90. Nanoparticle Delivery of PNA and Donor DNA for Genomic Modification and Inactivation of the CCR5 Gene

SMALL RNA AND OLIGONUCLEOTIDE BASED THERAPEUTICS Z138 cells) and unlike the BAFF ligand, they do not trigger cell proliferation. Importantly the aptamers are able to block BAFF ligand mediated cell proliferation and compete effectively with BAFF ligand for receptor binding, blocking. In an effort to enhance the functional utility of the aptamers, we created aptamer-STAT3 siRNA chimeras for cell-type specific siRNA delivery. The aptamer-siRNA chimears showed similar receptor binding profiles as the aptamer alone, and binding resulted in internalization of the aptamer and siRNA. The siRNA was processed by Dicer and incorporated in RISC where it triggered potent knockdown of the STAT3 mRNA and protein in Jeko-1 and Z138 B-cell lines. We also evaluated the aptamer delivered siRNA functional activity by carrying out RACE PCR analyses. The results showed that the target cleavage sites correlated with Dicer produced siRNAs as revealed by Illumina sequencing. Hence our results demonstrate that the dual-functional BAFF-R aptamer chimeras are able to deliver siRNAs and block ligand mediated processes. Our next step is to test the efficacy of the aptamer and aptamer chimeras in vivo in a humanized mouse model.

90. Nanoparticle Delivery of PNA and Donor DNA for Genomic Modification and Inactivation of the CCR5 Gene

Erica B. Schleifman,1 Nicole Ali McNeer,1 Andrew Jackson,1 Jean Leif,2 Christopher Cheng,1 Ranjit S. Bindra,1 Leonard D. Shultz,3 Dale L. Greiner,2 Priti Kumar,1 W. Mark Saltzman,1 Peter M. Glazer.1 1 Yale University, New Haven, CT; 2University of Massachusetts Medical School, Worcester, MA; 3The Jackson Laboratory, Bar Harbor, ME.

CCR5, chemokine receptor 5, encodes a major co-receptor for R5-tropic human immunodeficiency virus-1 (HIV-1) and must be present at the cell surface for R5-viral entry. Individuals who are homozygous for a 32 bp deletion mutation (delta32) in CCR5 are almost completely resistant to HIV-1 infection and show no significant adverse phenotypes. One therapeutic strategy for individuals with HIV infection is to use peptide nucleic acid (PNA)-induced homologous recombination to introduce mutations into the CCR5 gene in hematopoietic cells to mimic the effect of the naturally occurring delta32 mutation, thereby creating blood cells resistant to HIV. These triplex-forming PNAs bind sequence specifically to duplex DNA and, when combined with donor DNA molecules, stimulate recombination in mammalian cells. We have found that triplex-forming PNAs can enhance recombination in the CCR5 gene in a human cell line at a targeting frequency of 2.45%. To further enhance the efficiency and reduce the toxicity of gene targeting in primary human primary cells, we have tested the use of biodegradable poly (lactide-co-glycolide) (PLGA) nanoparticles (PLGA-NPs) that can encapsulate both the triplex-forming PNAs and the donor DNAs for delivery into various cell types. CCR5-targeted PLGA-NPs were formulated by a double-emulsion solvent evaporation technique and evaluated for their ability to enter CD34+ hematopoietic stem cells (HSCs) or peripheral blood mononuclear cells (PBMCs) and deliver PNA and donors for gene targeting. Efficient cellular uptake of the PLGA-NPs in both cell types was verified by FACS analysis, and targeted modification was observed at levels equal to or greater than after conventional nucleofection of the same molecules. Treatment of these cell types with NPs also led to a reduction in cellular toxicity compared to conventional nucleofection techniques. The addition of cell-penetrating peptides to the surface of the NPs led to an increase in targeted genomic modification in both PBMCs and CD34+ HSCs. PBMCs treated with CCR5-targeted NPs were injected into NOD-scid IL2rγnull adult mice and were able to engraft at levels equal to untreated cells and the presence of the targeted CCR5 modification was detected in the spleen of the engrafted mice four weeks post-transplantation. This work suggests a potential therapeutic strategy for permanently S36

inactivating the CCR5 co-receptor in human HSCs or PBMCs from HIV-1 infected individuals to create a reservoir of cells, which are virus-resistant, thus preserving immune system function.

91. In Vivo HIV Inhibition by CCR5 KnockDown by RNA Interference through Stem Cell Based Gene Therapy Approach in the Humanized NSG BLT Mouse Model

Saki Shimizu,1,2 Ruth Cortado,1,2 Emily Lowe,1,2 Anna Sahakyan,1,2 Joshua Boyer,1,2 Tom Suree,1,2 Dong Sung An.1,2 1 School of Nursing, University of California, Los Angeles, Los Angeles, CA; 2Department of Medicine, Division of HematologyOncology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA. Inhibiting the expression of the HIV-1 co-receptor CCR5 holds great promise for controlling HIV-1 infection in patients. Here we report HIV inhibition by stable knockdown of human-CCR5 by a short hairpin RNA (shRNA) in a humanized bone marrow/ liver/thymus (BLT) mouse model. To achieve stable knockdown of human-CCR5 by a short hairpin RNA (shRNA), we delivered a potent shRNA against CCR5 into human CD34+ hematopoietic progenitor/stem cells (HPSCs) by lentiviral vector transduction. We transplanted vector-transduced CD34+ cells and a thymus segment under the mouse kidney capsule to produce human T cells in the transplanted human thymus tissue. Vector-transduced autologous CD34+ cells were subsequently injected in the irradiated mouse intended to create systemic reconstitution. CCR5 expression was down-regulated in human T cells and monocytes/macrophages in systemic lymphoid tissues. The shRNA-mediated CCR5 knockdown had no apparent adverse effects on T cell development as assessed by polyclonal T cell receptor Vβfamily development and naive/memory T cell differentiation. We next examined HIV inhibition ex vivo and in vivo. CCR5 tropic HIV-1 infection was effectively inhibited in isolated human splenocytes ex vivo. When mice were challenged with CCR5 tropic HIV-1, we observed selective maintenance of the CD4/CD8 ratio in CCR5-shRNA expressing mouse PBMCs. This phenomenon was also confirmed in various mouse tissues, including gut associated lymphoid tissue (GALT) which is the major site of HIV-1 replication. Furthermore, CCR5 down-regulated memory CD4+ T cells were protected from depletion by HIV-1 infection. We measured the amount of HIV-1 DNA in isolated human splenocytes by Real time PCR. We confirmed the amount of HIV-1 DNA was low in CCR5 down-regulated cells compared to control cells. These results demonstrated that lentiviral vector delivery of shRNA into human HPSCs could stably down-regulate CCR5 and prevent CD4+ T cell and memory CD4+ T cell depletion by HIV-1 infection. Our current results provide further evidence that it may be possible to use of this technology in HIV-1 treatment.

92. Efficient In Vivo Delivery of Vitamin E-Conjugated siRNA from Colorectum to Liver

Kazutaka Nishina,1 Masahiro Murakami,2 Kie Yoshida,1 Hiroya Kuwahara,1 Hidehiro Mizusawa,1 Takanori Yokota.1 1 Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; 2Osaka Ohtani University, Osaka, Japan. RNA interference (RNAi) is a powerful tool of target-specific knockdown of gene expression. For a therapeutic approach, however, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ has not been established. Here, as a carrier molecule of siRNA to the liver, we use vitamin E (α-tocopherol) and its own physiological transport system. Orally intaken α-tocopherol is absorbed, assembled in chylomicron (CM) in enterocytes and excreted into the lymph duct. CM is metabolized into chylomicron remnant by lipoprotein lipase (LPL) which enters hepatocytes by remnant Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy