- Email: [email protected]
502. A Novel Viral-Mimetic Vector for HER2 Targeted Gene Therapy
Nonviral Gene Transfer - Targeting Strategies that can encapsulate plasmid DNA for targeted delivery to cancer cells. A poly DL-lactide co-glycolide (PLGA) nanoparticulate system (PLGA-NP) conjugated with transferrin (Tf) ligand was used as a delivery vector in lung cancer cell lines. The Tf-conjugated PLGANP contained a plasmid DNA encoding the Flt-23K intraceptor, a recombinant construct of the vascular endothelial growth factor receptor-1 (VEGFR-1) binding domains coupled with an endoplasmic reticulum (ER) retention signal. The Flt-23K intraceptor sequesters VEGF in the ER of tumor cells and facilitates its degradation resulting in downregulation of VEGF expression in tumor cells and inhibition of VEGF-mediated tumor angiogenesis. Transfection of transferrin positive A549 cells with Tf-PLGA-NP-Flt23K nanoparticles resulted in inhibition of VEGF in a time and dose-dependent manner as evidenced by Western blotting and enzyme-linked immunoassay (ELISA). Inhibition of VEGF was not observed in cells that were untreated or treated with control nanoparticles. We next determined the effects of VEGF-reduced conditioned media collected from Tf-PLGA-NP-Flt23K nanoparticles-treated A549 cells on human umbilical vein endothelial cell (HUVEC) proliferation. A significant (P<0.05) inhibition of HUVEC cell proliferation associated with activation of caspase-3 was observed when treated with conditioned medium from Tf-PLGA-NP-Flt23K nanoparticles-treated A549 cells compared to the inhibitory effects produced when treated with conditioned medium from untreated or control nanoparticle-treated A549 cells. Our in vitro studies show Tf-PLGA-Flt23K intraceptor nanoparticles can effectively inhibit VEGF and endothelial cell proliferation and warrant further testing in in vivo tumor xenograft models.
500. Introducing Multivalent Binding to NonViral Vectors Quinn K. T. Ng,1 Tatiana Segura.1 Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA.
1
Non-viral gene delivery has been widely investigated over the past decade as a means to guide tissue regeneration, treat disease and study gene function. However, low efficiencies of gene transfer and inability to target desired cell populations have limited the use of this approach. Although current non-viral delivery approaches utilize receptors that are uniquely express at the cell surface for targeting and to enhance gene transfer, strategies that target the density of receptors at the cell surface have not been explored. We believe that by targeting multiple ligands at the cell surface simultaneously (multivalent binding), we can enhance targeting of the desired cell type and enhance gene transfer through the engagement of biological pathways that are unique to clustered receptors. Interestingly adenovirus 2 and 12 utilize this approach to enhance cellular internalization through proteins at their surface that contain spatially constrained integrin binding peptides that can interact with multiple integrin receptors simultaneously. In our present studies, we investigated the effect of spatially constrained Arg-Gly-Asp (RGD) peptides on the surface of DNA/polyethylene imine (PEI) polyplexes on the efficiency of non-viral gene transfer. RGD peptides were spatially constrained through immobilizing them to nanoparticles (nano-RGD). The resulting nano-RGD nanoparticles were used to decorate DNA/PEI polyplexes such that the polyplexes contain nanoparticles at their surface. We synthesized 5-nm nanoRGDs with a theoretical density of 1.5 (low), 15 (med) or 30 (high) RGD peptides per particle. The nano-RGD nanoparticles were either covalently or electrostatically immobilized to the surface DNA/PEI polyplexes and the resulting nano-RGD modified polyplexes were used to transfect HeLa cells in the presence of 10% serum. We found that polyplexes that were modified with nano-RGD med resulted in a 5.4-fold increase of non-viral gene transfer over unmodified polyplexes or polyplexes modified with nano-RGD low or nanoRGD high, indicating that the presentation of the RGD peptides on S188
the surface of DNA/PEI polyplexes affects their ability to enhance gene transfer. Further, nano-RGD was able to enhance gene transfer only when the nanoparticles were covalently bound to the DNA/ PEI polyplexes, with polyplexes modified with nano-RGD through electrostatic interactions achieving no enhancement. Interestingly, experiments exploring the concentration of nano-RGDs that enhance gene transfer resulted in an optimal concentration of nano-RGD to enhance gene transfer, indicating that not only the presentation, but also density of RGD peptides affects gene transfer. Our goal is to explore the use of a non-viral gene delivery strategy that can engage multiple integrin receptors simultaneously to result in more effective non-viral gene delivery vectors.
501. Novel Receptor-Targeted Nanoparticles for Delivery and Magnetic Resonance Imaging of Gene Therapy In Vivo
Michele J. Writer,1 Panagiotis Kyrtatos,2 Pauliina Lehtolainen,2 Mark F. Lythgoe,2 Stephen L. Hart.1 1 Molecular Immunology Unit, Institute of Child Health, London, United Kingdom; 2Centre for Advanced Biomedical Imaging, UCL, London, United Kingdom.
The ability to non-invasively visualize vector distribution in vivo by techniques such as magnetic resonance imaging (MRI) is becoming increasingly important for the development and optimization of therapeutic gene delivery formulations. Non-viral formulations are particularly well suited to the development of such reagents. We are developing a novel synthetic gene delivery formulation that allows real time imaging of gene transfer in the brain by MRI. The vector comprises a cationic liposome (DOTAP/DOPE) formulated with a lipid chelating the MRI contrast agent gadolinium (Gd3+), a peptide with an oligolysine DNA-binding region and receptor-binding motif (LPHKSMP), and plasmid DNA, which self assemble to form receptor-targeted nanoparticles on mixing (RTNs). Nanoparticle formulations of these components were optimised and characterised for particle size, and found to form stable particles of about 60 nm by dynamic light scattering, a size ideally suited for diffusion through tissue. In transfections of U87-MG human glioblastoma cells we found that the targeting peptide doubled the efficiency of transfection with a luciferase reporter gene (RLU/mg) compared with a particle where a non-targeting sequence was used, providing a strong indication of targeting specificity. The incorporation of Gd into the nanoparticle did not significantly affect the transfection efficiency, and toxicity of complexes as determined by an MTT assay was minimal indicating their biocompatibility. MRI investigation of RTN-transfected U87-MG cell pellets showed significant contrast enhancement with Gd-containing complexes, which was greater in cells transfected with targeted compared to non-targeted particles. A comparison of cells incubated with Gd+ve complexes at 4oC, where cell entry is limited, compared to cells where the incubation was performed at 37oC, allowing cell uptake, showed a marked increase in contrast enhancement upon internalisation. This suggested that the increased MRI signal was associated with internalisation and disassembly of the nanoparticles within the cell. This effect may increase the contrast between transfected cells and background signal from free vector. In summary we have described a new non-viral vector formulation for real time imaging of gene delivery.
502. A Novel Viral-Mimetic Vector for HER2 Targeted Gene Therapy Yuhua Wang,1 Brenda F. Canine,1 Arash Hatefi.1 1 Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.
Background: For systemically administered therapeutic genes to successfully reach the target tumor cells, a carrier (vector) should be Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Nonviral Gene Transfer - Targeting Strategies designed to overcome cellular barriers. We have genetically engineered a non-viral vecto composed of: a) a multimerized histone-based peptide (HP) to condense pDNA into nanosize particles, b) a HER2 targeting peptide (TP) to target cancer cells over-expressing HER2, and c) a fusogenic peptide, namely GALA to disrupt endosomes and facilitate escape of cargo into cytosol. To simplify, the vector will be shown as GALA-HP-TP, or in short GHT. We hypothesized that the designed gene delivery system is able to: a) condense pDNA into nanoparticles, b) target cancer cells overexpressing HER2, c) disrupt endosomes, and d) transfect efficiently. Methods: Cloning, Expression and Purification: The GHT gene was synthesized, cloned into pET28b vector , transformed into BL21 pLysS cells, and induced by IPTG. The expressed vector was purified by Ni-NTA column chromatography. The purity and expression of the vector was confirmed by SDS-PAGE, western blot, and MALDI-TOF analysis respectively. Particle Characterization: GHT vector was used to condense pDNA and the mean hydrodynamic size of vector/pDNA complexes was determined by Photon Correlation Spectroscopy. Gel Retardation Assay: Complexes were formed at different N/P ratio and electrophoresed on an agarose gel and visualized by ethidium bromide staining. Cell culture and transfection: SK-OV-3 (HER2 positive) or MDA-MB-231 (HER2 negative) cells were seeded and transfected with vector/pEGFP complexes in the presence of serum. The green fluorescent protein was visualized using an epifluorescent microscope and percent transfected cells was determined by a flow cytometer. Results: SDS-PAGE and western blot analysis showed that GHT was expressed and the purity was estimated to be >95%. MALDI-TOF analysis also confirmed the molecular weight of the expressed vector. GHT vector was then used to complex with pDNA and the size of the particles was determined at best to be ca. 90nm. The gel retardation assay indicated that the vector does interact with pDNA retarding its migration in a dose dependent manner and at N/P ratio 1:1 the pDNA is fully neutralized. In the transfection assay, vector without GALA, namely HT, was used as a control to test the efficacy of GALA. pEGFP was complexed with GHT or HT and used to transfect SKOV-3 cells in the presence of chloroquine. The transfection mediated by GHT was significantly higher than HT. To examine the ability of the GHT in targeting HER2, SK-OV-3 and MB-231 cells were transfected with vector/pEGFP complexes. Almost no transfection was observed in MB-231 cells while significant numbers of SKOV-3 cells were transfected. Conclusion: In conclusion, it was demonstrated that GHT vector was able to condense pDNA into nanoparticles, promote endosome disruption, and target cancer cells overexpressing HER2.
503. Recombinant Lipoprotein Particles for Liver-Targeting Delivery of Polynucleotides
Jinliang Peng, Mengjie Rui, Yuhong Xu. 1 Shanghai Centre for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China; 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; 3School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. RNA interference (RNAi) technology has emerged as a potentially useful method for developing specific gene-silencing therapeutics, but the delivery obstacle is still the greatest barrier for siRNA based gene therapy. In this paper, we constructed a new recombinant lipoprotein particle that consisted of the Apo A-I lipoproteins and cationic lipids. The particles had small particle sizes (about 10nm diameter in size) and positive surface potential based on PCS measurements. Both plasmid DNA and RNAi molecules were loaded onto the particles via charge interactions. The resulted complexes were about 190nm diameter in size. Since ApoA-I lipoprotein can interact with the scavenger receptor SR-BI on the surface of hepatocytes, we would like to use such a system for the targeted delivery of gene therapeutics to the liver for therapeutic applications. Specifically, siRNA molecules Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
designed to inhibit HBV antigen expression will be used to study the delivery efficiency and efficacy in vivo.
504. Analysis of a Novel Folate Conjugated PEI Polyplex as a Potential Gene Transfer Agent Targeting Lung Epithelial Cells
David J. Lea-Smith,1,3 Asier Unciti-Broceta,2 Ian Anderson,1,3 Javier Parra-Leiton,1,3 David J. Porteous,1,3 Mark Bradley,2 Christopher Boyd.1,3 1 Medical Genetics, Molecular Medicine Centre, Edinburgh University, Edinburgh, United Kingdom; 2School of Chemistry, Edinburgh University, Edinburgh, United Kingdom; 3UK Cystic Fibrosis Gene Therapy Consortium, ., United Kingdom. Folate conjugated polyplexes represent promising vectors in gene therapy directed against cells expressing high levels of folate receptor α (FRα). Important clinical targets include many varieties of cancer and lung epithelial cells, which have recently been demonstrated to express FRα on the apical surface. The aim of this study was to test the efficacy and toxicity of a novel PEI-folate conjugate in two cell lines before further examining its potential for gene delivery as a treatment for cystic fibrosis. The procedure for the covalent conjugation between folic acid and PEI is usually achieved by the indiscriminate activation of the α and/or γ carboxylic groups from the folate moiety in order to facilitate amide formation with the amino groups of PEI. However, it has been reported that only γ-conjugates are recognized by FRα [Wang, S. et al. (1996), Bioconj Chem. 7:56–62] thus performing endocytosis. We conjugated instead PEI and folate through the amino group in the 2 position of the pteridine moiety of folate, a strategy that has been shown not to alter the endocytic properties of folate [Bharali, et al. (2005), JACS 127:11364-11371]. To achieve this, the amino group was first coupled with an activated spacer (disuccinimidyl adipate) and, subsequently, the corresponding amount of this activated Folate derivative (see Figure 1) was added to a solution of 25 kDa PEI in DMSO to give the final conjugate.
Figure. Activated Folate derivative. This polyplex was tested in HeLa and A549 cells, respectively expressing high and low levels of FRα. Transfection levels were significantly higher in HeLa cells. Competition with free folate reduced transfection to A549 levels, suggesting that the polyplex enters the cell via the FRα. This reagent demonstrated low levels of toxicity in both cell lines. Therefore this novel polyplex is a promising agent for in vivo gene transfer to cells expressing FRα. To test this we will assess FRα mediated transfection in primary lung epithelial cells as a stepping stone to in vivo studies.
505. Antitumor Effects in Rat C6 Glioma by Tumor-Targeted Folate-PEG-Grafted-HybranchedPEI Mediated Both Cytosine Deaminase and TRAIL Genes Ying Peng,1 Bing Liang,1 Yi Li,1 Xin-Tao Shuai.2 Department of Neurology, The Second Affiliated Hospital, Guangzhou, Guangdong, China.
1
Combined treatment using nonviral agent-mediated enzyme/ prodrug therapy and immunotherapy has the potential to become a powerful alternative therapy for cancer. A great challenge for gene therapy is to develop a highly efficient gene delivery system with S189
500. Introducing Multivalent Binding to NonViral Vectors Quinn K. T. Ng,1 Tatiana Segura.1 Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA.
1
Non-viral gene delivery has been widely investigated over the past decade as a means to guide tissue regeneration, treat disease and study gene function. However, low efficiencies of gene transfer and inability to target desired cell populations have limited the use of this approach. Although current non-viral delivery approaches utilize receptors that are uniquely express at the cell surface for targeting and to enhance gene transfer, strategies that target the density of receptors at the cell surface have not been explored. We believe that by targeting multiple ligands at the cell surface simultaneously (multivalent binding), we can enhance targeting of the desired cell type and enhance gene transfer through the engagement of biological pathways that are unique to clustered receptors. Interestingly adenovirus 2 and 12 utilize this approach to enhance cellular internalization through proteins at their surface that contain spatially constrained integrin binding peptides that can interact with multiple integrin receptors simultaneously. In our present studies, we investigated the effect of spatially constrained Arg-Gly-Asp (RGD) peptides on the surface of DNA/polyethylene imine (PEI) polyplexes on the efficiency of non-viral gene transfer. RGD peptides were spatially constrained through immobilizing them to nanoparticles (nano-RGD). The resulting nano-RGD nanoparticles were used to decorate DNA/PEI polyplexes such that the polyplexes contain nanoparticles at their surface. We synthesized 5-nm nanoRGDs with a theoretical density of 1.5 (low), 15 (med) or 30 (high) RGD peptides per particle. The nano-RGD nanoparticles were either covalently or electrostatically immobilized to the surface DNA/PEI polyplexes and the resulting nano-RGD modified polyplexes were used to transfect HeLa cells in the presence of 10% serum. We found that polyplexes that were modified with nano-RGD med resulted in a 5.4-fold increase of non-viral gene transfer over unmodified polyplexes or polyplexes modified with nano-RGD low or nanoRGD high, indicating that the presentation of the RGD peptides on S188
the surface of DNA/PEI polyplexes affects their ability to enhance gene transfer. Further, nano-RGD was able to enhance gene transfer only when the nanoparticles were covalently bound to the DNA/ PEI polyplexes, with polyplexes modified with nano-RGD through electrostatic interactions achieving no enhancement. Interestingly, experiments exploring the concentration of nano-RGDs that enhance gene transfer resulted in an optimal concentration of nano-RGD to enhance gene transfer, indicating that not only the presentation, but also density of RGD peptides affects gene transfer. Our goal is to explore the use of a non-viral gene delivery strategy that can engage multiple integrin receptors simultaneously to result in more effective non-viral gene delivery vectors.
501. Novel Receptor-Targeted Nanoparticles for Delivery and Magnetic Resonance Imaging of Gene Therapy In Vivo
Michele J. Writer,1 Panagiotis Kyrtatos,2 Pauliina Lehtolainen,2 Mark F. Lythgoe,2 Stephen L. Hart.1 1 Molecular Immunology Unit, Institute of Child Health, London, United Kingdom; 2Centre for Advanced Biomedical Imaging, UCL, London, United Kingdom.
The ability to non-invasively visualize vector distribution in vivo by techniques such as magnetic resonance imaging (MRI) is becoming increasingly important for the development and optimization of therapeutic gene delivery formulations. Non-viral formulations are particularly well suited to the development of such reagents. We are developing a novel synthetic gene delivery formulation that allows real time imaging of gene transfer in the brain by MRI. The vector comprises a cationic liposome (DOTAP/DOPE) formulated with a lipid chelating the MRI contrast agent gadolinium (Gd3+), a peptide with an oligolysine DNA-binding region and receptor-binding motif (LPHKSMP), and plasmid DNA, which self assemble to form receptor-targeted nanoparticles on mixing (RTNs). Nanoparticle formulations of these components were optimised and characterised for particle size, and found to form stable particles of about 60 nm by dynamic light scattering, a size ideally suited for diffusion through tissue. In transfections of U87-MG human glioblastoma cells we found that the targeting peptide doubled the efficiency of transfection with a luciferase reporter gene (RLU/mg) compared with a particle where a non-targeting sequence was used, providing a strong indication of targeting specificity. The incorporation of Gd into the nanoparticle did not significantly affect the transfection efficiency, and toxicity of complexes as determined by an MTT assay was minimal indicating their biocompatibility. MRI investigation of RTN-transfected U87-MG cell pellets showed significant contrast enhancement with Gd-containing complexes, which was greater in cells transfected with targeted compared to non-targeted particles. A comparison of cells incubated with Gd+ve complexes at 4oC, where cell entry is limited, compared to cells where the incubation was performed at 37oC, allowing cell uptake, showed a marked increase in contrast enhancement upon internalisation. This suggested that the increased MRI signal was associated with internalisation and disassembly of the nanoparticles within the cell. This effect may increase the contrast between transfected cells and background signal from free vector. In summary we have described a new non-viral vector formulation for real time imaging of gene delivery.
502. A Novel Viral-Mimetic Vector for HER2 Targeted Gene Therapy Yuhua Wang,1 Brenda F. Canine,1 Arash Hatefi.1 1 Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.
Background: For systemically administered therapeutic genes to successfully reach the target tumor cells, a carrier (vector) should be Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Nonviral Gene Transfer - Targeting Strategies designed to overcome cellular barriers. We have genetically engineered a non-viral vecto composed of: a) a multimerized histone-based peptide (HP) to condense pDNA into nanosize particles, b) a HER2 targeting peptide (TP) to target cancer cells over-expressing HER2, and c) a fusogenic peptide, namely GALA to disrupt endosomes and facilitate escape of cargo into cytosol. To simplify, the vector will be shown as GALA-HP-TP, or in short GHT. We hypothesized that the designed gene delivery system is able to: a) condense pDNA into nanoparticles, b) target cancer cells overexpressing HER2, c) disrupt endosomes, and d) transfect efficiently. Methods: Cloning, Expression and Purification: The GHT gene was synthesized, cloned into pET28b vector , transformed into BL21 pLysS cells, and induced by IPTG. The expressed vector was purified by Ni-NTA column chromatography. The purity and expression of the vector was confirmed by SDS-PAGE, western blot, and MALDI-TOF analysis respectively. Particle Characterization: GHT vector was used to condense pDNA and the mean hydrodynamic size of vector/pDNA complexes was determined by Photon Correlation Spectroscopy. Gel Retardation Assay: Complexes were formed at different N/P ratio and electrophoresed on an agarose gel and visualized by ethidium bromide staining. Cell culture and transfection: SK-OV-3 (HER2 positive) or MDA-MB-231 (HER2 negative) cells were seeded and transfected with vector/pEGFP complexes in the presence of serum. The green fluorescent protein was visualized using an epifluorescent microscope and percent transfected cells was determined by a flow cytometer. Results: SDS-PAGE and western blot analysis showed that GHT was expressed and the purity was estimated to be >95%. MALDI-TOF analysis also confirmed the molecular weight of the expressed vector. GHT vector was then used to complex with pDNA and the size of the particles was determined at best to be ca. 90nm. The gel retardation assay indicated that the vector does interact with pDNA retarding its migration in a dose dependent manner and at N/P ratio 1:1 the pDNA is fully neutralized. In the transfection assay, vector without GALA, namely HT, was used as a control to test the efficacy of GALA. pEGFP was complexed with GHT or HT and used to transfect SKOV-3 cells in the presence of chloroquine. The transfection mediated by GHT was significantly higher than HT. To examine the ability of the GHT in targeting HER2, SK-OV-3 and MB-231 cells were transfected with vector/pEGFP complexes. Almost no transfection was observed in MB-231 cells while significant numbers of SKOV-3 cells were transfected. Conclusion: In conclusion, it was demonstrated that GHT vector was able to condense pDNA into nanoparticles, promote endosome disruption, and target cancer cells overexpressing HER2.
503. Recombinant Lipoprotein Particles for Liver-Targeting Delivery of Polynucleotides
Jinliang Peng, Mengjie Rui, Yuhong Xu. 1 Shanghai Centre for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China; 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; 3School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. RNA interference (RNAi) technology has emerged as a potentially useful method for developing specific gene-silencing therapeutics, but the delivery obstacle is still the greatest barrier for siRNA based gene therapy. In this paper, we constructed a new recombinant lipoprotein particle that consisted of the Apo A-I lipoproteins and cationic lipids. The particles had small particle sizes (about 10nm diameter in size) and positive surface potential based on PCS measurements. Both plasmid DNA and RNAi molecules were loaded onto the particles via charge interactions. The resulted complexes were about 190nm diameter in size. Since ApoA-I lipoprotein can interact with the scavenger receptor SR-BI on the surface of hepatocytes, we would like to use such a system for the targeted delivery of gene therapeutics to the liver for therapeutic applications. Specifically, siRNA molecules Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
designed to inhibit HBV antigen expression will be used to study the delivery efficiency and efficacy in vivo.
504. Analysis of a Novel Folate Conjugated PEI Polyplex as a Potential Gene Transfer Agent Targeting Lung Epithelial Cells
David J. Lea-Smith,1,3 Asier Unciti-Broceta,2 Ian Anderson,1,3 Javier Parra-Leiton,1,3 David J. Porteous,1,3 Mark Bradley,2 Christopher Boyd.1,3 1 Medical Genetics, Molecular Medicine Centre, Edinburgh University, Edinburgh, United Kingdom; 2School of Chemistry, Edinburgh University, Edinburgh, United Kingdom; 3UK Cystic Fibrosis Gene Therapy Consortium, ., United Kingdom. Folate conjugated polyplexes represent promising vectors in gene therapy directed against cells expressing high levels of folate receptor α (FRα). Important clinical targets include many varieties of cancer and lung epithelial cells, which have recently been demonstrated to express FRα on the apical surface. The aim of this study was to test the efficacy and toxicity of a novel PEI-folate conjugate in two cell lines before further examining its potential for gene delivery as a treatment for cystic fibrosis. The procedure for the covalent conjugation between folic acid and PEI is usually achieved by the indiscriminate activation of the α and/or γ carboxylic groups from the folate moiety in order to facilitate amide formation with the amino groups of PEI. However, it has been reported that only γ-conjugates are recognized by FRα [Wang, S. et al. (1996), Bioconj Chem. 7:56–62] thus performing endocytosis. We conjugated instead PEI and folate through the amino group in the 2 position of the pteridine moiety of folate, a strategy that has been shown not to alter the endocytic properties of folate [Bharali, et al. (2005), JACS 127:11364-11371]. To achieve this, the amino group was first coupled with an activated spacer (disuccinimidyl adipate) and, subsequently, the corresponding amount of this activated Folate derivative (see Figure 1) was added to a solution of 25 kDa PEI in DMSO to give the final conjugate.
Figure. Activated Folate derivative. This polyplex was tested in HeLa and A549 cells, respectively expressing high and low levels of FRα. Transfection levels were significantly higher in HeLa cells. Competition with free folate reduced transfection to A549 levels, suggesting that the polyplex enters the cell via the FRα. This reagent demonstrated low levels of toxicity in both cell lines. Therefore this novel polyplex is a promising agent for in vivo gene transfer to cells expressing FRα. To test this we will assess FRα mediated transfection in primary lung epithelial cells as a stepping stone to in vivo studies.
505. Antitumor Effects in Rat C6 Glioma by Tumor-Targeted Folate-PEG-Grafted-HybranchedPEI Mediated Both Cytosine Deaminase and TRAIL Genes Ying Peng,1 Bing Liang,1 Yi Li,1 Xin-Tao Shuai.2 Department of Neurology, The Second Affiliated Hospital, Guangzhou, Guangdong, China.
1
Combined treatment using nonviral agent-mediated enzyme/ prodrug therapy and immunotherapy has the potential to become a powerful alternative therapy for cancer. A great challenge for gene therapy is to develop a highly efficient gene delivery system with S189