398 recent advances using small molecule targeting for bilamellar invaginated vesicle (BIV) delivery

398 Recent Advances Using Small Molecule Targeting for Bilamellar Invaginated Vesicle (BIV) Delivery Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell[.]

CHEMICAL AND MOLECULAR CONJUGATES I 396 Gene Expression from Linearized Gene Expression Cassettes Capped with Multi-Arm DNA Junctions Howard H Lou,1 Brian J Lewandowski,1 Philip L Leopold.1 Chemistry, Chemical Biology, and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ The plasmid has been the traditional unit of DNA used for transfer and expression of genes However, during transfection, naked plasmids exhibit poor efficiency in overcoming the cellular barriers to gene transfer including the plasma membrane, cytosol, and nuclear envelope A variety of modifications to plasmids have been made to increase gene transfer efficiency including association of targeting and/or trafficking molecules through the use of modified DNA binding proteins, peptide nucleic acids, covalent modifications, and/or complex formation with cationic compounds, an approach commonly associated with lipid- or polymer-mediated gene delivery Taking the virus capsid as a model, it is clear that optimal trafficking of DNA through the cytosol will require precise positioning of multiple sets of bioactive molecules and precise control of sequential interactions with cellular components Toward this goal, a design has been developed in which targeting and/or trafficking molecules can be associated with a complex DNA scaffold at the end of a linearized gene expression cassette The present work compares the transfection efficiency of gene expression cassettes in the form of circularized gene expression cassettes (minicircles), linearized gene expression cassettes, or linearized expression cassettes capped with complex DNA adapters The gene expression cassette in this study encoded beta-galactosidase, and all constructs were delivered using Lipofectamine™ with equal numbers of expression cassettes Expression of beta-galactosidase was evaluated using X-gal staining as well as a chemiluminescence enzyme activity assay Linearized expression cassettes exhibited slightly lower expression levels than minicircles (28% reduction in expression) Addition of complex DNA adapters to the ends of linearized expression cassettes reduced gene expression significantly (82% reduction) To test the use of complex DNA adapters as a platform for addition of trafficking moieties, a 72 bp DNA nuclear targeting sequence from an SV40 promoter, previously reported to enhance plasmid trafficking to the nucleus was ligated to the adapter The expression level of the linearized expression cassettes bearing the SV40 functionalized adapter exhibited a significantly higher level of gene expression than non-functionalized adapters (2.7 fold, p 0.9) Similarly, dAd5AM1 produced anti-nicotine antibody titers 5.7 x 105 ± 7.5 x 104 in naive mice and 4.2 x 105 ± 3.9 x 104 in Ad5immune mice (p> 0.09), while dAd5PCN mice elicited titers of 3.6 x 105 ± 6.6 x 104 in the naive group and 2.1 x 105 ± 6.6 x 104 in the Ad5-immune group at wk post-vaccination (p>0.1) Assessment of anti-Ad IgG isotypes in all vaccine-injected mice showed, for all vaccines, a dominant IgG1 subtype followed by IgG2a and IgG2b with no significant difference among naive and Ad5-immune groups To further assess that the dAd5GNE anti-cocaine vaccine could still be efficacious even in the presence of anti-Ad immunity, we evaluated the capacity of dAd5GNE vaccination to prevent cocaine from reaching the brain in Ad5 immune mice 3H-cocaine was administered intravenously to the dAd5GNE vaccinated mice (naive and Ad5 immune), and the blood-brain distribution of administered 3H-cocaine evaluated after The Ad5 immune and non-immune mice both Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... a vaccine platform against small molecules in which the small molecules or their analogs are covalently coupled to the capsid proteins of disrupted Ad Because this vaccine platform is based on... that meet the criteria for an appropriate binding constant could be used for a personalized, targeted therapeutic in the clinic Furthermore, small molecules that target delivery to melanoma cell...CHEMICAL AND MOLECULAR CONJUGATES I for small molecule ligand binding to specific target cells Binding constants of 108 M-1 and greater are associated with small molecule ligands that promote expression