349 Surface Stabilized Calcium Phosphate Nano Crystals for Efficient Gene Delivery Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S134 DNA[.]
DNA VECTOROLOGY & GENE TARGETING I different feedback loops in signalling pathways affected in BHD pathogenesis and provides further insight into the role of the FLCN in BHD More significantly, this study demonstrates the suitability of episomally maintained S/MAR DNA vectors to successfully model the persistent functional expression of a therapeutic gene in a cancer cell line It should also establish this class of vectors as effective tools for investigating signalling components differentially regulated in different cancers and to aid the identification of novel cancer markers for diagnosis and therapy Additionally, this study illustrates the utility of this class of vectors for the genetic modification of cells without the risk of genotoxicity 347 Collagen VII Gene Delivery Via Sleeping Beauty Transposon in COL7A1-Deficient Keratinocytes from Epidermolysis Bullosa Patients Maria Carmela Latella,1 Fabienne Cocchiarella,1 Giandomenico Turchiano,1 Zsuzsanna Izsvak,2 Zoltan Ivics,3 Manuel Gonỗalves,4 Fulvio Mavilio,1,5 Fernando Larcher,6 Alessandra Recchia.1 Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 2Max Delbruck Center for Molecular Medicine, Berlin, Germany; 3Paul Ehrlich Institute, Langen, Germany; 4Leiden University Medical Center, Leiden, Netherlands; 5Genethon, Evry, France; 6Epithelial Biomedicine Division, CIEMAT, Madrid, Spain Transplantation of autologous, genetically corrected epidermal stem cells was successfully used to treat junctional epidermolysis bullosa (EB), a genetic skin adhesion disorder Autosomal recessive epidermolysis bullosa (RDEB) is a genetic skin adhesion defect caused by mutations in the type VII collagen gene (COL7A1) Although full-length type-VII collagen is successfully produced in human keratinocytes by retroviral vectors, genetic instability due to the large size (9kb) and the highly repeated nature of the gene sequence is a persistent problem The Sleeping Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the wild-type (pT2) transposon or the “sandwich” version (pSA) that showed robust transposition efficiency in human cells By co-transfecting the SB100X transposase together with the pT2 or the pSA transposon carrying a reporter gene and non coding stuffer DNA to reach comparable size of the COL7A1 cDNA, we observed up to 40% of transposition in Hela and in primary keratinocytes cells with both transposons Based on these data we selected the pT2 backbone and we constructed the pT2 transposon carrying the COL7A1 cDNA driven by the PGK promoter between the terminal inverted repeat sequences of the transposon vector We observed up to 40% transposition efficiency of the collagen VII expression cassette in immortalized keratinocytes from RDEB patients Clonal analysis demonstrated that the transposition events occur with no rearrangements and the average copy number of pT2 transposon integrated is 1.5 copies/cell We also investigated the restoration of type VII collagen expression and production in RDEB cells Despite its enormous potential, the plasmid transfection procedure of the transposon/transposase integrating system in RDEB cells remains an obstacle to its practical application in gene therapy To overcome this limitation we are currently vectorizing the transposon/transposase system into first-generation and helper-dependent adenoviral vectors S134 348 DNAJB4, a Heat Shock Rotein hsp40 Homolog, Activates Dendritic Cells and Functions as a Molecular Adjuvant When Incorporated into Cancer DNA Vaccine Hsing-Yu Chen,1 Jeremy J.W Chen,2 Chi-Chen Lin.3 Institute of Biomedical Sciences, College of Life Science, National Chung Hsing University, Taichung, Taiwan; 2Institute of Biomedical Sciences, College of Life Science, National Chung Hsing University, Taichung, Taiwan; 3Institute of Biomedical Sciences, College of Life Science, National Chung Hsing University, Taichung, Taiwan DNAJB4, a member of the heat shock protein 40 chaperone family, is a newly identified tumor suppressor that has been implicated in tumorigenesis and is associated with reduced cancer recurrence and prolonged survival of NSCLC patients However, the role of DNAJB4 in the anti-tumor immune response remains unclear Using the bone marrow dendritic cells (BMDCs) isolated from DNAJB4 knockout mice, our preliminary study showed for the first time that DNAJB4 can influence dendritic cell maturation and function in vitro Therefore, based on the findings above, we also examined whether the DNAJB4 expression in dendritic cells can be as an adjuvant for DNA vaccine via gene gun delivery Bicistronic plasmids expressing the N-terminal extracellular domain of human HER-2/neu and the mouse DNAJB4 was subcutaneously injected into mice by gene gun administration to elicit antitumor immunity against p185neu-overexpressing MBT-2 bladder tumor cells We found that mice treated with hN’-neu-IRESDNAJB4 showed greater reductions in tumor growth and longer survival times than mice treated with either hN’-neu or DNAJB4 DNA plasmid alone, indicating that the co-expression of DNAJB4 enhanced the antitumor efficacy of the HER-2/neu cancer vaccine Cytotoxicity analyses further revealed that the DNAJB4-enhanced antitumor effect was associated with significant increases in the number of functional CD8+ T cells and in the levels of cytotoxic T lymphocytes activity Moreover, in vivo lymphocyte depletion analyses confirmed that the antitumor efficacy of the hN’-neu-IRESDNAJB4 vaccine depended on functional CD8+ T cells Overall, these results support co-expression DNAJB4 with tumor antigen can be as an effective strategy to enhance the therapeutic antitumor effect of Cancer DNA vaccines against tumors 349 Surface-Stabilized Calcium Phosphate Nano-Crystals for Efficient Gene Delivery Min Sang Lee,1 Kyuri Lee,1 Nak Won Kim,1 Ji Hoon Jeong.1 School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea Calcium phosphate (CAP) has a wide range of applications in biomedical systems due to its excellent biocompatibility and unique bio-functionality that is similar to natural inorganic components of the human body Although there is great potential for the use of CAP in the development of gene delivery systems, the uncontrollable growth of CAP crystal makes it difficult to use in a practical nano-gene delivery system Thus, it is necessary for the size of CAP to be well controlled and without irregular particle growth at the nano-level Recently, there have been multiple attempts to utilize the adhesive properties of 3,4-dihydroxy-L-phenylalanine (dopa) in various biomaterials The catechol moiety of the dopa structure has a strong affinity for many metal ions including iron and calcium In this study, we developed a new gene delivery system based on calcium phosphate/nucleic acid (e.g., plasmid DNA, siRNA) nano-crystals using a dopa-modified chitosan The dopa-modified chitosan adsorbed to the surface of growing CAP nano-crystals to act as both a gene carrier and a particle stabilizer The cellular uptake and gene transfection efficiency of CAP nano-crystals was evaluated using GFP plasmid DNA and siRNA for GFP inhibition The resulting the CAP nano-crystals (CAP/pDNA/ Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy DNA VECTOROLOGY & GENE TARGETING I dopa-Chi) were successfully prepared by serial addition of plasmid DNA (pDNA) and dopa (3,4-dihydroxy-L-phenylalanine) modified chitosan to growing CAP particles in calcium phosphate solution The addition of the dopa moiety is thought to enable chitosan adsorption onto the surface of forming calcium phosphate particles to prevent further growth The CAP/pDNA/dopa-Chi significantly increased the serum stability of pDNA, and showed high cellular uptake efficiency and trans-gene expression Additionally, the chitosan stabilized CAP nano-crystals were also prepared for siRNA delivery (CAP/siRNA/ dopa-Chi) via the same method as for CAP/pDNA/dopa-Chi A notable siRNA gene silencing effect of CAP/siRNA/dopa-Chi was exhibited without any sign of cytotoxicity These results demonstrate that the dopa-Chi conjugate-stabilized CAP for gene delivery can be considered as an alternative non-viral carrier for clinical gene therapy 350 Polymeric Micellar Nanocarriers for Gene and Oligonucleotide Delivery Kazunori Kataoka.1 Materials Engineering/Center for Disease Biology and Integrative Medicineg, University of Tokyo, Tokyo, Japan Poly (ethylene glycol)-modified polyplexes (polyplex micelles) have gained appreciable perspective as non-viral gene carriers upon their structural advantages; distinct core-shell architecture and outer hydrophilic PEG shell shielding inner polyplex core, achieving superior biocompatibility and stealth behaviors in physiological environment Furthermore, we developed a PEG-based block catiomer with a wealth of favorable features, e.g facilitated endosome escaping capacity due to increased protonation at acidic pH and negligible cytotoxic profiles due to biodegradable nature On the other hand, high PEGylation tends to minimize non-specific interactions of polyplex micelles with cellular membrane, therefore may elicit inefficient cellular uptake activity To overcome this issue, we installed cyclic RGD (Arg-Gly-Asp) peptide (cRGD) onto the polyplex micelle surface intended for promoting RGD-integrin mediated cellular endocytosis We selected sFlt-1 as soluble form of VEGF receptor, blocking VEGF as a potent anti-angiogenic gene to prepare polyplex micelle The systemic in vivo test revealed remarkably enhanced gene expression at intractable pancreatic tumor model, ultimately accounted for appreciable tumor growth suppression effect The formulation from newly designed PEG-based block catiomer with a repetitive array of aminoethylene units in the side chain was recently found to be effective for in vivo delivery of messenger RNA (mRNA) mRNA delivery is a promising approach to produce therapeutic proteins without any risk of insertion mutagenesis into the host genome Nevertheless, instability and immunogenicity of mRNA are the major obstacles in practical application of mRNA-based therapeutics The use of the PEG-based block catiomer to encapsulate mRNA into the core of the polyplex micelles successfully overcome these obstacles, achieving significant increase in stability as well as almost complete prevention from the recognition by Toll-like receptors of mRNA Eventually, mRNA-loaded polyplex micelles administered into the central nervous system of mice successfully provided the sustained expression of reporter proteins for more than a week This result indicates the feasibility of mRNA-loaded polyplex micelles in molecular therapy of various intractable diseases Polyplex micelles are also applicable for delivery of oligonucleotides, including siRNA Due to the relatively small size of siRNA, additional function is needed to further increase the stability of polyplex micelles typically during the blood circulation We introduced disulfide linkage into the micelle core together with an increased hydrophobicity by side chain modification of the cationic segment These disulfide-stabilized micelles loaded with siRNA successfully deliver siRNA into solid tumors through systemic route by installation of ligand molecules on the periphery of the micelles, such as cRGD The polyplex micelles loaded with the cocktail of siVEGF and siVEGFR completely Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy suppress the growth of HeLa tumor through anti-angiogenic effect More recently, polyplex micelles loaded with siRNA directly targeting tumor cell were prepared, and were verified the growth inhibition of targeted tumor cells by intra venous administration 351 Methods of Facilitating Patient Participation in DNA Based Experimental Therapeutic Phase I Cancer Studies Wanda Strange,1 Cynthia Bedell,1 Shannon Cagnina,1 Jeanne Jones,1 Patricia Brown,1 Gladice Wallraven,4 John Nemunaitis.1,2,3,4 Mary Crowley Cancer Research Centers, Dallas; 2Texas Oncology, P.A, Dallas; 3Medical City HCA, Dallas; 4Gradalis, Inc, Dallas Phase I clinical trials provide a unique opportunity to cancer patients who have failed standard of care Expansion to clinical trial opportunities with DNA based technology has been increasing However, patient management aspects often not addressed involve cost, unique education and complex multi-specialty care needs Patient navigation programs have been established to assist patient needs Internists, oncologists, surgeons and often other clinical subspecialties require coordination for patient management involving standard medical issues (response, toxicity), as well as, protocol design issues (biopsy for molecular signal expression/knockdown) Both medical and protocol research management involve multiple clinics, procedure rooms, and hospital services for which IRB, IBC, hospital administration and clinic administration require FDA supported standard operating procedures (SOPs) While the sponsor of the clinical trial accrues the cost of the study drug and research related expenses, other expenses are the responsibility of the patient or their insurance carrier Some insurance plans not cover standard non-research costs if patients participate in research Medicare has a policy of covering standard costs of patients enrolled in research trials, and 29 states have enacted legislation mandating such coverage for all health insurance plans governed by their state (Klamerus et al CCR 2010) However, state laws not impact plans governed by the federal Employee Retirement Income Security ACT (ERISA) Certain employers can operate under a self-insurance scheme in which they are only governed by ERISA, and not by state law This has proven a popular business decision to reduce costs for employers due to recent premium increases On January 1, 2014, the minimum coverage mandated by the Patient Protection and Affordable Care Act will eliminate this loophole, and all plans will be required to cover non-research costs for patients enrolled in clinical trials (Klamerus et al CCR 2010) Until that time, clinical research centers must precertify all standard procedures before enrolling patient into a trial Pre-certification can cause a delay of up to six weeks, a time period that can be fatal to a late stage cancer patient seeking to enroll on a trial and receive an investigational agent as a last option, after standard therapies have failed In the current economic climate Oncology Nurse Navigators can play an integral part in health care reform and cost containment Specifically in assisting 1) education of patients , family and standard of care medical team involving unique DNA related questions and Phase I trial study questions; 2) financial guidance to cancer patient financial assistance programs (Strange et al CCO 2012) including travel assistance; and 3) coordination of multispecialty management involving both standard of care and research needed care Methods of facilitating participation and management of patients entered into DNA based experimental therapeutic trials will be presented S135 ... efficiency and trans -gene expression Additionally, the chitosan stabilized CAP nano- crystals were also prepared for siRNA delivery (CAP/siRNA/ dopa-Chi) via the same method as for CAP/pDNA/dopa-Chi... alternative non-viral carrier for clinical gene therapy 350 Polymeric Micellar Nanocarriers for Gene and Oligonucleotide Delivery Kazunori Kataoka.1 Materials Engineering/Center for Disease Biology and... notable siRNA gene silencing effect of CAP/siRNA/dopa-Chi was exhibited without any sign of cytotoxicity These results demonstrate that the dopa-Chi conjugate -stabilized CAP for gene delivery can