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271. The Airway Epithelium Differentiation State Affects Efficiency of siRNA Oligonucleotide Delivery
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY 269. Safety and Clonality Analyses for a New Vector for CGD Clinical Gene Therapy
Simone Scholz,1 Margarita Diaz,2 Stefan Stein,2 Axel Schambach,3 Hanno Glimm,1 Christof von Kalle,1 Manuel Grez,2 Manfred Schmidt.1 1 Translational Oncology, German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany; 2 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany; 3Experimental Hematology, Hannover Medical School, Hannover, Germany. Several (pre)clinical gene therapy trials have shown that integrating full-LTR driven gammaretroviral vectors can successfully be used to treat monogenetic diseases. However, with increasing efficiency of retroviral gene transfer severe side effects occurred. We have previously reported on a severe side effect in two patients enrolled in a clinical trial for the treatment of X-linked chronic granulomatous disease (X-CGD). Insertional activation of MDS1/EVI1 mediated by the used SFFV vector has led to clonal expansion of gene marked myeloid progenitor cells, triggering the development of a myelodysplastic syndrome with monosomy 7. Therefore, a new self-inactivating (SIN) gammaretroviral vector with an optimized design (SINfes.gp91s) was developed aiming to decrease vector-host interactions and associated side effects (Manuel Grez, Georg-SpeyerHaus, Frankfurt). In the course of the preclinical testing we performed safety and clonality analyses in vitro and in vivo. Samples from mice transplanted with gene modified hematopoietic stem cells were analyzed by LAM- and LM-PCR and subsequent high-throughput integration site (IS) analysis in combination with deep sequencing (454/Roche). As a control, a full LTR SFFV-promoter driven vector (SF91eGFP) similar to the one used in the previous clinical trial was analyzed in parallel. We could detect a total of 1938 unique IS in SF91eGFP (n = 4) and 3516 in SINfes.gp91s mice (n = 5). In average, we retrieved 530 unique IS in SF91eGFP mice, showing a less polyclonal IS pattern than SINfes.gp91s transplanted mice (754 unique IS/mouse). Moreover, (pre)dominant clones were found more frequently in mice transplanted with the SF91eGFP vector than with SINfes.gp91s. While in SF91eGFP mice we could detect 13 IS in/ near the EVI1/MDS1 locus with relative sequence counts (SC) up to 31%, in SINfes.gp91s mice only 5 IS with relative SC ≤ 0,2% were detectable in that locus. Overall, the SINfes.gp91s vector showed no obvious signs of vector-induced side effects.
Lung and Respiratory Disease Gene & Cell Therapy 270. Adeno-Associated Virus Transduction in the Ferret Airway
Ziying Yan,1,2 Diana C. M. Lei-Butters,1 Xingshen Sun,1 Xiaoming Liu,1,2 Nicholas W. Keiser,1 John Fisher,1 Hongshu Sui,1 John F. Engelhardt.1,2 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City; 2Center for Gene Therapy, The University of Iowa, Iowa City. Cystic fibrosis (CF) is an inherent disease caused by a single gene defect of cystic fibrosis transmembrane conductance regulator (CFTR). Since lung disease is the most life-threatening pathology in CF, the airways are currently the primary target for therapy. Since CF mouse models do not contract spontaneous lung disease, we recently generated a CFTR-KO ferret. This model develops neonatal and adult lethal lung infections that closely mirror pathology seen in CF patients. In an effort to develop CF lung gene therapy approaches with rAAV in this model, we have screened rAAV serotypes for their ability to infect the ferret airways. Previous studies demonstrated that apical transduction of AAV1 in ferret primary polarized airway epithelia culture is more efficient than that of AAV2 and AAV5 vectors. CoMolecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
administration with proteasome inhibitors doxorubicin and LLnL during the infection period dramatically enhanced the transduction for these AAV serotypes. To develop a lung-directed protocol for the ferret, we screened four AAV serotypes (AAV1, 2, 5, 6) for efficacy of gene transfer to the lungs of 14 day-old ferrets. Surprisingly, we found little gene transfer with any of these serotypes using an alkaline phosphatase reporter to allow for cellular localization. Application of 200 uM doxorubicin at the time of vector administration gave rise to the highest level of transduction with rAAV1 (with the majority of expression in the intra-lobar airways and alveolar regions of the lung and little expression in the trachea). These findings demonstrated that the tropism of various AAV serotypes tested to infect polarized tracheal ferret airway epithelia was substantially different than in vivo infection of more distal airways epithelia. To better determine the level of proteasome inhibitor augmentation of AAV1 infection in vivo, we infected 14 day-old ferrets by intratracheal instillation (n=4 for each group) with rAAV1-luciferase virus in 600ul saline or saline formulated with doxorubicin at the concentration of 7.4uM, 22uM, 67uM and 200uM. Luciferase reporter assays were performed 8 days after infection and demonstrated a >2000-fold induction by doxorubicin. We further compared the AAV1 and AAV2 transduction in newborn ferret pups (2 day old). Virus administration was performed by trans-tracheal injection. Each ferret was infected with 20ul virus solution containing 6x1010 AAV1-luciferase or AAV2luciferase and proteasome inhibitors (2ug doxorubicin (175uM) and 3ug LLnL (400uM)). Reporter assays were performed on 4 days after infection (n=6 for both groups). Results demonstrated that the AAV1 transduction efficiencies were 7.4 fold higher in trachea and 11.2 fold higher in lung than that of AAV2. These studies suggest that the AAV1 is the best candidate AAV vector for ferret airway transduction.
271. The Airway Epithelium Differentiation State Affects Efficiency of siRNA Oligonucleotide Delivery
Sateesh Krishnamurthy,1 Ashley Jacobi,2 Scott D. Rose,2 Mark A. Behlke,2 Paul B. McCray, Jr.,1,3 Beverely L. Davidson.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Integrated DNA Technologies, Coralville, IA; 3Pediatrics, University of Iowa, Iowa City, IA. The delivery of siRNA oligos to the surface epithelium of the airway offers the opportunity to manipulate host or pathogen gene expression for therapeutic or biologic purposes. However, welldifferentiated epithelia present several barriers to the successful delivery of nucleic acid. Here we used primary pig airway epithelial cells grown at the air-liquid interface as a model for the delivery of dicer-substrate siRNA duplexes against pig IL-8 and HPRT. In well-differentiated epithelia (> 2 weeks in culture), the application of oligos to the apical (mucosal) surface using lipid reagents or several different cationic polymers yielded no knockdown. Furthermore, transduction of siRNA with a peptide-delivery vehicle (PTDDRBD) or transfection with siRNA coupled to a ligand failed to knockdown the target. Neither increasing oligo dosages, the oligo residence time, or enhancing the permeability of epithelial junctions during transfection resulted in knockdown. In contrast, in poorly differentiated cells (1-4 days in culture), transfection or transduction of siRNA with any of the reagents resulted in approximately 30-70% knockdown of the target. We used DIG-labeled siRNA to follow the fate of the oligos under various experimental conditions. Using the PTD-DRBD formulation, we observed that internalization of the oligo was significantly reduced in well-differentiated cells compared to the poorly differentiated cells. These results indicate that airway epithelial cell differentiation is accompanied by the development of a significant barrier for oligo entry. An improved understanding of the differentiation process may yield insights that facilitate oligo delivery to well-differentiated airway epithelia. S105
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY 272. Strain-Specific Differences in Pulmonary Gene Transfer Efficiency: Relevance for Toxicology Studies in Mice
Giannoulis Legakis,1 Benjamin Causton,2 Jie Zhu,1 Nikki Newman,1 Raymond Farley,1 Cuixiang Meng,1 Clare M. Lloyd,2 Eric W. F. W. Alton,1 Uta Griesenbach.1 1 Department of Gene Therapy, Imperial College and the UK Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom; 2 Leukocyte Biology Section, NHLI, Imperial College, London, United Kingdom. As part of our ongoing clinical trial programme aimed at assessing, if non-viral gene transfer improves lung disease severity in cystic fibrosis (CF) patients, the UK CF Gene Therapy Consortium is currently conducting toxicology studies to support a multi-dose nonviral gene therapy trial in CF subjects. As part of this work, preliminary experiments determined significantly lower (approximately one log) lung transfection efficiency in outbred ICR(CD-1) mice (commonly used for toxicology studies conducted by contract research organisations) compared to inbred Balb/C mice (commonly used for pre-clinical gene transfer experiments) following nebulisation with pCIKLuc (regulated by a CMV promoter/enhancer). Here, we reproduced and confirmed these initial findings. We next extended these shssowing that strain-specific differences were maintained when using other non-viral vectors such as PEI, plasmids regulated by eukaryotic promoters or when transfecting the mouse nasal epithelium, which is a good model for human airways. To understand these strain specific-differences we first compared inflammatory responses after bolus administration of GL67A/pCIKlux complexes to the mouse lung by nasal sniffing. As previously reported nasal sniffing of GL67A/pDNA caused lung inflammation, but on histological assessment 24 hr after transfection there were no differences between the two strains. The pro-inflammatory chemokines KC and MIP1a were significantly (p<0.05) increased in broncho-alveolar lavage fluid of both strains 6 and 24 hrs after gene transfer. Interestingly, levels of both cytokines were significantly higher in Balb/C compared to CD-1 mice at both time-points (KC (pg/ml) shown as example, Balb/C 6 hr: 896±438, CD1 6 hr: 438±67, Balb/C 24 hr: 644±97, CD1 24 hr: 255±33, n=16/group). It is currently unclear if and how differences in cytokine expression relate to differences in transfection efficiency and additional studies have to be performed. We next compared gene expression in CD1/Balb/C F1 hybrids to the parental strains. Interestingly, the F1 hybrids had intermediate levels of gene expression and were significantly different from both parental strains. In conclusion, the mouse strain affects transfection efficiency. In the short-term, these underline the importance of careful selection of suitable mouse strains before embarking on toxicology studies and has led us to perform our toxicology study in Balb/C mice rather than CD1 mice. In the longer-term, understanding the reasons for these differences may inform key factors involved in airway gene transfer and may be informative for improving gene transfer in man.
S106
273. Reporter Gene Expression Following Repeat Administration of a HIV-1 Lentiviral Vector in Mouse Airways
Patricia L. Cmielewski,1,3 Don S. Anson,2,4 David W. Parsons.1,3,4,5 Respiratory and Sleep Medicine, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 2Gene Technology Unit, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 3School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia, Australia; 4 Centre for Stem Cell Research, University of Adelaide, Adelaide, South Australia, Australia; 5Women’s and Children’s Health Research Institute, Adelaide, South Australia, Australia. 1
Introduction: Repeat dosing may be necessary to ensure long term correction for cystic fibrosis airway disease. Using luciferase (Luc) bioluminescence imaging, we have examined in-vivo the persistence of gene expression in individual animals over time to determine if repeat dosing with a VSV-G pseudotyped lentiviral (LV) gene vector maintained gene expression in airways of normal mice. Methods: Three groups of mice (n=8/group) received a single VSV-G pseudotyped LV gene vector delivered intra-nasally (4µl of lysophosphatidylcholine (LPC, 0.3%) followed 1 hour later by a 20µl bolus of the LV-Luc gene vector). Four weeks later 1 group of mice was re-dosed with a different reporter-gene (LPC/LV-LacZ), while another group was re-dosed with the same LPC/LV-Luc gene. Luc bioluminescence was measured 1, 4, 5, 8 and 12 weeks later (IVIS, Xenogen) 10 mins after intranasally delivered substrate D-luciferin (50µl, 15mg/ml). Results: Nasal bioluminescence was the same across all 3 groups at 1-5 weeks post initial LV instillation (n.s., ANOVA). Those mice that received a different transgene at re-dose (i.e. LPC/LV-LacZ) displayed similar gene expression compared to the single dose group at both later time points. However at 8 and 12 weeks after the 2nd dose, mice that received two doses of LPC/LVLuc showed significantly less gene expression compared those given a single dose of LPC/LV-Luc (p<0.001, ANOVA). Conclusions: These results indicate that re-administration of the same LV vector transgene can reduce subsequent gene expression. This effect is likely to be due to a cell-mediated immune response directed against the specific transgene.
274. Gene Transfer by Influenza HA Pseudotyped EIAV Lentiviral Vectors to the Airways of Neonatal Mice
Manij Patel,1 John C. Olsen.1 Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC. 1
CF lung disease starts early in childhood and so phenotypic correction at the beginning of life could be a very desirable option for early treatment or amelioration of early onset lung disease. We have been investigating the use of influenza hemagglutinin (HA) pseudotyped lentiviral vectors to transfer genes to the airway epithelium of mice. Previously, we reported that nasal inhalation of EIAV vectors pseudotyped with influenza HA resulted in high levels of gene transfer to nasal, tracheal, and lung epithelial cells of adult mice. To determine if these vectors would be suitable for transferring genes to newborn mice, we intra-nasally administered HA pseudotyped EIAV vectors to mouse pups 24 to 48 hours after birth. For these studies, we used vectors encoding luciferase, lacZ, or dual luciferase-lacZ expression cassettes. Bioluminescence in the noses and lungs of the pups from luciferase expression could be detected within 48 hour of vector administration. Measurement over a 10-day period in the nose and ultimately luciferase activity in lung homogenates showed that gene transfer was dose dependent. In other studies, pups were dosed with 6, 12, or 18 microliters of concentrated (2E8 IU/ml) vectors encoding lacZ. Histochemical analysis revealed, Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Simone Scholz,1 Margarita Diaz,2 Stefan Stein,2 Axel Schambach,3 Hanno Glimm,1 Christof von Kalle,1 Manuel Grez,2 Manfred Schmidt.1 1 Translational Oncology, German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany; 2 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany; 3Experimental Hematology, Hannover Medical School, Hannover, Germany. Several (pre)clinical gene therapy trials have shown that integrating full-LTR driven gammaretroviral vectors can successfully be used to treat monogenetic diseases. However, with increasing efficiency of retroviral gene transfer severe side effects occurred. We have previously reported on a severe side effect in two patients enrolled in a clinical trial for the treatment of X-linked chronic granulomatous disease (X-CGD). Insertional activation of MDS1/EVI1 mediated by the used SFFV vector has led to clonal expansion of gene marked myeloid progenitor cells, triggering the development of a myelodysplastic syndrome with monosomy 7. Therefore, a new self-inactivating (SIN) gammaretroviral vector with an optimized design (SINfes.gp91s) was developed aiming to decrease vector-host interactions and associated side effects (Manuel Grez, Georg-SpeyerHaus, Frankfurt). In the course of the preclinical testing we performed safety and clonality analyses in vitro and in vivo. Samples from mice transplanted with gene modified hematopoietic stem cells were analyzed by LAM- and LM-PCR and subsequent high-throughput integration site (IS) analysis in combination with deep sequencing (454/Roche). As a control, a full LTR SFFV-promoter driven vector (SF91eGFP) similar to the one used in the previous clinical trial was analyzed in parallel. We could detect a total of 1938 unique IS in SF91eGFP (n = 4) and 3516 in SINfes.gp91s mice (n = 5). In average, we retrieved 530 unique IS in SF91eGFP mice, showing a less polyclonal IS pattern than SINfes.gp91s transplanted mice (754 unique IS/mouse). Moreover, (pre)dominant clones were found more frequently in mice transplanted with the SF91eGFP vector than with SINfes.gp91s. While in SF91eGFP mice we could detect 13 IS in/ near the EVI1/MDS1 locus with relative sequence counts (SC) up to 31%, in SINfes.gp91s mice only 5 IS with relative SC ≤ 0,2% were detectable in that locus. Overall, the SINfes.gp91s vector showed no obvious signs of vector-induced side effects.
Lung and Respiratory Disease Gene & Cell Therapy 270. Adeno-Associated Virus Transduction in the Ferret Airway
Ziying Yan,1,2 Diana C. M. Lei-Butters,1 Xingshen Sun,1 Xiaoming Liu,1,2 Nicholas W. Keiser,1 John Fisher,1 Hongshu Sui,1 John F. Engelhardt.1,2 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City; 2Center for Gene Therapy, The University of Iowa, Iowa City. Cystic fibrosis (CF) is an inherent disease caused by a single gene defect of cystic fibrosis transmembrane conductance regulator (CFTR). Since lung disease is the most life-threatening pathology in CF, the airways are currently the primary target for therapy. Since CF mouse models do not contract spontaneous lung disease, we recently generated a CFTR-KO ferret. This model develops neonatal and adult lethal lung infections that closely mirror pathology seen in CF patients. In an effort to develop CF lung gene therapy approaches with rAAV in this model, we have screened rAAV serotypes for their ability to infect the ferret airways. Previous studies demonstrated that apical transduction of AAV1 in ferret primary polarized airway epithelia culture is more efficient than that of AAV2 and AAV5 vectors. CoMolecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
administration with proteasome inhibitors doxorubicin and LLnL during the infection period dramatically enhanced the transduction for these AAV serotypes. To develop a lung-directed protocol for the ferret, we screened four AAV serotypes (AAV1, 2, 5, 6) for efficacy of gene transfer to the lungs of 14 day-old ferrets. Surprisingly, we found little gene transfer with any of these serotypes using an alkaline phosphatase reporter to allow for cellular localization. Application of 200 uM doxorubicin at the time of vector administration gave rise to the highest level of transduction with rAAV1 (with the majority of expression in the intra-lobar airways and alveolar regions of the lung and little expression in the trachea). These findings demonstrated that the tropism of various AAV serotypes tested to infect polarized tracheal ferret airway epithelia was substantially different than in vivo infection of more distal airways epithelia. To better determine the level of proteasome inhibitor augmentation of AAV1 infection in vivo, we infected 14 day-old ferrets by intratracheal instillation (n=4 for each group) with rAAV1-luciferase virus in 600ul saline or saline formulated with doxorubicin at the concentration of 7.4uM, 22uM, 67uM and 200uM. Luciferase reporter assays were performed 8 days after infection and demonstrated a >2000-fold induction by doxorubicin. We further compared the AAV1 and AAV2 transduction in newborn ferret pups (2 day old). Virus administration was performed by trans-tracheal injection. Each ferret was infected with 20ul virus solution containing 6x1010 AAV1-luciferase or AAV2luciferase and proteasome inhibitors (2ug doxorubicin (175uM) and 3ug LLnL (400uM)). Reporter assays were performed on 4 days after infection (n=6 for both groups). Results demonstrated that the AAV1 transduction efficiencies were 7.4 fold higher in trachea and 11.2 fold higher in lung than that of AAV2. These studies suggest that the AAV1 is the best candidate AAV vector for ferret airway transduction.
271. The Airway Epithelium Differentiation State Affects Efficiency of siRNA Oligonucleotide Delivery
Sateesh Krishnamurthy,1 Ashley Jacobi,2 Scott D. Rose,2 Mark A. Behlke,2 Paul B. McCray, Jr.,1,3 Beverely L. Davidson.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Integrated DNA Technologies, Coralville, IA; 3Pediatrics, University of Iowa, Iowa City, IA. The delivery of siRNA oligos to the surface epithelium of the airway offers the opportunity to manipulate host or pathogen gene expression for therapeutic or biologic purposes. However, welldifferentiated epithelia present several barriers to the successful delivery of nucleic acid. Here we used primary pig airway epithelial cells grown at the air-liquid interface as a model for the delivery of dicer-substrate siRNA duplexes against pig IL-8 and HPRT. In well-differentiated epithelia (> 2 weeks in culture), the application of oligos to the apical (mucosal) surface using lipid reagents or several different cationic polymers yielded no knockdown. Furthermore, transduction of siRNA with a peptide-delivery vehicle (PTDDRBD) or transfection with siRNA coupled to a ligand failed to knockdown the target. Neither increasing oligo dosages, the oligo residence time, or enhancing the permeability of epithelial junctions during transfection resulted in knockdown. In contrast, in poorly differentiated cells (1-4 days in culture), transfection or transduction of siRNA with any of the reagents resulted in approximately 30-70% knockdown of the target. We used DIG-labeled siRNA to follow the fate of the oligos under various experimental conditions. Using the PTD-DRBD formulation, we observed that internalization of the oligo was significantly reduced in well-differentiated cells compared to the poorly differentiated cells. These results indicate that airway epithelial cell differentiation is accompanied by the development of a significant barrier for oligo entry. An improved understanding of the differentiation process may yield insights that facilitate oligo delivery to well-differentiated airway epithelia. S105
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY 272. Strain-Specific Differences in Pulmonary Gene Transfer Efficiency: Relevance for Toxicology Studies in Mice
Giannoulis Legakis,1 Benjamin Causton,2 Jie Zhu,1 Nikki Newman,1 Raymond Farley,1 Cuixiang Meng,1 Clare M. Lloyd,2 Eric W. F. W. Alton,1 Uta Griesenbach.1 1 Department of Gene Therapy, Imperial College and the UK Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom; 2 Leukocyte Biology Section, NHLI, Imperial College, London, United Kingdom. As part of our ongoing clinical trial programme aimed at assessing, if non-viral gene transfer improves lung disease severity in cystic fibrosis (CF) patients, the UK CF Gene Therapy Consortium is currently conducting toxicology studies to support a multi-dose nonviral gene therapy trial in CF subjects. As part of this work, preliminary experiments determined significantly lower (approximately one log) lung transfection efficiency in outbred ICR(CD-1) mice (commonly used for toxicology studies conducted by contract research organisations) compared to inbred Balb/C mice (commonly used for pre-clinical gene transfer experiments) following nebulisation with pCIKLuc (regulated by a CMV promoter/enhancer). Here, we reproduced and confirmed these initial findings. We next extended these shssowing that strain-specific differences were maintained when using other non-viral vectors such as PEI, plasmids regulated by eukaryotic promoters or when transfecting the mouse nasal epithelium, which is a good model for human airways. To understand these strain specific-differences we first compared inflammatory responses after bolus administration of GL67A/pCIKlux complexes to the mouse lung by nasal sniffing. As previously reported nasal sniffing of GL67A/pDNA caused lung inflammation, but on histological assessment 24 hr after transfection there were no differences between the two strains. The pro-inflammatory chemokines KC and MIP1a were significantly (p<0.05) increased in broncho-alveolar lavage fluid of both strains 6 and 24 hrs after gene transfer. Interestingly, levels of both cytokines were significantly higher in Balb/C compared to CD-1 mice at both time-points (KC (pg/ml) shown as example, Balb/C 6 hr: 896±438, CD1 6 hr: 438±67, Balb/C 24 hr: 644±97, CD1 24 hr: 255±33, n=16/group). It is currently unclear if and how differences in cytokine expression relate to differences in transfection efficiency and additional studies have to be performed. We next compared gene expression in CD1/Balb/C F1 hybrids to the parental strains. Interestingly, the F1 hybrids had intermediate levels of gene expression and were significantly different from both parental strains. In conclusion, the mouse strain affects transfection efficiency. In the short-term, these underline the importance of careful selection of suitable mouse strains before embarking on toxicology studies and has led us to perform our toxicology study in Balb/C mice rather than CD1 mice. In the longer-term, understanding the reasons for these differences may inform key factors involved in airway gene transfer and may be informative for improving gene transfer in man.
S106
273. Reporter Gene Expression Following Repeat Administration of a HIV-1 Lentiviral Vector in Mouse Airways
Patricia L. Cmielewski,1,3 Don S. Anson,2,4 David W. Parsons.1,3,4,5 Respiratory and Sleep Medicine, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 2Gene Technology Unit, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 3School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia, Australia; 4 Centre for Stem Cell Research, University of Adelaide, Adelaide, South Australia, Australia; 5Women’s and Children’s Health Research Institute, Adelaide, South Australia, Australia. 1
Introduction: Repeat dosing may be necessary to ensure long term correction for cystic fibrosis airway disease. Using luciferase (Luc) bioluminescence imaging, we have examined in-vivo the persistence of gene expression in individual animals over time to determine if repeat dosing with a VSV-G pseudotyped lentiviral (LV) gene vector maintained gene expression in airways of normal mice. Methods: Three groups of mice (n=8/group) received a single VSV-G pseudotyped LV gene vector delivered intra-nasally (4µl of lysophosphatidylcholine (LPC, 0.3%) followed 1 hour later by a 20µl bolus of the LV-Luc gene vector). Four weeks later 1 group of mice was re-dosed with a different reporter-gene (LPC/LV-LacZ), while another group was re-dosed with the same LPC/LV-Luc gene. Luc bioluminescence was measured 1, 4, 5, 8 and 12 weeks later (IVIS, Xenogen) 10 mins after intranasally delivered substrate D-luciferin (50µl, 15mg/ml). Results: Nasal bioluminescence was the same across all 3 groups at 1-5 weeks post initial LV instillation (n.s., ANOVA). Those mice that received a different transgene at re-dose (i.e. LPC/LV-LacZ) displayed similar gene expression compared to the single dose group at both later time points. However at 8 and 12 weeks after the 2nd dose, mice that received two doses of LPC/LVLuc showed significantly less gene expression compared those given a single dose of LPC/LV-Luc (p<0.001, ANOVA). Conclusions: These results indicate that re-administration of the same LV vector transgene can reduce subsequent gene expression. This effect is likely to be due to a cell-mediated immune response directed against the specific transgene.
274. Gene Transfer by Influenza HA Pseudotyped EIAV Lentiviral Vectors to the Airways of Neonatal Mice
Manij Patel,1 John C. Olsen.1 Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC. 1
CF lung disease starts early in childhood and so phenotypic correction at the beginning of life could be a very desirable option for early treatment or amelioration of early onset lung disease. We have been investigating the use of influenza hemagglutinin (HA) pseudotyped lentiviral vectors to transfer genes to the airway epithelium of mice. Previously, we reported that nasal inhalation of EIAV vectors pseudotyped with influenza HA resulted in high levels of gene transfer to nasal, tracheal, and lung epithelial cells of adult mice. To determine if these vectors would be suitable for transferring genes to newborn mice, we intra-nasally administered HA pseudotyped EIAV vectors to mouse pups 24 to 48 hours after birth. For these studies, we used vectors encoding luciferase, lacZ, or dual luciferase-lacZ expression cassettes. Bioluminescence in the noses and lungs of the pups from luciferase expression could be detected within 48 hour of vector administration. Measurement over a 10-day period in the nose and ultimately luciferase activity in lung homogenates showed that gene transfer was dose dependent. In other studies, pups were dosed with 6, 12, or 18 microliters of concentrated (2E8 IU/ml) vectors encoding lacZ. Histochemical analysis revealed, Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy