- Email: [email protected]
436. Dual pH Response Nanoparticles for Gene Delivery
DNA VECTOROLOGY & GENE TARGETING - I 433. Octaarginine- and pH Sensitive Fusogenic Peptide-Modified Nanoparticles for Liver Gene Delivery
Yasuhiro Hayashi,1 Ryoichi Mizuno,1 Khalil A. Ikramy,1 Hideyoshi Harashima.1 1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. We previously reported that octaarginine peptide modified liposomes (R8-liposomes) largely accumulated in the liver after intravenous administration and that this is dependent on the R8density. We report herein on the development of a Multifunctional Envelope-type Nano Device modified with R8 and GALA, as a pH-sensitive fusogenic peptide (R8-GALA-MEND) for liver gene delivery. An R8-MEND encapsulating pDNA prepared using two different cores (negatively or positively charged pDNA/polyethylene imine condensed particles) failed to produce a high gene expression in the liver. Modification with GALA dramatically increased gene expression particularly in the liver only in the case of a negative core R8-MEND. Quantification of the delivered gene copies to the liver nucleus revealed that the amount of pDNA was comparable in all 4 cases (positive or negative cores, absence or presence of GALA). Gene expression efficiencies per nucleus-delivered pDNA were greatly improved in the case of the negative core R8-GALA-MEND, suggesting that the substantial improvement in gene expression can be explained by an improved gene expression efficiency per pDNA in the presence of GALA. A comparative study between the developed R8GALA-MEND and a similar system containing DOTAP, a commonly used cationic lipid, instead of R8 showed that gene expression of the R8-GALA-MEND was 29 times higher than that of the DOTAPGALA-MEND and is more selective for the liver. Collectively, these results suggest that the combination of a negatively charged core system and GALA modification of the R8-MEND is useful system for efficiently delivering genes to the liver.
434. Oligonucleotides Delivery Using HighPressurized Lipoplexes
Tsuyoshi Kimura,1 Asami Sano,1 Kwangwoo Nam,1 Yoshihiro Sasaki,1 Kazunari Akiyoshi,2 Akio Kishida.1 1 Tokyo Medical and Dental University, Tokyo, Japan; 2Kyoto University, Kyoto, Japan. Regulation of gene expression using oligonucleotides (ONs) is one of attractive methods for gene therapy. Cationic liposomes (CLs) are used to deliver ONs into cells effectively, and to inhibit expression of target gene selectively because stable and small complexes of CL and ON, called lipoplex, are formed by electrostatic interaction. Although lipoplex could be introduced into cell effectively, the efficiency and selectivity of gene suppression are insufficient. Previously, we reported that high hydrostatic pressurization (HHP) treatment for the complex of CL and plasmid DNA could improve the transfection efficiency. In this study, we investigated antisense-ODNs (AS-ODNs) delivery using lipoplex treated with HHP to inhibit a target gene effectively and selectively. Further, physicochemical properties of the HHP-treated lipoplex were examined in order to elucidate relationship between the structure and the transfection efficiency of the HHP treated lipoplex. Some researchers reported that the supramolecular structure of lipoplex strongly affected the efficiency of transfection. The pressurized lipoplexes with AS-ODNs were added to HEK293 cells expressing luc2 stably. The activity of luciferase was measured by using luminometer. Although the levels of gene suppression were different between AS-ONs used, the efficiency of gene silencing was enhanced by using the HHP-treated lipoplex compared to the nonpressurized lipoplex. For TEM observation, the lamellar structure of the non-treated lipoplexes was observed, whereas the amorphous structure including small and regular structure was observed for Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
the HHP-treated lipoplexes. The hydrophobic microenvironment of lipoplex was investigated by using fluorescent hydrophobic probes, such as Laurdan and pyrene. The fluorescent spectrum and intensity of these probes differed between lipoplexes with/without pressurization. These results suggest that the structure of lipoplex was changed by the HHP treatment. In conclusion, we successfully improved the efficiency of gene suppression by using the HHP treated lipoplex. It was found that the change of the structure of lipoplex was induced by the HHP treatment. We believe that this change of lipoplex structure by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and ON delivery.
435. Aptamer Conjugated Liposomes (Aptamosomes) as an Efficient Detection/Delivery Nano-Vehicle toward Prostate Cancer Cells
Keun-Sik Kim,1 Seho Kim,2 Hyun-Mi Kwon,2,3 Gwang-Hyun Lee,2 Yong Serk Park,4 Dong-Eun Kim.2,3 1 Department of Biomedical Laboratory Science, Konyang University, Daejeon, Korea; 2Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea; 3WCU and BRL Program, Konkuk University, Seoul, Korea; 4Department of Biomedical Labolatory Science, Yonsei University, Wonju, Korea. Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug or gene encapsulated liposomes particles in a cell- or tissue-specific manner. In this study, we report the design of aptamer conjugated POPC (1-parmitonyl-2-oleoyl-sn-glycerol-3phosphocholine)-liposomes (aptamosomes) that achieves selective and strong binding of otherwise low-affinity aptamers at physiological conditions. Published PSMA RNA aptamer A9 which was well known PSMA (prostate-specific membrane antigen) specific RNA sequences was extended for annealing with complementary DNA linker which was labeled with thiol group and FITC. The modified PSMA aptamers were conjugated with maleimide group on the surface of PEGylated micelles. Specifically, we prepared the PSMA-specific aptamosomes by a new post-insertion method. These nanoparticle-aptamosomes of the nano size (90∼100 nm) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells resulting in significantly enhanced in vitro cellular binding as compared with nontargeted nanoparticles that lack the PSMA aptamer. The doxorubicin (Dox)encapsulated aptamosomes were significantly more cytotoxic towered targeted LNCaP cells than the nontargeted PC3 and A549 cells. The FITC-labeled siRNA encapsulated aptamosomes also exhibited remarkable transfection efficacy of siRNA into LNCap cells as compared with nontargeted cells (PC3 and A549). Furthermore, we are currently preparing for the Gd- aptamosomes containing 10 mol % Gadolinium-phosphatidylethanolamine (Gd-DTPA-PE) as MRI agents for MR imaging to LNCaP tumor in LNCaP xenograft nude mouse. These results will show our PSMA-aptamosome has high potential for in vivo imaging of prostate cancer cell recognition and for in vivo drug or gene delivery application.
436. Dual pH Response Nanoparticles for Gene Delivery
Jose M. Morachis,1 Enas A. Mahmoud,1 Jagadis Sankaranarayanan,1 Jenny Huang,1 Adah Almutairi.1,2 1 Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA; 2Materials Science and Engineering, University of California at San Diego, La Jolla, CA.
Emerging gene delivery tools offer novel therapeutic approaches to address several types of diseases including progeria, cystic fibrosis, parkinsons, and multiple types of cancers. We investigated a novel gene delivery system using logic gate nanoparticles developed with a dual pH responsive random co-polymer (poly-ß-aminoester ketal-2). S167
DNA VECTOROLOGY & GENE TARGETING - I The random co-polymer is unique because the amine backbone undergoes a sharp hydrophobic-hydrophilic switch at acidic pH. This leads to an increase in uptake of water (bulk dissolution) and hence an increase in ketal hydrolysis (surface and bulk degradation). We hypothesized that these nanoparticles would be able to increase gene delivery and expression efficiency by providing effective endosomal release and expression of DNA due to its ability to quickly “burst” and release the DNA content into the cytoplasm. Passive nanoparticle entry into cells was demonstrated by delivering Cy-5 labeled pDNA econding EGFP into HCT116, a colon carcinoma cell line. Flow cytometry analysis showed cells to be positive for Cy5DNA-nanoparticles and later produced EGFP expression superior to PLGA nanoparticles. Inhibition of VATPases using bafilomycin A1 demonstrates that expression of EGFP is dependent on the “proton sponge effect”. Further experiments are underway to test if siRNA can also be encapsulated and delivered for fast release and gene knockdown. The advanced dual response nanoparticles offer new therapeutic possibilities and can be combined with cell type specific peptides for improved cellular entry and specificity.
437. The Interaction between SP100 and Rep78 Inhibits Rep-Dependent Integration and Mediates SP100 Degradation
Jinzhong Chen,1 Yuan-Yuan Xue,1 Ran Wang,1 Yang-bo Yue,1 Jinglun Xue.1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China. The adeno-associated virus type 2 (AAV-2) Rep78 is a multifunctional protein that is required for viral transcription, replication, and sitespecific integration. Rep-dependent integration is not related to any known disease; therefore, this type of integration may be a potential tool for carrying therapeutic genes to the human genome. However, the intracellular process of Rep-dependent integration is still unclear. As a result, improving the Rep-dependent integration system is difficult. In the present study, we revealed the interaction between Rep78 and the cellular protein SP100, a member of the PML nuclear bodies(PML-NBs). SP100 and Rep78 are located in the nucleolus, which provides the spatial possibility for their interaction. In addition, Rep78 was shown to co-immunoprecipitate with the EGFP-SP100 fusion protein but not with EGFP, which provided evidence for the interaction between SP100 and Rep78. A knock-down of the endogenous SP100 expression using siRNA resulted in an increased efficiency of the Rep-dependent integration.
Conversely, the transient expression of Rep78 resulted in an increased degradation of cellular SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome.
These results have furthered our knowledge about Rep78 and the cellular protein SP100. These results may also lead to an improvement in the Rep78-related transgene method and in the selection of target cells for gene therapy.
438. Rep of AAV Protects p53 from E1B Mediated p53 Nucleolus Egress and Degradation Xu Wang,1 Jing-lun Xue,1 Wenjuan Li,1 Jinzhong Chen,1 Jinjin Wang.1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
Adenoviral E1B mediated P53 degradation is key step of adenoviral early process. It also involved in regulating AAV infection cycle. We identified a C terminal fragment of rep78 is a p53 binding domain by protein array. The result was further verified by coIP assay, rep78 but not rep68 binds to p53. As a p53 binding protein, rep78 protects p53 from E1B mediated protein degradation.
S168
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Yasuhiro Hayashi,1 Ryoichi Mizuno,1 Khalil A. Ikramy,1 Hideyoshi Harashima.1 1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. We previously reported that octaarginine peptide modified liposomes (R8-liposomes) largely accumulated in the liver after intravenous administration and that this is dependent on the R8density. We report herein on the development of a Multifunctional Envelope-type Nano Device modified with R8 and GALA, as a pH-sensitive fusogenic peptide (R8-GALA-MEND) for liver gene delivery. An R8-MEND encapsulating pDNA prepared using two different cores (negatively or positively charged pDNA/polyethylene imine condensed particles) failed to produce a high gene expression in the liver. Modification with GALA dramatically increased gene expression particularly in the liver only in the case of a negative core R8-MEND. Quantification of the delivered gene copies to the liver nucleus revealed that the amount of pDNA was comparable in all 4 cases (positive or negative cores, absence or presence of GALA). Gene expression efficiencies per nucleus-delivered pDNA were greatly improved in the case of the negative core R8-GALA-MEND, suggesting that the substantial improvement in gene expression can be explained by an improved gene expression efficiency per pDNA in the presence of GALA. A comparative study between the developed R8GALA-MEND and a similar system containing DOTAP, a commonly used cationic lipid, instead of R8 showed that gene expression of the R8-GALA-MEND was 29 times higher than that of the DOTAPGALA-MEND and is more selective for the liver. Collectively, these results suggest that the combination of a negatively charged core system and GALA modification of the R8-MEND is useful system for efficiently delivering genes to the liver.
434. Oligonucleotides Delivery Using HighPressurized Lipoplexes
Tsuyoshi Kimura,1 Asami Sano,1 Kwangwoo Nam,1 Yoshihiro Sasaki,1 Kazunari Akiyoshi,2 Akio Kishida.1 1 Tokyo Medical and Dental University, Tokyo, Japan; 2Kyoto University, Kyoto, Japan. Regulation of gene expression using oligonucleotides (ONs) is one of attractive methods for gene therapy. Cationic liposomes (CLs) are used to deliver ONs into cells effectively, and to inhibit expression of target gene selectively because stable and small complexes of CL and ON, called lipoplex, are formed by electrostatic interaction. Although lipoplex could be introduced into cell effectively, the efficiency and selectivity of gene suppression are insufficient. Previously, we reported that high hydrostatic pressurization (HHP) treatment for the complex of CL and plasmid DNA could improve the transfection efficiency. In this study, we investigated antisense-ODNs (AS-ODNs) delivery using lipoplex treated with HHP to inhibit a target gene effectively and selectively. Further, physicochemical properties of the HHP-treated lipoplex were examined in order to elucidate relationship between the structure and the transfection efficiency of the HHP treated lipoplex. Some researchers reported that the supramolecular structure of lipoplex strongly affected the efficiency of transfection. The pressurized lipoplexes with AS-ODNs were added to HEK293 cells expressing luc2 stably. The activity of luciferase was measured by using luminometer. Although the levels of gene suppression were different between AS-ONs used, the efficiency of gene silencing was enhanced by using the HHP-treated lipoplex compared to the nonpressurized lipoplex. For TEM observation, the lamellar structure of the non-treated lipoplexes was observed, whereas the amorphous structure including small and regular structure was observed for Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
the HHP-treated lipoplexes. The hydrophobic microenvironment of lipoplex was investigated by using fluorescent hydrophobic probes, such as Laurdan and pyrene. The fluorescent spectrum and intensity of these probes differed between lipoplexes with/without pressurization. These results suggest that the structure of lipoplex was changed by the HHP treatment. In conclusion, we successfully improved the efficiency of gene suppression by using the HHP treated lipoplex. It was found that the change of the structure of lipoplex was induced by the HHP treatment. We believe that this change of lipoplex structure by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and ON delivery.
435. Aptamer Conjugated Liposomes (Aptamosomes) as an Efficient Detection/Delivery Nano-Vehicle toward Prostate Cancer Cells
Keun-Sik Kim,1 Seho Kim,2 Hyun-Mi Kwon,2,3 Gwang-Hyun Lee,2 Yong Serk Park,4 Dong-Eun Kim.2,3 1 Department of Biomedical Laboratory Science, Konyang University, Daejeon, Korea; 2Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea; 3WCU and BRL Program, Konkuk University, Seoul, Korea; 4Department of Biomedical Labolatory Science, Yonsei University, Wonju, Korea. Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug or gene encapsulated liposomes particles in a cell- or tissue-specific manner. In this study, we report the design of aptamer conjugated POPC (1-parmitonyl-2-oleoyl-sn-glycerol-3phosphocholine)-liposomes (aptamosomes) that achieves selective and strong binding of otherwise low-affinity aptamers at physiological conditions. Published PSMA RNA aptamer A9 which was well known PSMA (prostate-specific membrane antigen) specific RNA sequences was extended for annealing with complementary DNA linker which was labeled with thiol group and FITC. The modified PSMA aptamers were conjugated with maleimide group on the surface of PEGylated micelles. Specifically, we prepared the PSMA-specific aptamosomes by a new post-insertion method. These nanoparticle-aptamosomes of the nano size (90∼100 nm) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells resulting in significantly enhanced in vitro cellular binding as compared with nontargeted nanoparticles that lack the PSMA aptamer. The doxorubicin (Dox)encapsulated aptamosomes were significantly more cytotoxic towered targeted LNCaP cells than the nontargeted PC3 and A549 cells. The FITC-labeled siRNA encapsulated aptamosomes also exhibited remarkable transfection efficacy of siRNA into LNCap cells as compared with nontargeted cells (PC3 and A549). Furthermore, we are currently preparing for the Gd- aptamosomes containing 10 mol % Gadolinium-phosphatidylethanolamine (Gd-DTPA-PE) as MRI agents for MR imaging to LNCaP tumor in LNCaP xenograft nude mouse. These results will show our PSMA-aptamosome has high potential for in vivo imaging of prostate cancer cell recognition and for in vivo drug or gene delivery application.
436. Dual pH Response Nanoparticles for Gene Delivery
Jose M. Morachis,1 Enas A. Mahmoud,1 Jagadis Sankaranarayanan,1 Jenny Huang,1 Adah Almutairi.1,2 1 Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA; 2Materials Science and Engineering, University of California at San Diego, La Jolla, CA.
Emerging gene delivery tools offer novel therapeutic approaches to address several types of diseases including progeria, cystic fibrosis, parkinsons, and multiple types of cancers. We investigated a novel gene delivery system using logic gate nanoparticles developed with a dual pH responsive random co-polymer (poly-ß-aminoester ketal-2). S167
DNA VECTOROLOGY & GENE TARGETING - I The random co-polymer is unique because the amine backbone undergoes a sharp hydrophobic-hydrophilic switch at acidic pH. This leads to an increase in uptake of water (bulk dissolution) and hence an increase in ketal hydrolysis (surface and bulk degradation). We hypothesized that these nanoparticles would be able to increase gene delivery and expression efficiency by providing effective endosomal release and expression of DNA due to its ability to quickly “burst” and release the DNA content into the cytoplasm. Passive nanoparticle entry into cells was demonstrated by delivering Cy-5 labeled pDNA econding EGFP into HCT116, a colon carcinoma cell line. Flow cytometry analysis showed cells to be positive for Cy5DNA-nanoparticles and later produced EGFP expression superior to PLGA nanoparticles. Inhibition of VATPases using bafilomycin A1 demonstrates that expression of EGFP is dependent on the “proton sponge effect”. Further experiments are underway to test if siRNA can also be encapsulated and delivered for fast release and gene knockdown. The advanced dual response nanoparticles offer new therapeutic possibilities and can be combined with cell type specific peptides for improved cellular entry and specificity.
437. The Interaction between SP100 and Rep78 Inhibits Rep-Dependent Integration and Mediates SP100 Degradation
Jinzhong Chen,1 Yuan-Yuan Xue,1 Ran Wang,1 Yang-bo Yue,1 Jinglun Xue.1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China. The adeno-associated virus type 2 (AAV-2) Rep78 is a multifunctional protein that is required for viral transcription, replication, and sitespecific integration. Rep-dependent integration is not related to any known disease; therefore, this type of integration may be a potential tool for carrying therapeutic genes to the human genome. However, the intracellular process of Rep-dependent integration is still unclear. As a result, improving the Rep-dependent integration system is difficult. In the present study, we revealed the interaction between Rep78 and the cellular protein SP100, a member of the PML nuclear bodies(PML-NBs). SP100 and Rep78 are located in the nucleolus, which provides the spatial possibility for their interaction. In addition, Rep78 was shown to co-immunoprecipitate with the EGFP-SP100 fusion protein but not with EGFP, which provided evidence for the interaction between SP100 and Rep78. A knock-down of the endogenous SP100 expression using siRNA resulted in an increased efficiency of the Rep-dependent integration.
Conversely, the transient expression of Rep78 resulted in an increased degradation of cellular SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome.
These results have furthered our knowledge about Rep78 and the cellular protein SP100. These results may also lead to an improvement in the Rep78-related transgene method and in the selection of target cells for gene therapy.
438. Rep of AAV Protects p53 from E1B Mediated p53 Nucleolus Egress and Degradation Xu Wang,1 Jing-lun Xue,1 Wenjuan Li,1 Jinzhong Chen,1 Jinjin Wang.1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
Adenoviral E1B mediated P53 degradation is key step of adenoviral early process. It also involved in regulating AAV infection cycle. We identified a C terminal fragment of rep78 is a p53 binding domain by protein array. The result was further verified by coIP assay, rep78 but not rep68 binds to p53. As a p53 binding protein, rep78 protects p53 from E1B mediated protein degradation.
S168
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy