- Email: [email protected]
141. A Rapid, PCR-Based Method for Direct In-Vitro Cloning into Recombinant Adenoviral Vectors
ADENOVIRUS AND OTHER DNA VIRUS VECTORS I with the addition of antibody. This cell line has lower levels of αvβ3 than OSW. This data suggests that, while these vectors may be able to utilize alternative receptors with the potential to bypass the normal Ad entry mechanisms, they may continue to utilize integrin binding as at least a portion of their entry mechanism. Additionally, stimulation of canine lymphoma cells with appropriate mitogens may enhance transduction by retargeted virus in a CAR-independent manner. Thus, integrin expression remains critical to this process.
139. Simultaneous Genetic Peptide Targeting of Adenovirus Subtype 5 towards Two Different Receptors
Michael Behr,1,2 Maximilian Richter,1,2 Martin A. Häusl,3 Philipp Fieger,1,2 Sarah Engelhardt,1,2 Carolin Stegmüller,1,2 Alexander H. Enk,2 Anja Ehrhardt,3 Dirk M. Nettelbeck.1,2 1 Helmholtz-University Group Oncolytic Adenoviruses, German Cancer Research Center, Heidelberg, Germany; 2Dept. of Dermatology, Heidelberg University Hospital, Heidelberg, Germany; 3Max von Pettenkofer-Institute, München, Germany. Adenoviral oncolysis is a promising new modality for the treatment of cancer. Safety and proof-of-principle of oncolytic adenoviruses were shown in clinical trials, but lack until now therapeutic efficiency. Perhaps one of the most important limitations is the poor efficiency and specificity of cell entry. We genetically incorporated small peptide ligands with specificity towards receptors which are preferentially overexpressed on cancer cells into different loops of a chimeric fiber, containing knob- and shaft-domains of the Ad41 small fiber gene fused to the Ad5 fiber tail-domain. We have previously shown that this fiber format is receptor blind and serves as a detargeted scaffold for the insertion of targeting ligands. The retargeted chimeric fibers were incorporated in a genomically fiberless Ad5 reporter virus using an established transient transfection/infection system. De- and retargeting capacity of the chimeric fibers were analysed in vitro. We found, that the published YSA-peptide, incorporated in our chimeric fiber, is capable to mediate specific transduction of cells expressing the receptor tyrosine kinase EphA2, which is present on tumor endothelium and overexpressed on a large panel of cancer cells. However, not all tumor cells within a tumor have the same expression profile of individual receptors. In consequence tumor cells lacking the targeting receptor cannot be efficiently transduced. To overcome this possible limitation we incorporated simultaneously besides the YSA-peptide further ligands into other positions of the Ad41 short fiber knob. On the one hand we combined the YSA-peptide with the well characterized RGD-peptide that targets integrins. On the other hand we combined the YSA-peptide with a transferrin receptor targeting ligand successfully. Resulting fibers were able to trimerize and were incorporated into the genomically fiberless Ad5 reporter viruses. Due to the presence of the additional ligand, these viruses were able to transduce EphA2-negative cancer cells in vitro efficiently. Currently we are generating Ad5 based reporter viruses with genomically modified chimeric fiber genes carrying the most promising ligand combinations. Towards this end we are using a versatile cloning strategy based on a BAC-recombineering system that allows for rapid modifications of the Ad genome.In conclusion, our data indicate that targeting towards two different receptors with a single agent is possible via genetic insertion of peptide ligands, opening new avenues for therapeutic applications.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
140. Augmentation of Rat Skin Flap Viability by Relaxin-Expressing Adenovirus
Il-Kyu Choi,1,3 Won Jai Lee,2 In Sik Yun,2 Yong-Oock Kim,2 Tae Jin Yun,1 Dong Kyun Rah,2 Chae-Ok Yun.1,3 1 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Severance Biomedical Science Institute, Seoul, Republic of Korea; 2Institute for Human Tissue Restoration, Department of Plastic & Reconstructive Surgery, Seoul, Republic of Korea; 3 KOSEF through National Core Research Center for Nanomedical Technology, Seoul, Republic of Korea. Relaxin (RLX) has multiple vascular actions such as vasodilatation, angiogenesis, and production of a vascular endothelial growth factor (VEGF). We have generated a RLX-expressing (dE1-RGD/lacZ/ RLX) adenovirus, and investigated whether or not it enhances the skin flap survival. A total of 30 Sprangue-Dawley rats were divided into three groups: RLX-expressing adenovirus group, control virus group, and PBS group. On the distally based flap of 3×9 cm in size, it was subdermally injected with the dE1-RGD/lacZ/RLX (107 PFU), dE1-RGD/lacZ virus, and PBS, which were administered 48 hours prior and immediately before flap elevation. A survival area of a flap and the amount of blood flow were measured. On postoperative day 10, the CD31 positively stained vessels and VEGF protein expression were examined. There was a significant increase in the survival area of the flap in the RLX group. The Doppler measurement also showed significantly increased blood flow immediately after the operation and on postoperative days (day) 7 and 10. The CD31 (the number of the CD31) positively stained vessels and VEGF protein expression were significantly larger (increased) in the RLX group. Administrated RLX-expressing adenovirus into the elevated skin flaps increased VEGF expression, number of capillaries, and blood flow to the flap, thereby improving the skin flap survival.
141. A Rapid, PCR-Based Method for Direct InVitro Cloning into Recombinant Adenoviral Vectors
Thomas P. Quinn,1 Lily Lee,1 Mei Fong,1 Michael Haugwitz,1 Hiroaki Sagawa.1 1 Clontech Laboratories Inc., A Takara Bio Company, Mountain View, CA.
Adenoviral vectors are widely accepted for gene delivery because they can be cultured to high titers and efficiently transduce a wide variety of cell types in vitro and in vivo. Previous methods of construction have been troubled by inefficient recombination efficiencies, the need for shuttle vectors, plaque purifications, and labor intensive methods. Here, we present a simple and more streamlined method to produce recombinant adenoviral constructs using a 30 minute, ligation-independent and sequence-independent reaction to seamlessly and directionally clone PCR fragments directly into a linearized adenoviral vector. Moreover, no additional treatment of the PCR fragment is required, such as restriction enzyme digestion, phosphorylation, or blunt-end polishing. This rapid approach requires only 3 days to generate a transfectable adenoviral vector compared to the 6 to 8 days needed by other cloning methods that use a donor or shuttle vector. Using an I-Ceu I and PI-Sce I linearized adenoviral backbone with numerous PCR amplimers, we observed >80% cloning efficiency and nearly 100% cloning success with little or no background as measured by colony PCR screening and endonuclease restriction analysis. We also observed strong correlation between PCR positive clones and intact adenoviral DNA suggesting that the vectors remained stable throughout the cloning process, showing minimal rearrangements. Additionally, we sequenced clones to confirm both the insert orientation and the integrity of cloning junctions and found that 100% were in proper orientation with no sequence changes at the junctions. The flexibility of the method was confirmed by cloning into numerous vector formats including constitutive and promoterless types both with and without fluorescent proteins expressed from S55
ADENOVIRUS AND OTHER DNA VIRUS VECTORS I the E3 region. To demonstrate complete functionality, we cloned several cDNAs into an adenoviral vector containing the complete 3rd generation tetracycline-inducible expression system that was optimized for low basal and high maximal expression. These Pac I linearized recombinant adenoviral vectors were efficiently transfected into 293 cells using a biodegradable polymer and an optimized 4hr protocol. Rescue was deemed complete when cytopathic effect was observed 7 days following transfection. Adenoviral stocks produced from these vectors, were able to produce high titer virus (1e9 IFU/ ml) and achieve induced expression levels that were >1300 fold over the uninduced state in HeLa cells. We believe this cloning system will ultimately increase the utility of adenoviral vectors by allowing rapid, accurate, and directional high-throughput cloning, further facilitating the production of functional adenoviral stocks for gene therapy development and gene function studies.
142. Differential Effects of Murine and Human Factor X on Adenovirus Transduction Via Cell Surface Heparan Sulfate
Anne K. Zaiss,1,2 Roger Lawrence,3 David Elashoff,4 Jeffrey D. Esko,3 Harvey R. Herschman.1,2 1 Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 2Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 3Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, CA; 4Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA. Serum factor X (FX) is proposed to play a major role in Adenovirus tropism, binding to the virus hexon protein and promoting transduction by bridging the virus to cell surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction of an Adenovirus vector 2-4 fold in murine hepatocytelike cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary cells. mFX was not only unable to promote transduction in these cells; mFX competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggest increased transduction by both mFX and hFX in hepatocyte-derived cells and increased transduction mediated by hFX in epithelial cells were dependent on HSPGs. Hepatocyte-derived cell lines express substantially more HSPGs than the cancer cell lines studied. Dose response curves suggest that Ad:hFX has a greater affinity for HSPGs than does Ad:mFX. In coagulation factor-depleted mice, hFX showed a similar enhanced ability, compared to mFX, to reconstitute hepatic Adenovirus transduction. These results suggest that differences in Ad:hFX and Ad:mFX affinity to HSPGs might result in differences in their ability to enhance Adenovirus transduction of many cell types. These findings may have implications for murine models of adenovirus vector targeting.
143. Improved Tropism of Adenoviral Vectors for Muscle Cells Using an IGF-1R Targeted Bispecific Ligand
Maxime Pinard,1,2 Birgit Kastberger,1 Yue Zeng,1 Nancy Larochelle,3 Josephine Nalbantoglu,3 Bernard Massie,1,2 Rénald Gilbert.1,3 1 Biotechnology Research Institute, Montreal, Canada; 2University of Montreal, Montreal, Canada; 3Montreal Neurological Institute, Montreal, Canada. Adenoviral vector (AdV) is being intensively investigated as a tool to deliver exogenous sequences to different tissues in order to correct S56
multiple genetic disorders. Limited expression of the Coxsackie and Adenovirus Receptor (CAR), the primary receptor for AdV, has been a major drawback in gene transfer approaches. To compensate for the paucity of CAR, multiple strategies have been proposed. Some of them consist to alter the AdV fiber to enable recognition of new receptors at the cellular surface, such as PEGylation or using fibers derived from other serotypes. Our group has developed a bispecific ligand approach coupled with Ad5 fiber modification. The interaction between the Ad5 and the ligand is generated by using an artificial interaction domain, the E-Coil/K-Coil. The K-Coil is inserted within the HI loop domain of the Ad5 fiber and its counter part, the E-Coil, is fused with a ligand of interest. The insulin-like growth factor-1, (IGF-1) recognizes the IGF-1 receptor (IGF-1R) expresses on many cell lines such as muscle. Indeed, we demonstrated expression of IGF-1R by western blot in C2C12 myoblasts and in myotubes. We also showed that it was expressed on normal and dystrophin deficient (mdx) mice by immunochemistry and by western blot. Therefore, to increase the transduction efficacy of AdV for muscle we constructed a bispecific ligand (IGF-E5) made of IGF-1 fused to five repeats of the E-Coil and to a His-Tag for protein purification. Binding of IGF-E5 to the fiber of an AdV encoding GFP and containing the K-Coil within the HI-loop (Ad5HIK5cDm/GFP) was shown by ELISA. Using flow cytometry we also demonstrated that IGF-E5 could increase the transduction efficacy of Ad5HIK5cDm/GFP by 16-fold in myoblasts. In the absence of ligand, the transduction efficacy of Ad5HIK5cDm/GFP was comparable to an AdV with wild type fiber. Importantly, incubation with recombinant IGF-1 did not increase the transduction of Ad5HIK5cDm/GFP in myoblasts. A 6-fold increase of GFP expression was also measured by western blot when myotubes were transduced at a MOI of 300 with Ad5HIK5cDm/GFP associated with IGF-E5.
144. An Alternate Method for Efficient Delivery of Catalyzing Enzymes
Andrew D. Fontes,1 Rene Quintanilla,1 Michael Poderycki,2 Jon Chesnut,2 Uma Lakshmipathy.1 1 Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA; 2Global Science and Innovation Office, Life Technologies, Carlsbad, CA.
We had earlier reported the development of integrase-based JumpIn platform for site-specific integration and stable expression of transgenes in human embryonic stem cells. This method utilizes vectors with special integrase recognizing sequences that facilitate the insertion of the construct carrying the transgene into the host genome catalyzed by the expression of the appropriate enzyme. Current methods utilize cotransfection of the integrational plasmid carrying the transgene of interest with the appropriate catalyzing enzyme expression vector. To overcome any adverse effect resulting from random integration of the catalyzing enzyme expression vectors into the host genome, we utilized a non-integrating transient expression system for delivery. BacMam, a baculovirus based gene delivery method is known to facilitate high efficiency gene transfer into hard-to-transfect cell types such as primary and stem cells. PhiC31 Integrase, R4 Integrase and NLS-Cre Recombinase were cloned into the BacMam vector and high titer virus generated. Expression of the Integrases and Recombinase was confirmed by qRTPCR or immunochemical analysis of BacMam transduced HEK293 cells. Functionality was evaluated by the emergence of cells expressing a visual marker signifying successful reaction by the introduced enzymes. Our results indicate that catalyzing enzymes can be delivered at high efficiency via BacMam and the frequency of obtaining genetically modified cells is comparable or better than reaction catalyzed with plasmid DNA. Given the efficiency of BacMam to facilitate high efficiency gene transfer into adult stem cells such as mesenchymal stem cells and neural stem cells that are Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
139. Simultaneous Genetic Peptide Targeting of Adenovirus Subtype 5 towards Two Different Receptors
Michael Behr,1,2 Maximilian Richter,1,2 Martin A. Häusl,3 Philipp Fieger,1,2 Sarah Engelhardt,1,2 Carolin Stegmüller,1,2 Alexander H. Enk,2 Anja Ehrhardt,3 Dirk M. Nettelbeck.1,2 1 Helmholtz-University Group Oncolytic Adenoviruses, German Cancer Research Center, Heidelberg, Germany; 2Dept. of Dermatology, Heidelberg University Hospital, Heidelberg, Germany; 3Max von Pettenkofer-Institute, München, Germany. Adenoviral oncolysis is a promising new modality for the treatment of cancer. Safety and proof-of-principle of oncolytic adenoviruses were shown in clinical trials, but lack until now therapeutic efficiency. Perhaps one of the most important limitations is the poor efficiency and specificity of cell entry. We genetically incorporated small peptide ligands with specificity towards receptors which are preferentially overexpressed on cancer cells into different loops of a chimeric fiber, containing knob- and shaft-domains of the Ad41 small fiber gene fused to the Ad5 fiber tail-domain. We have previously shown that this fiber format is receptor blind and serves as a detargeted scaffold for the insertion of targeting ligands. The retargeted chimeric fibers were incorporated in a genomically fiberless Ad5 reporter virus using an established transient transfection/infection system. De- and retargeting capacity of the chimeric fibers were analysed in vitro. We found, that the published YSA-peptide, incorporated in our chimeric fiber, is capable to mediate specific transduction of cells expressing the receptor tyrosine kinase EphA2, which is present on tumor endothelium and overexpressed on a large panel of cancer cells. However, not all tumor cells within a tumor have the same expression profile of individual receptors. In consequence tumor cells lacking the targeting receptor cannot be efficiently transduced. To overcome this possible limitation we incorporated simultaneously besides the YSA-peptide further ligands into other positions of the Ad41 short fiber knob. On the one hand we combined the YSA-peptide with the well characterized RGD-peptide that targets integrins. On the other hand we combined the YSA-peptide with a transferrin receptor targeting ligand successfully. Resulting fibers were able to trimerize and were incorporated into the genomically fiberless Ad5 reporter viruses. Due to the presence of the additional ligand, these viruses were able to transduce EphA2-negative cancer cells in vitro efficiently. Currently we are generating Ad5 based reporter viruses with genomically modified chimeric fiber genes carrying the most promising ligand combinations. Towards this end we are using a versatile cloning strategy based on a BAC-recombineering system that allows for rapid modifications of the Ad genome.In conclusion, our data indicate that targeting towards two different receptors with a single agent is possible via genetic insertion of peptide ligands, opening new avenues for therapeutic applications.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
140. Augmentation of Rat Skin Flap Viability by Relaxin-Expressing Adenovirus
Il-Kyu Choi,1,3 Won Jai Lee,2 In Sik Yun,2 Yong-Oock Kim,2 Tae Jin Yun,1 Dong Kyun Rah,2 Chae-Ok Yun.1,3 1 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Severance Biomedical Science Institute, Seoul, Republic of Korea; 2Institute for Human Tissue Restoration, Department of Plastic & Reconstructive Surgery, Seoul, Republic of Korea; 3 KOSEF through National Core Research Center for Nanomedical Technology, Seoul, Republic of Korea. Relaxin (RLX) has multiple vascular actions such as vasodilatation, angiogenesis, and production of a vascular endothelial growth factor (VEGF). We have generated a RLX-expressing (dE1-RGD/lacZ/ RLX) adenovirus, and investigated whether or not it enhances the skin flap survival. A total of 30 Sprangue-Dawley rats were divided into three groups: RLX-expressing adenovirus group, control virus group, and PBS group. On the distally based flap of 3×9 cm in size, it was subdermally injected with the dE1-RGD/lacZ/RLX (107 PFU), dE1-RGD/lacZ virus, and PBS, which were administered 48 hours prior and immediately before flap elevation. A survival area of a flap and the amount of blood flow were measured. On postoperative day 10, the CD31 positively stained vessels and VEGF protein expression were examined. There was a significant increase in the survival area of the flap in the RLX group. The Doppler measurement also showed significantly increased blood flow immediately after the operation and on postoperative days (day) 7 and 10. The CD31 (the number of the CD31) positively stained vessels and VEGF protein expression were significantly larger (increased) in the RLX group. Administrated RLX-expressing adenovirus into the elevated skin flaps increased VEGF expression, number of capillaries, and blood flow to the flap, thereby improving the skin flap survival.
141. A Rapid, PCR-Based Method for Direct InVitro Cloning into Recombinant Adenoviral Vectors
Thomas P. Quinn,1 Lily Lee,1 Mei Fong,1 Michael Haugwitz,1 Hiroaki Sagawa.1 1 Clontech Laboratories Inc., A Takara Bio Company, Mountain View, CA.
Adenoviral vectors are widely accepted for gene delivery because they can be cultured to high titers and efficiently transduce a wide variety of cell types in vitro and in vivo. Previous methods of construction have been troubled by inefficient recombination efficiencies, the need for shuttle vectors, plaque purifications, and labor intensive methods. Here, we present a simple and more streamlined method to produce recombinant adenoviral constructs using a 30 minute, ligation-independent and sequence-independent reaction to seamlessly and directionally clone PCR fragments directly into a linearized adenoviral vector. Moreover, no additional treatment of the PCR fragment is required, such as restriction enzyme digestion, phosphorylation, or blunt-end polishing. This rapid approach requires only 3 days to generate a transfectable adenoviral vector compared to the 6 to 8 days needed by other cloning methods that use a donor or shuttle vector. Using an I-Ceu I and PI-Sce I linearized adenoviral backbone with numerous PCR amplimers, we observed >80% cloning efficiency and nearly 100% cloning success with little or no background as measured by colony PCR screening and endonuclease restriction analysis. We also observed strong correlation between PCR positive clones and intact adenoviral DNA suggesting that the vectors remained stable throughout the cloning process, showing minimal rearrangements. Additionally, we sequenced clones to confirm both the insert orientation and the integrity of cloning junctions and found that 100% were in proper orientation with no sequence changes at the junctions. The flexibility of the method was confirmed by cloning into numerous vector formats including constitutive and promoterless types both with and without fluorescent proteins expressed from S55
ADENOVIRUS AND OTHER DNA VIRUS VECTORS I the E3 region. To demonstrate complete functionality, we cloned several cDNAs into an adenoviral vector containing the complete 3rd generation tetracycline-inducible expression system that was optimized for low basal and high maximal expression. These Pac I linearized recombinant adenoviral vectors were efficiently transfected into 293 cells using a biodegradable polymer and an optimized 4hr protocol. Rescue was deemed complete when cytopathic effect was observed 7 days following transfection. Adenoviral stocks produced from these vectors, were able to produce high titer virus (1e9 IFU/ ml) and achieve induced expression levels that were >1300 fold over the uninduced state in HeLa cells. We believe this cloning system will ultimately increase the utility of adenoviral vectors by allowing rapid, accurate, and directional high-throughput cloning, further facilitating the production of functional adenoviral stocks for gene therapy development and gene function studies.
142. Differential Effects of Murine and Human Factor X on Adenovirus Transduction Via Cell Surface Heparan Sulfate
Anne K. Zaiss,1,2 Roger Lawrence,3 David Elashoff,4 Jeffrey D. Esko,3 Harvey R. Herschman.1,2 1 Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 2Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 3Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, CA; 4Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA. Serum factor X (FX) is proposed to play a major role in Adenovirus tropism, binding to the virus hexon protein and promoting transduction by bridging the virus to cell surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction of an Adenovirus vector 2-4 fold in murine hepatocytelike cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary cells. mFX was not only unable to promote transduction in these cells; mFX competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggest increased transduction by both mFX and hFX in hepatocyte-derived cells and increased transduction mediated by hFX in epithelial cells were dependent on HSPGs. Hepatocyte-derived cell lines express substantially more HSPGs than the cancer cell lines studied. Dose response curves suggest that Ad:hFX has a greater affinity for HSPGs than does Ad:mFX. In coagulation factor-depleted mice, hFX showed a similar enhanced ability, compared to mFX, to reconstitute hepatic Adenovirus transduction. These results suggest that differences in Ad:hFX and Ad:mFX affinity to HSPGs might result in differences in their ability to enhance Adenovirus transduction of many cell types. These findings may have implications for murine models of adenovirus vector targeting.
143. Improved Tropism of Adenoviral Vectors for Muscle Cells Using an IGF-1R Targeted Bispecific Ligand
Maxime Pinard,1,2 Birgit Kastberger,1 Yue Zeng,1 Nancy Larochelle,3 Josephine Nalbantoglu,3 Bernard Massie,1,2 Rénald Gilbert.1,3 1 Biotechnology Research Institute, Montreal, Canada; 2University of Montreal, Montreal, Canada; 3Montreal Neurological Institute, Montreal, Canada. Adenoviral vector (AdV) is being intensively investigated as a tool to deliver exogenous sequences to different tissues in order to correct S56
multiple genetic disorders. Limited expression of the Coxsackie and Adenovirus Receptor (CAR), the primary receptor for AdV, has been a major drawback in gene transfer approaches. To compensate for the paucity of CAR, multiple strategies have been proposed. Some of them consist to alter the AdV fiber to enable recognition of new receptors at the cellular surface, such as PEGylation or using fibers derived from other serotypes. Our group has developed a bispecific ligand approach coupled with Ad5 fiber modification. The interaction between the Ad5 and the ligand is generated by using an artificial interaction domain, the E-Coil/K-Coil. The K-Coil is inserted within the HI loop domain of the Ad5 fiber and its counter part, the E-Coil, is fused with a ligand of interest. The insulin-like growth factor-1, (IGF-1) recognizes the IGF-1 receptor (IGF-1R) expresses on many cell lines such as muscle. Indeed, we demonstrated expression of IGF-1R by western blot in C2C12 myoblasts and in myotubes. We also showed that it was expressed on normal and dystrophin deficient (mdx) mice by immunochemistry and by western blot. Therefore, to increase the transduction efficacy of AdV for muscle we constructed a bispecific ligand (IGF-E5) made of IGF-1 fused to five repeats of the E-Coil and to a His-Tag for protein purification. Binding of IGF-E5 to the fiber of an AdV encoding GFP and containing the K-Coil within the HI-loop (Ad5HIK5cDm/GFP) was shown by ELISA. Using flow cytometry we also demonstrated that IGF-E5 could increase the transduction efficacy of Ad5HIK5cDm/GFP by 16-fold in myoblasts. In the absence of ligand, the transduction efficacy of Ad5HIK5cDm/GFP was comparable to an AdV with wild type fiber. Importantly, incubation with recombinant IGF-1 did not increase the transduction of Ad5HIK5cDm/GFP in myoblasts. A 6-fold increase of GFP expression was also measured by western blot when myotubes were transduced at a MOI of 300 with Ad5HIK5cDm/GFP associated with IGF-E5.
144. An Alternate Method for Efficient Delivery of Catalyzing Enzymes
Andrew D. Fontes,1 Rene Quintanilla,1 Michael Poderycki,2 Jon Chesnut,2 Uma Lakshmipathy.1 1 Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA; 2Global Science and Innovation Office, Life Technologies, Carlsbad, CA.
We had earlier reported the development of integrase-based JumpIn platform for site-specific integration and stable expression of transgenes in human embryonic stem cells. This method utilizes vectors with special integrase recognizing sequences that facilitate the insertion of the construct carrying the transgene into the host genome catalyzed by the expression of the appropriate enzyme. Current methods utilize cotransfection of the integrational plasmid carrying the transgene of interest with the appropriate catalyzing enzyme expression vector. To overcome any adverse effect resulting from random integration of the catalyzing enzyme expression vectors into the host genome, we utilized a non-integrating transient expression system for delivery. BacMam, a baculovirus based gene delivery method is known to facilitate high efficiency gene transfer into hard-to-transfect cell types such as primary and stem cells. PhiC31 Integrase, R4 Integrase and NLS-Cre Recombinase were cloned into the BacMam vector and high titer virus generated. Expression of the Integrases and Recombinase was confirmed by qRTPCR or immunochemical analysis of BacMam transduced HEK293 cells. Functionality was evaluated by the emergence of cells expressing a visual marker signifying successful reaction by the introduced enzymes. Our results indicate that catalyzing enzymes can be delivered at high efficiency via BacMam and the frequency of obtaining genetically modified cells is comparable or better than reaction catalyzed with plasmid DNA. Given the efficiency of BacMam to facilitate high efficiency gene transfer into adult stem cells such as mesenchymal stem cells and neural stem cells that are Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy