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574. Improved Lentiviral Formats for Genome Editing Using Zinc Finger Nucleases
DNA VECTOROLOGY & GENE TARGETING II 572. Poly(2-Dimethylaminoethyl Methacrylate)Functionalized Graphene Oxide Nanoflakes for Efficient siRNA Delivery Jung Eun Lee,1 Min Sang Lee,1 Jeong A. Nam,2 Nak Won Kim,1 Sung Young Park,2 Ji Hoon Jeong.1 1 School of Pharmacy, Sungkyunkwan University, Suwon-si, Republic of Korea; 2Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju-si, Chungbuk, Republic of Korea.
RNA interference (RNAi)-based gene therapy has been considered a promising approach for the treatment of hard-to-cure diseases and gene-related disorders in gene therapy. Intracellular delivery of synthetic small interfering RNA (siRNA), a double stranded RNA having 19 - 23 base pairs, can induce sequence-specific gene silencing via the RNAi phenomenon. Due to its charged nature and degradation in physiological solutions, development of efficient and safe delivery vehicle is one of the most critical prerequisite for the use of siRNA in clinical settings. However, siRNA cannot be readily formulated with conventional cationic polymers since the length of siRNA is too short and rigid to act as an efficient cross-linker for the formation of tight polyelectrolyte complex structure. Graphene oxide (GO), a 2-dimmensional nano-sized sheet composed of carbon atoms, has been popularly studied in biomedical fields such as drug delivery, cellular imaging, and bio sensors owing to its physical, chemical and mechanical properties. The flexible 2D GO sheet would be an ideal carrier for siRNA since short and rigid siRNA molecules can be aligned and adsorbed onto the surface of GO sheet. Although hydrophobic drugs and single-stranded nucleic acids (DNA or RNA) can be adsorbed on GO surface through - stacking and other non-covalent molecular interactions, double stranded siRNA cannot be readily adsorbed onto GO via the interactions. In this study, we developed a facile method to functionalize and stabilize the surface of graphene oxide (GO) with a cationic polymer, poly(2-dimethylaminoethyl methacrylate) (poly(DMAEMA)), using a biomimetic adhesive molecule, 3,4-dihydroxy-L-phenylalanine (dopa). The surface of GO can be readily modified with dopa-functionalized poly(DMAEMA). The resulting GO-dopa-poly(DMAEMA) interacted with siRNA to form multi-layered nano-structures, which provided excellent protection of siRNA form the attack of serum-driven nucleases. In addition, GO-dopa-poly(DMAEMA) demonstrated significantly improved cellular uptake and enhanced gene silencing efficiency, compared to unmodified GO and PEI 25k. Considering its low toxicity and desirable transfection efficiency, GO-dopa-poly(DMAEMA) would be potential candidate for siRNA delivery.
573. Efficient Delivery DNA Vaccine for Alzheimer’s Disease by Triggered Release of Polyplexes from Microneedles
Kim Nak Won,1 Lee Kyuri,1 Lee Min Sang,1 Jeong Ji Hoon.1 1 School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea. DNA vaccines have attracted a lot of attention for last decades due to their easy and inexpensive production and superior stability in ambient temperature. The DNA vaccination approaches also give an opportunity of overcoming safety concerns of conventional live vaccines. Since DNA vaccination mainly relies on cellular delivery of the highly negatively charged macromolecule, selecting delivery system and route of administration is considered crucial prerequisite for effective vaccination. Microneedle is a promising candidate for DNA vaccine delivery since they can mediate efficient and minimally invasive delivery of DNA into subcutaneum where a number of antigen presenting cells such as macrophages, dendritic and Langerhans cells exist. Several approaches using microneedles coated with naked plasmid DNA have been introduced, but the efficacy of S220
DNA delivery to target cells is highly limited due to biodegradable and charged nature of DNA molecules. In this study, microneedle-based gene delivery system which allows triggered release of DNA/polymer complexes (polyplexes) upon the pH changes was developed using the layer-by-layer assembly of multilayered polyelectrolytes composed of negatively charged heparin and cationized albumin which performs pH-dependent charge-inversion. Mannosylate polyethyleniminedeoxycholic acid conjugate was synthesized and used as a non-toxic cationic carrier for the formation of polyplex with plasmid DNA. The mannosylated cationic polymer was designed to perform targeted gene delivery to resident macrophages in subcutaneum. The polyplexes was placed onto the multilayered microneedles as an outer most layer via electrostatic interactions. The pH transition to neutral pH allows accelerated release of polyplexes in vitro, suggesting the efficient release of polyplexes after injection. Immunization of plasmid DNA encoding amyloid beta monomer(A1-42) using the multilayered microneedle system demonstrated superior antigen expression and antibody (anti-A1-42 IgG) production in vivo, compared to conventional subcutaneous injection with syringe. In addition, the microneedle-based vaccination can also mediate highly elevated recall immune response, an immune memory response, after re-challenging of the antigen.
574. Improved Lentiviral Formats for Genome Editing Using Zinc Finger Nucleases
Monika Gjoka,1 Kevin Kang,1 Yuping Huang,1 Gregory D. Davis.1 1 ZFN Group, Sigma-Aldrich, St. Louis, MO. Despite major advances in zinc finger nuclease (ZFN) design and engineering, ZFN delivery and expression remain challenging for particular cell types. Genetic tools such as shRNA and microRNA have benefited greatly from lentiviral gene expression, enabling manipulation of primary cells, non-dividing cells, and a variety of other difficult cell types. Due to their compact DNA binding protein structure and low content of repetitive sequence, ZFNs have been successfully expressed and implemented using lentiviral vectors. While lentiviral technology supports functional delivery of ZFNs, integration-deficient lentivirus (IDLV) has been required to avoid permanent integration of ZFN transgenes into the host genome. In general, relative to integration competent lentivirus, IDLV-formatted gene expression has suffered from significantly lower transgene expression levels. Lower nuclease expression levels are a concern for challenging genome editing scenarios in which the genomic context or other factors make targeting less efficient than average. To address these expression limitations, we have constructed a series of new lentiviral vectors, some of which link ZFN expression directly to fluorescent protein reporters. Lentiviral titers are also known to be negatively impacted by large genetic payloads, and the insertion of reporter genes into the lentiviral ZFN expression cassette is facilitated by the relatively small size of ZFN genes. This reporterlinked expression format enables enrichment of cell populations that have undergone both efficient transduction and subsequent high level expression of ZFN transgenes and can substantially increase genome editing frequencies in clonal cell populations requiring lentiviral delivery.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
RNA interference (RNAi)-based gene therapy has been considered a promising approach for the treatment of hard-to-cure diseases and gene-related disorders in gene therapy. Intracellular delivery of synthetic small interfering RNA (siRNA), a double stranded RNA having 19 - 23 base pairs, can induce sequence-specific gene silencing via the RNAi phenomenon. Due to its charged nature and degradation in physiological solutions, development of efficient and safe delivery vehicle is one of the most critical prerequisite for the use of siRNA in clinical settings. However, siRNA cannot be readily formulated with conventional cationic polymers since the length of siRNA is too short and rigid to act as an efficient cross-linker for the formation of tight polyelectrolyte complex structure. Graphene oxide (GO), a 2-dimmensional nano-sized sheet composed of carbon atoms, has been popularly studied in biomedical fields such as drug delivery, cellular imaging, and bio sensors owing to its physical, chemical and mechanical properties. The flexible 2D GO sheet would be an ideal carrier for siRNA since short and rigid siRNA molecules can be aligned and adsorbed onto the surface of GO sheet. Although hydrophobic drugs and single-stranded nucleic acids (DNA or RNA) can be adsorbed on GO surface through - stacking and other non-covalent molecular interactions, double stranded siRNA cannot be readily adsorbed onto GO via the interactions. In this study, we developed a facile method to functionalize and stabilize the surface of graphene oxide (GO) with a cationic polymer, poly(2-dimethylaminoethyl methacrylate) (poly(DMAEMA)), using a biomimetic adhesive molecule, 3,4-dihydroxy-L-phenylalanine (dopa). The surface of GO can be readily modified with dopa-functionalized poly(DMAEMA). The resulting GO-dopa-poly(DMAEMA) interacted with siRNA to form multi-layered nano-structures, which provided excellent protection of siRNA form the attack of serum-driven nucleases. In addition, GO-dopa-poly(DMAEMA) demonstrated significantly improved cellular uptake and enhanced gene silencing efficiency, compared to unmodified GO and PEI 25k. Considering its low toxicity and desirable transfection efficiency, GO-dopa-poly(DMAEMA) would be potential candidate for siRNA delivery.
573. Efficient Delivery DNA Vaccine for Alzheimer’s Disease by Triggered Release of Polyplexes from Microneedles
Kim Nak Won,1 Lee Kyuri,1 Lee Min Sang,1 Jeong Ji Hoon.1 1 School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea. DNA vaccines have attracted a lot of attention for last decades due to their easy and inexpensive production and superior stability in ambient temperature. The DNA vaccination approaches also give an opportunity of overcoming safety concerns of conventional live vaccines. Since DNA vaccination mainly relies on cellular delivery of the highly negatively charged macromolecule, selecting delivery system and route of administration is considered crucial prerequisite for effective vaccination. Microneedle is a promising candidate for DNA vaccine delivery since they can mediate efficient and minimally invasive delivery of DNA into subcutaneum where a number of antigen presenting cells such as macrophages, dendritic and Langerhans cells exist. Several approaches using microneedles coated with naked plasmid DNA have been introduced, but the efficacy of S220
DNA delivery to target cells is highly limited due to biodegradable and charged nature of DNA molecules. In this study, microneedle-based gene delivery system which allows triggered release of DNA/polymer complexes (polyplexes) upon the pH changes was developed using the layer-by-layer assembly of multilayered polyelectrolytes composed of negatively charged heparin and cationized albumin which performs pH-dependent charge-inversion. Mannosylate polyethyleniminedeoxycholic acid conjugate was synthesized and used as a non-toxic cationic carrier for the formation of polyplex with plasmid DNA. The mannosylated cationic polymer was designed to perform targeted gene delivery to resident macrophages in subcutaneum. The polyplexes was placed onto the multilayered microneedles as an outer most layer via electrostatic interactions. The pH transition to neutral pH allows accelerated release of polyplexes in vitro, suggesting the efficient release of polyplexes after injection. Immunization of plasmid DNA encoding amyloid beta monomer(A1-42) using the multilayered microneedle system demonstrated superior antigen expression and antibody (anti-A1-42 IgG) production in vivo, compared to conventional subcutaneous injection with syringe. In addition, the microneedle-based vaccination can also mediate highly elevated recall immune response, an immune memory response, after re-challenging of the antigen.
574. Improved Lentiviral Formats for Genome Editing Using Zinc Finger Nucleases
Monika Gjoka,1 Kevin Kang,1 Yuping Huang,1 Gregory D. Davis.1 1 ZFN Group, Sigma-Aldrich, St. Louis, MO. Despite major advances in zinc finger nuclease (ZFN) design and engineering, ZFN delivery and expression remain challenging for particular cell types. Genetic tools such as shRNA and microRNA have benefited greatly from lentiviral gene expression, enabling manipulation of primary cells, non-dividing cells, and a variety of other difficult cell types. Due to their compact DNA binding protein structure and low content of repetitive sequence, ZFNs have been successfully expressed and implemented using lentiviral vectors. While lentiviral technology supports functional delivery of ZFNs, integration-deficient lentivirus (IDLV) has been required to avoid permanent integration of ZFN transgenes into the host genome. In general, relative to integration competent lentivirus, IDLV-formatted gene expression has suffered from significantly lower transgene expression levels. Lower nuclease expression levels are a concern for challenging genome editing scenarios in which the genomic context or other factors make targeting less efficient than average. To address these expression limitations, we have constructed a series of new lentiviral vectors, some of which link ZFN expression directly to fluorescent protein reporters. Lentiviral titers are also known to be negatively impacted by large genetic payloads, and the insertion of reporter genes into the lentiviral ZFN expression cassette is facilitated by the relatively small size of ZFN genes. This reporterlinked expression format enables enrichment of cell populations that have undergone both efficient transduction and subsequent high level expression of ZFN transgenes and can substantially increase genome editing frequencies in clonal cell populations requiring lentiviral delivery.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy