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163. Hepatocyte Targeted Endosomal Escape Agents
Chemical/Molecular Conjugates References: [1] C. H. Ryu, S. H. Park, S. A. Park, S. M. Kim, J. Y. Lim, C. H. Jeong, W. S. Yoon, W. I. Oh, Y. C. Sung, S. S. Jeun, Human gene therapy 2011, 22, 733. [2] M. A. Mintzer, E. E. Simanek, Chemical reviews 2009, 109, 259. [3] P. Xu, G. K. Quick, Y. Yeo, Biomaterials 2009, 30, 5834.
161. Directing Integration of SB Transposons Into Selected Genomic Sites in Liver for Non-Viral Gene Therapy
Bryan C. Hall,1 Jason B. Bell,1 Elena L. Aronovich,1 Allicia Gunderman,1 Shayan Farahani,1 Chelsea Fortin,1 Perry B. Hackett.1 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN. Insertional mutagenesis is a concern for those practicing gene therapy. Several methods that employ modified recombinases, integrases, and transposases have been proposed and tested to direct integration of transgenic expression cassettes to specific sites in mammalian genomes. Site-specific nucleases employing zincfingers (ZFNs), transcription activator-like coupled to endonucleases (TALENs), and RNA-guided nucleases (RGNs, which include the CRISPR/Cas systems) are effective to various extents for site-specific recombination to repair genes as well as direct insertion of new genetic sequences. There are some concerns with cleavage of non-targeted sites that have yet to be resolved using site-specific nucleases. We have been developing an alternative method for directing integration of the Sleeping Beauty (SB) transposon system for non-viral gene therapy. SB transposons nearly randomly integrate into TA sites in genomes (about 2X106 in mammalian genomes) and are agnostic regarding the transcriptional state or purpose of a given region. Our targeting procedure employs the RecA protein from E. coli that is involved with DNA repair and recombination. Single-stranded DNA oligonucleotides (ODNs) complexed with RecA protein can interact with genomic DNA to form triple DNA helices. These can be recognized by some transposases for selective transposition (Fig. 1). We tested whether site-specific integration by SB transposase could be achieved by targeting the MIA14 locus in mouse liver. There are about 2098 copies of the repetitive element in benign locations in the mouse genome. By targeting a repetitive element, the chances of avoiding integration into non-targeted sites theoretically would be reduced. If effective, RecA-mediated targeting for a given transposon (transgene) to any site could be achieved by just specifying a single 40-mer ODN. To test our proposed method for targeting integration, we hydrodynamically infused pT2 transposons carrying a luciferase expression cassette, SB100 transposase, and RecA-40mer filaments into mice. Mice were sacked four days post-infusion, livers were harvested, genomic DNA isolated, and selected regions amplified using bar-coded, transposon-specific primers for analysis of integration sites by Illumina sequencing. Our initial results indicate that this method of targeting integration without using site-specific nucleases has potential.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
162. Crossing the Blood-Cerebrospinal Fluid Barrier in the Mouse Choroid Plexus With an Engineered Receptor/Ligand System
Hector R. Mendez-Gomez,1,4 Albert Galera-Prat,2,3 Craig Meyers,1,4 Weijun Chen,1,4 Mariano Carrion-Vazquez,2,3 Nicholas Muzyczka.1,4 1 Molecular Genetics and Microbioloby, University of Florida, Gainesville, FL; 2Cajal Institute, Madrid, Spain; 3Instituto Madrileño de Estudios Avanzados en Neurociencia, Madrid, Spain; 4Powell Gene Therapy Center and UF Genetics Institute, Gainesville. Crossing the blood-brain and the blood-cerebrospinal fluid barriers are one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because they prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Mimicking nature, we engineered a receptor/ligand system to cross the brain barrier by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested it in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the blood-cerebrospinal fluid barrier, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.
163. Hepatocyte Targeted Endosomal Escape Agents
Christopher W. White,1 Nicholas J. Baumhover,1 Kevin G. Rice.1 1 Medicinal Natural Products Chemistry, University of Iowa, Iowa City. The successful development of potent non-viral DNA and mRNA delivery agents to transfect liver hepatocytes in vivo requires the use of an endosomal escape molecule. Two major strategies have emerged, one that utilizes membrane lytic peptides, and a second that uses proton sponge polymers. Both approaches would require administration of excess endosomal release agent with a smaller dose of targeted polyplex. The present study compared these approaches by conducting 384-well in vitro transfections on primary hepatocytes, as previously described.1 An N-glycan targeted melittin was designed to target hepatocytes via the asialoglycoprotein receptor. The synthesis used a purified triantennary N-glycan from bovine fetuin as an potent high affinity asialoglycoprotein receptor targeting ligand that was conjugated by disulfide bound to an N-terminal Cys on melittin, a 26 amino acid membrane lytic peptide. Following targeted entry into hepatocytes via receptor mediated endocytosis, melittin is bioactivated by reduction of the disulfide bond to release the N-glycan, leading to endosomal lysis. RBC hemolysis assays established the membrane lytic activity of triantennary-melittin was muted prior to reduction, which allowed it to be safely i.v. dosed in mice. A second strategy involved the modification of PEI by reductive amination with lactose followed by acetylation, resulting in an asialoglycoprotein receptor targeted proton sponge polymer. The resulting LacAcyl-PEI was purified by RP-HPLC and characterized by NMR. The i.v. dosing of LacAcyl PEI in mice demonstrated that it possessed a much greater safety margin than unmodified PEI. These novel endosomal escape agents are being compared for their ability to mediate in vitro gene transfer of glycan targeted DNA and mRNA in miniaturized primary hepatocyte transfection assays. 1. Li J, Crowley ST, Duskey J, Khangharia S, Wu M, Rice KG. Miniaturization of gene transfection assays in 384- and 1536-well microplates. Analytical Biochem. 2015; 470: 14-21. S65
161. Directing Integration of SB Transposons Into Selected Genomic Sites in Liver for Non-Viral Gene Therapy
Bryan C. Hall,1 Jason B. Bell,1 Elena L. Aronovich,1 Allicia Gunderman,1 Shayan Farahani,1 Chelsea Fortin,1 Perry B. Hackett.1 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN. Insertional mutagenesis is a concern for those practicing gene therapy. Several methods that employ modified recombinases, integrases, and transposases have been proposed and tested to direct integration of transgenic expression cassettes to specific sites in mammalian genomes. Site-specific nucleases employing zincfingers (ZFNs), transcription activator-like coupled to endonucleases (TALENs), and RNA-guided nucleases (RGNs, which include the CRISPR/Cas systems) are effective to various extents for site-specific recombination to repair genes as well as direct insertion of new genetic sequences. There are some concerns with cleavage of non-targeted sites that have yet to be resolved using site-specific nucleases. We have been developing an alternative method for directing integration of the Sleeping Beauty (SB) transposon system for non-viral gene therapy. SB transposons nearly randomly integrate into TA sites in genomes (about 2X106 in mammalian genomes) and are agnostic regarding the transcriptional state or purpose of a given region. Our targeting procedure employs the RecA protein from E. coli that is involved with DNA repair and recombination. Single-stranded DNA oligonucleotides (ODNs) complexed with RecA protein can interact with genomic DNA to form triple DNA helices. These can be recognized by some transposases for selective transposition (Fig. 1). We tested whether site-specific integration by SB transposase could be achieved by targeting the MIA14 locus in mouse liver. There are about 2098 copies of the repetitive element in benign locations in the mouse genome. By targeting a repetitive element, the chances of avoiding integration into non-targeted sites theoretically would be reduced. If effective, RecA-mediated targeting for a given transposon (transgene) to any site could be achieved by just specifying a single 40-mer ODN. To test our proposed method for targeting integration, we hydrodynamically infused pT2 transposons carrying a luciferase expression cassette, SB100 transposase, and RecA-40mer filaments into mice. Mice were sacked four days post-infusion, livers were harvested, genomic DNA isolated, and selected regions amplified using bar-coded, transposon-specific primers for analysis of integration sites by Illumina sequencing. Our initial results indicate that this method of targeting integration without using site-specific nucleases has potential.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
162. Crossing the Blood-Cerebrospinal Fluid Barrier in the Mouse Choroid Plexus With an Engineered Receptor/Ligand System
Hector R. Mendez-Gomez,1,4 Albert Galera-Prat,2,3 Craig Meyers,1,4 Weijun Chen,1,4 Mariano Carrion-Vazquez,2,3 Nicholas Muzyczka.1,4 1 Molecular Genetics and Microbioloby, University of Florida, Gainesville, FL; 2Cajal Institute, Madrid, Spain; 3Instituto Madrileño de Estudios Avanzados en Neurociencia, Madrid, Spain; 4Powell Gene Therapy Center and UF Genetics Institute, Gainesville. Crossing the blood-brain and the blood-cerebrospinal fluid barriers are one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because they prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Mimicking nature, we engineered a receptor/ligand system to cross the brain barrier by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested it in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the blood-cerebrospinal fluid barrier, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.
163. Hepatocyte Targeted Endosomal Escape Agents
Christopher W. White,1 Nicholas J. Baumhover,1 Kevin G. Rice.1 1 Medicinal Natural Products Chemistry, University of Iowa, Iowa City. The successful development of potent non-viral DNA and mRNA delivery agents to transfect liver hepatocytes in vivo requires the use of an endosomal escape molecule. Two major strategies have emerged, one that utilizes membrane lytic peptides, and a second that uses proton sponge polymers. Both approaches would require administration of excess endosomal release agent with a smaller dose of targeted polyplex. The present study compared these approaches by conducting 384-well in vitro transfections on primary hepatocytes, as previously described.1 An N-glycan targeted melittin was designed to target hepatocytes via the asialoglycoprotein receptor. The synthesis used a purified triantennary N-glycan from bovine fetuin as an potent high affinity asialoglycoprotein receptor targeting ligand that was conjugated by disulfide bound to an N-terminal Cys on melittin, a 26 amino acid membrane lytic peptide. Following targeted entry into hepatocytes via receptor mediated endocytosis, melittin is bioactivated by reduction of the disulfide bond to release the N-glycan, leading to endosomal lysis. RBC hemolysis assays established the membrane lytic activity of triantennary-melittin was muted prior to reduction, which allowed it to be safely i.v. dosed in mice. A second strategy involved the modification of PEI by reductive amination with lactose followed by acetylation, resulting in an asialoglycoprotein receptor targeted proton sponge polymer. The resulting LacAcyl-PEI was purified by RP-HPLC and characterized by NMR. The i.v. dosing of LacAcyl PEI in mice demonstrated that it possessed a much greater safety margin than unmodified PEI. These novel endosomal escape agents are being compared for their ability to mediate in vitro gene transfer of glycan targeted DNA and mRNA in miniaturized primary hepatocyte transfection assays. 1. Li J, Crowley ST, Duskey J, Khangharia S, Wu M, Rice KG. Miniaturization of gene transfection assays in 384- and 1536-well microplates. Analytical Biochem. 2015; 470: 14-21. S65