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333. Acetylation of Microtubules Leads to Greater Cytoplasmic Plasmid Trafficking and Enhanced Transfection Efficiency
DNA Vectorology: Non-Viral Vector Engineering lines; however, canine isolates of the EPO and SEAP genes have not been developed as reporters. Canine EPO was cloned from cDNA, incorporating an amino terminal synthetic graft according to the published sequence. We introduced several point mutations in the cEPO sequence to generate protein that maintained immunoreactivity in an ELISA while reducing biological activity, to avoid inducing erythrocytosis in test animals. Upon further sequence analysis we found that the 5’ end of the reported cEPO coding sequence was in fact an unspliced variant containing 54 bp of intron 1. Exon 1 (missing from the published dog genome sequence) was amplified, cloned and sequenced, allowing us to determine the authentic 5’ terminus and graft this sequence onto the cEPO coding sequence. CMV-regulated expression plasmids and CAGS-regulated SB constructs containing cEPO were tested by hydrodynamic injection into C57BL/6 mice. Plasma contained substantial levels (up to 104 miU/mL) of immunoreactive canine EPO, assayed by ELISA after one day, and blood samples collected one week post-injection showed an increase in hematocrit (up to 73), demonstrating the biological activity of cloned cEPO. An intestinal canine AP was identified by sequence comparison with hSEAP as a likely heat stable and secretable protein. PCR cloning of the canine AP cDNA yielded an interior fragment of 1100 bp. The 5’ end of the coding sequence was synthetically extended by PCR. The 3’ end of the coding sequence was restored through a combination of synthetic extension and PCR cloning of cDNA and genomic DNA (due again to gaps in the published dog genomic sequence). The resulting 3’ end encoded an 18 AA Thr/Arg/ Pro repeat which did not correspond with any known AP sequences. The impact of this repeat region and a truncation required for secretion were tested by electroporation of CMV-regulated constructs in 293T cells. Truncation of the sequence was required for secretion of heatstable AP, while the T/R/P repeat had no effect on cSEAP activity in a chemiluminescent assay. These CMV-cSEAP constructs are currently being tested in vivo in mice for the in vivo utility of this reporter system. Because these cEPO and cSEAP sequences encode non-immunogenic and secretable proteins, we anticipated that they will be highly useful in long term expression studies using the SB transposon system.
333. Acetylation of Microtubules Leads to Greater Cytoplasmic Plasmid Trafficking and Enhanced Transfection Efficiency
Erin E. Vaughan,1,2 Melissa A. Badding,1 Aaron M. Miller,1,2 R. C. Geiger,2,3 David A. Dean.1,2 1 Pediatrics, University of Rochester, Rochester, NY; 2Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL; 3Biomedical Engineering, Florida Gulf Coast University, Ft. Myers, FL. The success of viral and non-viral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm to reach the nucleus. Although much work has been directed at understanding how plasmids enter the nucleus, relatively little is known about trafficking in the cytoplasm. We and others have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules using nocodazole greatly inhibited cytoplasmic plasmid trafficking, it did not abolish it. Assuming that diffusion cannot occur and that the microtubule network was disrupted via drug treatment, we sought to determine how the plasmids were able to reach the nucleus. Interestingly, there is a subset of modified microtubules that remain intact even after nocodazole treatment which are posttranslationally acetylated. Intriguingly, it has been demonstrated recently by others that acetylated microtubules promote binding of the molecular motors kinesin and dynein as well as their transport, suggesting a role for acetylated microtubules in trafficking. To determine whether this modification may account for the residual Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
DNA trafficking in nocodazole-treated cells, we inhibited or knocked down levels of the tubulin deacetylase, HDAC6, thus generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not due to increased transcriptional activity, but rather to increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild type cells. Since it has been shown that motor proteins load more efficiently onto acetylated rather than unmodified microtubules, we acetylated microtubules in vitro and assessed DNA binding in the presence of cell extracts. Plasmids preferentially bound to acetylated microtubules compared to unmodified microtubules. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.
334. Enhancement of Reporter Gene Detection Sensitivity and Therapeutic Gene Function by Insertion of Specific Mini-Peptide-Coding Sequences Jeffry Cutrera, Denada Dibra, Shulin Li. 1 Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA.
Achieving high levels of gene expression is one of the primary limiting factors for successfully treating malignancies with gene therapy, as well as monitoring tumor regression and gene distributions in gene-therapy-treated animals using reporter genes. The conventional approaches for elevating the level of gene expression include enhancing gene delivery efficiency, gene transcriptional level, and gene product stability. Here we report a novel concept to complement the conventional approaches—increasing the activity of the gene product. A group of peptides have been isolated from tumor-targeting phage display studies. It was assumed that these peptides have tumor-targeting properties; however, most of them failed to demonstrate such a function. We hypothesized that these peptides may play a role in stabilizing the gene products rather than targeting them to tissues. To test this hypothesis, we inserted mini-peptide-coding sequences directly before the stop codons in protein-coding plasmids. Based on the in vitro and in vivo reporter gene data, the peptide CWDDWLC increases the enzymatic activity, but not the stability, of Secreted Alkaline Phosphatase (SEAP) more than any other tested peptide. After testing the most promising peptide constructs in several different cell lines, SEAP-CWDDWLC proved to increase the SEAP activity level up to 11 fold compared to the control. The in vivo results show a trend similar to that seen in the in vitro results with a 6-fold increase compared to the control. The western blot analysis revealed that there is no difference in the amount of protein in the serum samples, so the increase in activity is due to an increase in sensitivity of the SEAP-CWDDWLC enzyme and not stability of the protein. For the first time, we show that inserting peptide-coding sequences into reporter gene plasmid DNA can increase the reporter gene activity. Also, inserting the CWDDWLC peptide into Interferon α (IFNα) encoding plasmid DNA increased the biological activity of IFNα as evidenced through flow cytometry by an average 11-fold upregulation of MHC class I expression per unit IFNα compared to unmodified IFNα plasmid DNA. Interestingly, insertion of the peptide into IL-12 and IFNγ plasmids did not alter the biological activity of either cytokine which suggests that this effect is gene-specific rather than universal. In conclusion, inserting the CWDDWLC-coding sequence into SEAP plasmid DNA increases the reporter gene sensitivity which could lead to a broader range of reporter gene applications including large animal models. Likewise, the increase in the biological activity of the IFNα-CWDDWLC could lead to a stronger therapeutic effect. S125
333. Acetylation of Microtubules Leads to Greater Cytoplasmic Plasmid Trafficking and Enhanced Transfection Efficiency
Erin E. Vaughan,1,2 Melissa A. Badding,1 Aaron M. Miller,1,2 R. C. Geiger,2,3 David A. Dean.1,2 1 Pediatrics, University of Rochester, Rochester, NY; 2Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL; 3Biomedical Engineering, Florida Gulf Coast University, Ft. Myers, FL. The success of viral and non-viral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm to reach the nucleus. Although much work has been directed at understanding how plasmids enter the nucleus, relatively little is known about trafficking in the cytoplasm. We and others have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules using nocodazole greatly inhibited cytoplasmic plasmid trafficking, it did not abolish it. Assuming that diffusion cannot occur and that the microtubule network was disrupted via drug treatment, we sought to determine how the plasmids were able to reach the nucleus. Interestingly, there is a subset of modified microtubules that remain intact even after nocodazole treatment which are posttranslationally acetylated. Intriguingly, it has been demonstrated recently by others that acetylated microtubules promote binding of the molecular motors kinesin and dynein as well as their transport, suggesting a role for acetylated microtubules in trafficking. To determine whether this modification may account for the residual Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
DNA trafficking in nocodazole-treated cells, we inhibited or knocked down levels of the tubulin deacetylase, HDAC6, thus generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not due to increased transcriptional activity, but rather to increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild type cells. Since it has been shown that motor proteins load more efficiently onto acetylated rather than unmodified microtubules, we acetylated microtubules in vitro and assessed DNA binding in the presence of cell extracts. Plasmids preferentially bound to acetylated microtubules compared to unmodified microtubules. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.
334. Enhancement of Reporter Gene Detection Sensitivity and Therapeutic Gene Function by Insertion of Specific Mini-Peptide-Coding Sequences Jeffry Cutrera, Denada Dibra, Shulin Li. 1 Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA.
Achieving high levels of gene expression is one of the primary limiting factors for successfully treating malignancies with gene therapy, as well as monitoring tumor regression and gene distributions in gene-therapy-treated animals using reporter genes. The conventional approaches for elevating the level of gene expression include enhancing gene delivery efficiency, gene transcriptional level, and gene product stability. Here we report a novel concept to complement the conventional approaches—increasing the activity of the gene product. A group of peptides have been isolated from tumor-targeting phage display studies. It was assumed that these peptides have tumor-targeting properties; however, most of them failed to demonstrate such a function. We hypothesized that these peptides may play a role in stabilizing the gene products rather than targeting them to tissues. To test this hypothesis, we inserted mini-peptide-coding sequences directly before the stop codons in protein-coding plasmids. Based on the in vitro and in vivo reporter gene data, the peptide CWDDWLC increases the enzymatic activity, but not the stability, of Secreted Alkaline Phosphatase (SEAP) more than any other tested peptide. After testing the most promising peptide constructs in several different cell lines, SEAP-CWDDWLC proved to increase the SEAP activity level up to 11 fold compared to the control. The in vivo results show a trend similar to that seen in the in vitro results with a 6-fold increase compared to the control. The western blot analysis revealed that there is no difference in the amount of protein in the serum samples, so the increase in activity is due to an increase in sensitivity of the SEAP-CWDDWLC enzyme and not stability of the protein. For the first time, we show that inserting peptide-coding sequences into reporter gene plasmid DNA can increase the reporter gene activity. Also, inserting the CWDDWLC peptide into Interferon α (IFNα) encoding plasmid DNA increased the biological activity of IFNα as evidenced through flow cytometry by an average 11-fold upregulation of MHC class I expression per unit IFNα compared to unmodified IFNα plasmid DNA. Interestingly, insertion of the peptide into IL-12 and IFNγ plasmids did not alter the biological activity of either cytokine which suggests that this effect is gene-specific rather than universal. In conclusion, inserting the CWDDWLC-coding sequence into SEAP plasmid DNA increases the reporter gene sensitivity which could lead to a broader range of reporter gene applications including large animal models. Likewise, the increase in the biological activity of the IFNα-CWDDWLC could lead to a stronger therapeutic effect. S125