. Greene,1 Brian P. Sorrentino.1 Division of Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TN. 1

The literature contains apparently conflicting reports in regard to the relative benefit of additional lentiviral vector exposures when seeking to achieve greater transduction efficiency of human CD34+ cells. Numerous groups, including our own, have demonstrated that a second vector exposure provides no additional benefit using rigorous assays such as CFU-C in vitro analysis and NSG repopulating cell in vivo analysis, while other groups have provided evidence that a second exposure does improve transduction. The differences in the protocols of the groups reporting benefit was that they employed a wash/rest phase between vector exposures and they used higher dose cytokine cocktails similar to those used for gamma-retroviral vectors. It has been reported that cytokine doses higher than the standard 100/100/100 ng/ml cytokine cocktail of SCF, Flt-3L, and TPO or the addition of IL-3 or IL-6 can impair the engraftment of NSG repopulating cells derived from CD34+ grafts. To determine if the wash/rest phase and/or high dose cytokine cocktails were necessary to realize the transduction benefit of a second vector exposure, we compared the transduction efficiency of a 100/100/100 standard dose cytokine cocktail to a 300/300/100/60 ng/ml high dose cocktail of SCF, Flt-3L, TPO, and IL-6 in our standard serum-free media paired with the use of a single 24 hour vector exposure, a double 24 hour exposure without a wash/rest phase, or a double 12 hour exposure separated by a 12 hour wash/rest phase transduction protocol. We evaluated these protocols using a stably produced clinical grade SCID-X1 lentiviral preparation to transduce G-CSF mobilized peripheral blood CD34+ cell grafts derived from three normal human donors which were then transplanted into busulfan conditioned NSG mice and harvested at a minimum of 12 weeks post-transplant. Bone marrow was analyzed for human CD45+ engraftment by flow cytometry and transduction efficiency of the NSG repopulating cell compartment was determined by real-time quantitative PCR. With our standard transduction protocol of standard dose cytokine cocktail with S203