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794. Use of Near Infrared Imaging To Determine Injection Success
Adenovirus Vectors: Biology and Pharmacology vectors in murine c-kit+, lin- cells, yield efficient expression of the β-globin protein.
(A) Schematic structures of scAAV-HS2-βp-β87+-globin (i), and scAAV-B19p6-β87+-globin (ii) vectors. (B) Fluorescence-activated cell sorting (FACS) analyses of expression of the β-globin protein in human K562 cells. (C) Quantitation of the data in (B), corrected for the human δ-globin protein expression. (D) FACS analyses of expression of the β-globin protein in primary murine c-kit+, lincells ±EPO. (E) Quantitation of the data from (D). These optimized AAV vectors should prove useful in achieving therapeutic levels the β-globin protein in human erythroid cells, which has implications in the use of these vectors in gene therapy of β-thalassemia and sickle cell disease.
Adenovirus Vectors: Biology and Pharmacology 793. Loss of Interaction between HexonModified Adenovirus and Blood Factors Impaired Liver Transgene Expression
Frédéric Vigant,1 Delphyne Descamps,1 Betsy Jullienne,1 Elisabeth Connault,1 Paule Opolon,1 Thierry Tordjman,2 Emmanuelle Vigne,3 Michel Perricaudet,1 Karim Benihoud.1,4 1 CNRS UMR8121, Institut Gustave Roussy, Villejuif, France; 2 INSERM U442, Orsay, France; 3Sanofi-Aventis, Vitry-sur-Seine, France; 4Faculté des Sciences, Université Paris-Sud, Orsay, France. Adenovirus (Ad) are valuable tools for gene therapy because they are easy to construct, are produced at high titers, and display high transduction efficiencies. However, their use in gene therapy is confronted with different hurdles. In particular, following systemic delivery, different untargeted tissues and particularly the liver are transduced, leading to a high risk of toxicity. Even after local injections, Ad leakage through the bloodstream can occur leading to liver uptake and toxicity. During biodistribution studies of capsidmodified Ad performed in mice, we observed that Ad which hexon HVR5 region was replaced by different peptides displayed a dramatic decrease in liver transgene expression (>95%) after intravenous injection when compared to unmodified Adwt. This observation was confirmed in different strains of mice. This reduction of transgene expression was correlated with a reduction of liver viral genome content. Mice injected with hexon-modifed Ad or with Adwt displayed a comparable level of Ad DNA at an earlier time point (30 min), thereby demonstrating that hexon-modifed Ad are cleared more rapidly from liver. Pretreatment of mice with clodronate, a drug able to deplete Kuppfer cells, led to a comparable increase of both liver transgene expression and Ad DNA content in mice injected with Adwt or hexon-modified Ad, ruling out a higher uptake of hexonMolecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
modifed Ad by kupffer cells. Moreover, measurement of Ad DNA content in purified liver sub-populations confirmed no difference 30 min post-injection in binding to (transduction of) non-parenchymal or parenchymal cells compared to Adwt-injected mice. Since vitamin K-dependent coagulation factors were previously shown to promote Ad transduction of liver and refractory cells in vitro, we examined whether impairment of interactions between hexon-modifed Ad and these blood factors could be responsible of Ad faster liver clearance. Indeed, some hexon-modified vectors were no longer able to bind with blood factors (Factors IX and mostly FX) immobilized on membrane while Adwt was. In addition, whereas CHO cells transduction by Adwt, as measured by transgene expression, was enhanced by coincubation of the virus with blood factors, most of hexon-modified Ad were not able to improve CHO transduction. Measurement of Ad DNA content in CHO-infected cells 1h post infection showed that hexon-modified Ad were not internalized more efficiently in presence of blood factors into CHO cells contrary to Adwt. Altogether these data show that substitution of hexon HVR5 region reduce Ad interaction with blood factors, and constitutes a very effective way to reduce liver transgene expression and associated toxicity. Studies are currently performed to better understand how modification of hexon protein impairs Ad binding to blood factors.
794. Use of Near Infrared Imaging To Determine Injection Success
Sean E. Hofherr,1 Michael A. Barry.2 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Mayo Clinic, Internal Medicine, Immunovirology and Biodefense, Rochester, MN.
Some injection techniques can be technically difficult. There is also the uncertainty of whether the injection was successful or unsuccessful. We demonstrate that adding a nonreactive near infrared dye Angiosense to the sample being injected, and then imaging the mice directly following injection, the success of the injection can be determined. In this study we inject adenovirus expressing the dsRed transgene in neonatal pups by the intravenous, intracardiac and intrahepatic routes. For intravenous injection, the scalp vein in pups is very difficult to inject when the pups are so young. For intracardiac injection, the needle can miss the heart, or inject other parts of the cardiac cavity instead. In the case of the intrahepatic injection the liver can easily be missed, or passed through with the needle before the sample is injected. We added Angiosense to the virus sample and injected the vector. Immediately following injection we imaged the pups in the near infrared range and determined which pups were successfully injected and which pups were not. We then imaged the mice at 3 days post-injection for the dsRed expression. By comparing the near infrared imaging with the expression of the transgene, we could determine how effective the various injection methods are at getting the vector into the blood stream.
795. Fluorescent Labelling of Adenovirus Capsids with Quantum Dots for Imaging
Stuart A. Nicklin, Andrew H. Baker. 1 BHF GCRC, University of Glasgow, Glasgow, United Kingdom. Imaging adenovirus (Ad) trafficking has been studied in vitro using fluorescent dyes, e.g. Cy3, conjugated to capsid proteins. Although in vivo imaging of Cy3 labelled Ad has been achieved, the technology is limited by the wide emission bandwidth of Cy3, potentially hampering resolution of labelled Ad in individual cells in tissues. For high resolution in vivo imaging we investigated the potential of labelling Ad using quantum dots, semi-conductor crystals that emit light with high energy in a narrow wavelength band and provide stable, high signals for imaging. Two methods were used. First a 13 amino acid acceptor peptide1 for biotin was cloned into S297
(A) Schematic structures of scAAV-HS2-βp-β87+-globin (i), and scAAV-B19p6-β87+-globin (ii) vectors. (B) Fluorescence-activated cell sorting (FACS) analyses of expression of the β-globin protein in human K562 cells. (C) Quantitation of the data in (B), corrected for the human δ-globin protein expression. (D) FACS analyses of expression of the β-globin protein in primary murine c-kit+, lincells ±EPO. (E) Quantitation of the data from (D). These optimized AAV vectors should prove useful in achieving therapeutic levels the β-globin protein in human erythroid cells, which has implications in the use of these vectors in gene therapy of β-thalassemia and sickle cell disease.
Adenovirus Vectors: Biology and Pharmacology 793. Loss of Interaction between HexonModified Adenovirus and Blood Factors Impaired Liver Transgene Expression
Frédéric Vigant,1 Delphyne Descamps,1 Betsy Jullienne,1 Elisabeth Connault,1 Paule Opolon,1 Thierry Tordjman,2 Emmanuelle Vigne,3 Michel Perricaudet,1 Karim Benihoud.1,4 1 CNRS UMR8121, Institut Gustave Roussy, Villejuif, France; 2 INSERM U442, Orsay, France; 3Sanofi-Aventis, Vitry-sur-Seine, France; 4Faculté des Sciences, Université Paris-Sud, Orsay, France. Adenovirus (Ad) are valuable tools for gene therapy because they are easy to construct, are produced at high titers, and display high transduction efficiencies. However, their use in gene therapy is confronted with different hurdles. In particular, following systemic delivery, different untargeted tissues and particularly the liver are transduced, leading to a high risk of toxicity. Even after local injections, Ad leakage through the bloodstream can occur leading to liver uptake and toxicity. During biodistribution studies of capsidmodified Ad performed in mice, we observed that Ad which hexon HVR5 region was replaced by different peptides displayed a dramatic decrease in liver transgene expression (>95%) after intravenous injection when compared to unmodified Adwt. This observation was confirmed in different strains of mice. This reduction of transgene expression was correlated with a reduction of liver viral genome content. Mice injected with hexon-modifed Ad or with Adwt displayed a comparable level of Ad DNA at an earlier time point (30 min), thereby demonstrating that hexon-modifed Ad are cleared more rapidly from liver. Pretreatment of mice with clodronate, a drug able to deplete Kuppfer cells, led to a comparable increase of both liver transgene expression and Ad DNA content in mice injected with Adwt or hexon-modified Ad, ruling out a higher uptake of hexonMolecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
modifed Ad by kupffer cells. Moreover, measurement of Ad DNA content in purified liver sub-populations confirmed no difference 30 min post-injection in binding to (transduction of) non-parenchymal or parenchymal cells compared to Adwt-injected mice. Since vitamin K-dependent coagulation factors were previously shown to promote Ad transduction of liver and refractory cells in vitro, we examined whether impairment of interactions between hexon-modifed Ad and these blood factors could be responsible of Ad faster liver clearance. Indeed, some hexon-modified vectors were no longer able to bind with blood factors (Factors IX and mostly FX) immobilized on membrane while Adwt was. In addition, whereas CHO cells transduction by Adwt, as measured by transgene expression, was enhanced by coincubation of the virus with blood factors, most of hexon-modified Ad were not able to improve CHO transduction. Measurement of Ad DNA content in CHO-infected cells 1h post infection showed that hexon-modified Ad were not internalized more efficiently in presence of blood factors into CHO cells contrary to Adwt. Altogether these data show that substitution of hexon HVR5 region reduce Ad interaction with blood factors, and constitutes a very effective way to reduce liver transgene expression and associated toxicity. Studies are currently performed to better understand how modification of hexon protein impairs Ad binding to blood factors.
794. Use of Near Infrared Imaging To Determine Injection Success
Sean E. Hofherr,1 Michael A. Barry.2 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Mayo Clinic, Internal Medicine, Immunovirology and Biodefense, Rochester, MN.
Some injection techniques can be technically difficult. There is also the uncertainty of whether the injection was successful or unsuccessful. We demonstrate that adding a nonreactive near infrared dye Angiosense to the sample being injected, and then imaging the mice directly following injection, the success of the injection can be determined. In this study we inject adenovirus expressing the dsRed transgene in neonatal pups by the intravenous, intracardiac and intrahepatic routes. For intravenous injection, the scalp vein in pups is very difficult to inject when the pups are so young. For intracardiac injection, the needle can miss the heart, or inject other parts of the cardiac cavity instead. In the case of the intrahepatic injection the liver can easily be missed, or passed through with the needle before the sample is injected. We added Angiosense to the virus sample and injected the vector. Immediately following injection we imaged the pups in the near infrared range and determined which pups were successfully injected and which pups were not. We then imaged the mice at 3 days post-injection for the dsRed expression. By comparing the near infrared imaging with the expression of the transgene, we could determine how effective the various injection methods are at getting the vector into the blood stream.
795. Fluorescent Labelling of Adenovirus Capsids with Quantum Dots for Imaging
Stuart A. Nicklin, Andrew H. Baker. 1 BHF GCRC, University of Glasgow, Glasgow, United Kingdom. Imaging adenovirus (Ad) trafficking has been studied in vitro using fluorescent dyes, e.g. Cy3, conjugated to capsid proteins. Although in vivo imaging of Cy3 labelled Ad has been achieved, the technology is limited by the wide emission bandwidth of Cy3, potentially hampering resolution of labelled Ad in individual cells in tissues. For high resolution in vivo imaging we investigated the potential of labelling Ad using quantum dots, semi-conductor crystals that emit light with high energy in a narrow wavelength band and provide stable, high signals for imaging. Two methods were used. First a 13 amino acid acceptor peptide1 for biotin was cloned into S297