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872. Efficient Gene Delivery to Pig Airways Using Helper-Dependent Adenoviral Vectors
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY 869. Myelospecic Self-Inactivating Gammaretroviral Vectors for p47phox CGD Gene Therapy
Vital Wohlgensinger,1 Christian Brendel,2 Axel Schambach,3 Manuel Grez,2 Janine Reichenbach,1 Reinhard A. Seger,1 Ulrich Siler.1 1 Immunology/Hematology/BMT, University Children’s Hospital Zürich, Zürich, Switzerland; 2Georg-Speyer Research Institute, Frankfurt A.M., Germany; 3Experimental Hematology, Hannover Medical School, Hannover, Germany. First clinical success in gene therapy could be achieved e.g. for X-CGD and X-SCID using retroviral LTR-driven vectors. Unfortunately these results were accompanied with clonal dominance or leukemic progression as long term side effects of transactivation events. To develop a safety-optimized vector for the p47phox-decient form of CGD we cloned various myelospecic promoter candidates driving p47phox expression into a gammaretroviral self-inactivating (SIN) vector. The constructs were screened for myelospecicity and functional reconstitution in transduced murine p47phox -/- HSC upon ex vivo propagation to granulocytes. This screen demarcated the panel of promoter candidates. One of the candidates was tested in vivo in p47phox -/- mouse model and compared to SFFV driven p47phox positive control. Six weeks after reinfusion of transduced murine HSC, mice treated with the vector containing a myeloid-specic promoter driving p47phox expression showed enhanced p47phox levels in granulocytes and low/no p47phox expression in monocytes, lymphocytes and hematopoietic stem/progenitor cells in contrast to the SFFV control. Mice treated with this vector showed functional reconstitution of the NADPH-oxidase function in granulocytes as revealed by a DHR assay. p47phox CGD gene therapy using SIN vectors with a myeloid specic promoters results in tissue-specic expression of the p47phox transgene and restoration of NADPH function in granulocytes in the murine p47phox -/- CGD model.
870. Delivery of Soluble ICAM-1 in NOD Salivary Glands Can Result in B Cell Proliferation and Immunoglobulin Production
Jelle L. Vosters,1,2 Nienke Roescher,1,2 Paul P. Tak,1 John A. Chiorini.2 1 Division of Clinical Immunology and Rheumatology, Academic Medical Center, Amsterdam, Netherlands; 2Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, MD.
Objective: Sjögren’s syndrome (SS) is an autoimmune disease with unknown etiology. Accumulation and activation of lymphocytes in the salivary glands of SS patients is an important hallmark of the disease. Lymphocyte function-associated antigen-1 (LFA-1) is the receptor on lymphocytes, which binds endothelial or lymphocyte expressed Intercellular Adhesion Molecule-1 (ICAM-1), resulting in lymphocyte migration, co-stimulation and activation. Previously, a clinical trial in our department using anti-LFA-1 in SS patients resulted in exacerbated SS and induced signicant increase in local inammation and systemic autoimmunity [1]. The aim of this study is to elucidate the role of ICAM-1/LFA-1 in an autoimmune animal model by testing the effect on inammation by local expression of soluble ICAM-1. Methods and Materials: Using in vivo adeno associated viral gene transfer, we stably expressed soluble ICAM1:Fc fusion protein locally in the salivary glands in the Non-Obese Diabetic animal model of SS. Pilocarpine stimulated saliva ow was measured to address the salivary gland function and salivary glands were analyzed for focus score, composition of the inltrate, and immunoglobulins (Ig’s). Results: Local expression of ICAM1:Fc resulted in a signicant increase in B220-positive cells in the salivary glands. Also, trends towards decreased plasma cells (CD138), S336
increased CD4+ and CD8+ T cells were detected. While no change in salivary ow, focus score, or autoantibody levels was detected, statistically signicant increased levels of IgM were detected in both the salivary glands and serum. IgG and IgA also increased in salivary glands and serum, but were not statistically signicance. Conclusion: Our ndings suggest that expression of soluble ICAM-1 in the salivary gland can have a stimulatory effect on B lymphocytes. So far, this and recent clinical data suggest that these observations are induced by activated T cells. Further research will be required to understand the mechanism of ICAM-1/LFA-1 interaction blockade on lymphocyte activation and the role of ICAM-1/LFA-1 interaction in the induction of SS pathogenesis. [1] Nikolay P. Nikolov, et al. Blocking LFA-1 with Efalizumab Promotes Inammation, T Cell Activation and Autoimmunity in Primary Sjögren’s Syndrome (pSS). Arthritis Rheum., 60 suppl 10: 499 (2009).
871. Modulation of CXCR4 Expression by Novel Growth Factor Cocktail on Stem Cells
Somayeh Shahrokhi,1 Kamran Alimoghaddam,2 Massoumeh Ebtekar,1 Zohre Shari,3 Seyed Hamidolah Ghaffari,2 Ali Akbar Pourfathollah,1 Mahboobeh Mohseni,1 Ardeshir Ghavamzadeh.2 1 Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran; 2 Hematology, Oncology and Stem Cell Transplantation Research Center of Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; 3Research Center, Department of Immunology, Iranian Blood Transfusion Organization, Tehran, Islamic Republic of Iran. Background: CXCR4 is critical adhesion molecule in homing process. Modulation of its expression on cord blood (CB) CD34+ could overcome delay following cord blood transplantation. Complicated network of growth factors including cytokines and neuropeptides in microenvironment has important role in regulation of this adhesion molecule. So, we aimed to assess role of 2 neuropeptide substance p (SP) and Calcitonin gene related peptide (CGRP) in addition to cytokine cocktail on CXCR4. Material and Methods: CD34+cells puried from CB were cultured in a serum-free liquid culture system. Different concentrations of SP and/or CGRP were used in combination with cytokine cocktail. Protein and genomic levels of CXCR4 was assessed by owcytometry and real time RT-PCR. Results: Our data show increased CXCR4+CD34+ cells cultured with SP and/or CGRP by day 7. Concentration 10-9M either SP or CGRP increased genomic expression of CXCR4 molecule, by day 11 compare to control group. Conclusions: Our experiment indicates that SP and CGRP induce CXCR4 protein expression in 7 days culture and enhance its genomic expression. Consequently, overexpression of CXCR4 improves engraftment of CB CD34+ cells.
Lung and Respiratory Disease Gene & Cell Therapy 872. Efcient Gene Delivery to Pig Airways Using Helper-Dependent Adenoviral Vectors
Huibi Cao,1 Jonathan C. Yeung,2 Cathleen Duan,1 Allan Coates,1,3 Kitty Leung,1 Shaf Keshavjee,2 Jim Hu.1,3,4 1 Physiology & Experimental Medicine, The Hospital for Sick Children, Toronto, ON, Canada; 2Thoracic Surgery Research Laboratories, Division of Thoracic Surgery, The Toronto General Hospital Research Institute, Toronto, ON, Canada; 3Departments of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 4Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Gene therapy is currently being developed for respiratory diseases such as Cystic Fibrosis. While various gene therapy vectors have been successfully used to deliver genes to mouse lungs, gene transfer to the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY airway epithelium of humans has proven to be inefcient. Because of signicant differences in anatomic and physiologic features between mouse and human lungs, development of large animal models for gene transfer is critical for the development of clinical lung gene therapy. Pigs share many features of lung biology with humans, including anatomy, histology, electrolyte transport, and immune responses. Their bronchi show similar patterns of branching and histology, possess similar abundance of submucosal glands and have similar products of secretion. Recently, a porcine CFTR-/- model has been created and the neonatal CF pigs displayed a high degree of similarity in electrophysiological and pathological changes to human neonates with CF (Rogers et al, Science, 2008). Therefore, pigs could become an important pre-clinical model for lung gene therapy. In this study, we used wild-type pigs as a model to investigate the technique of viral vector delivery, to explore the distribution and expression of foreign genes, and to analyze the host immune responses to Ad vector and transgene products in pig airways. Using an Aeroprobe catheter under exible bronchoscopic guidance, we aerosolized 5x1012 particles of HD-K18-LacZ in 5 ml of normal saline containing 0.1% L-a-lysophosphatidylcholine into the left lung. X-gal staining of reporter gene expression and histological analyses were performed 8 days post gene delivery. A robust level of reporter expression was detected by X-gal staining in tissue blocks from the left lung, but not the control right lung. The nuclear staining of the transduced cells in bronchial and alveolar epithelia was characteristic of the expression of the nuclear-targeted beta-galactosidase. More interestingly, many submucosal gland cells, the major CFTR-expressing cells in the airway, were transduced. Pigs tolerated the procedure and the vector very well. These results suggest that highly efcient delivery of HD-Ad vectors to the airway of large animals can be achieved and will provide important insights into the design of clinical studies for lung gene therapy.
873. SIV Vector Pseudotyped with SeV-F/ HN Envelope Proteins Produces Long Lasting Expression in the Murine Lung, Is Readministrable and Transfects Human Airway Models
Uta Griesenbach,1 Makoto Inoue,2 Cuixiang Meng,1 Andrea M. Brum,1 Raymond Farley,1 Nikki Newman,1 Eiji Akiba,2 Mamoru Hasegawa,2 Eric W. F. W. Alton.1 1 Department of Gene Therapy and UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, United Kingdom; 2 DNAVEC Coorporation, Tskuba, Japan.
We have previously shown that simian immunodeciency virus pseudotyped with F and HN protein from Sendai virus (F/HN-SIV) transduces murine nasal epithelium efciently (3-5% of respiratory epithelial cells after perfusion with 5x108 TU) and importantly can be repeatedly administered. We now show that expression in the nasal epithelium persists for the life-time of the animals (16-25 months, 9 of 9 C57Bl/6 mice). The vast majority of published studies have assessed lentiviral vectors in mouse nasal epithelium, and transduction of lung epithelium, particularly without pre-conditioning through polidocanol treatment or tissue damage, remains challenging. Here, we show that F/HN-SIV transduces lung epithelium efciently and dose-dependently. In contrast to other studies, we show that lentivirus-mediated gene expression in the lung is stable for at least 13 months (latest time-point currently analyzed) (month 2: 362660±63922photons/sec, month 13: 431454±65647photons/sec, n=5-8 mice). We also show, for the rst time, that lentivirus can be repeatedly administered to the lung (2 doses of F/HN-SIV-GFP followed by a 3rd dose of F/HN-SIV-Lux at monthly interval) without loss of activity compared to a single dose of F/HN-SIV-Lux (1 Dose: 40178±4843, 3 Doses: 39080±7490 RLU/mg protein, n=21/ group). Importantly, we also show that gene expression increases with increasing number of doses (10 doses at daily interval) (Dose 1: Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
4693± 899, Dose 10: 239212±48362 RLU/mg protein, n=8/group, p< 0.0005). Importantly, we have not observed acute or chronic toxicity in 12 months follow-up studies in mice. We have also assessed the performance of F/HN-SIV in various airway ex vivo models. (a) The virus transduces human air liquid interface cultures efciently with expression persisting for at least 4 months. (b) The virus transduces freshly obtained human nasal brushings dose-dependently. (c) The virus transduces human lung slices efciently and expression persists for the life-span of the slices. These data suggest that, F/HN-SIV may be a suitable vector for cystic brosis gene therapy.
874. Airway Lentiviral Gene Transfer In Marmosets
David Parsons,1,2,4 Richard Bright,2,4 Darren Miller,2 Don Anson.3,4 Respiratory and Sleep Medicine, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 2Women’s and Children’s Health Resarch Insitute, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 3SA Pathology, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 4Centre for Stem Cell Research, University of Adelaide, Adelaide, South Australia, Australia. 1
Preclinical studies examining effectiveness and safety in nonhuman primates (NHP) will be important in developing clinicallyappropriate gene transfer protocols to treat cystic brosis (CF) airway disease. Extended in-vivo lentiviral (LV) gene expression suited to long lasting CF airway correction has been described in mice (Stocker et al 2009, J Gene Med). We report ndings from the rst studies of LV gene transfer in lungs of the marmoset (Callithrix jacchus), a small NHP where lung anatomy and physiology is similar to humans. Methods: Pretreatment with lysophosphatidylcholine (LPC, 0.1%, 200-350 ul) was followed one hour later by LV-LacZ vector pseudotyped with vesicular stomatitis virus G (∼ 1.2 x109 TU/ ml, 350-500ul), each delivered via an ET tube into the mid-trachea of four anaesthetised animals (2 M, 2F). After 7 days trachea, lungs and other organs in two animals were examined for LacZ reporter gene expression via standard Xgal staining protocols. Regular blood samples, secretions and tissue samples were collected and examined for presence of vector particles. Results: A rapid transient O 2 desaturation was noted in some animals after LPC administration, but behavioural and physiological indices were normal post-procedure. Body weights followed usual post-anaesthesia trajectories. Patchy epithelial cell LacZ gene expression was evident en face and in cross-sections primarily in conducting airways. Discrete and limited patches of haemorrhage and neutrophil/mononuclear cell inltration were deemed unremarkable by a veterinary pathologist. No evidence of lacZ gene expression was observed in any other tissue. LV vector capsid protein levels (p24) were present in serum at Day 1 but absent from Day 2 onwards. Further histological, immunological and RTPCR analyses are in progress. Conclusions: These initial studies suggest LPC/LV dosing procedures are well-tolerated and are able to induce target-cell gene expression in a non-human primate. The remaining 2 animals are being maintained for longer-term assessment of the effects of the single-dose lentiviral lung gene transfer on lung gene expression.
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Vital Wohlgensinger,1 Christian Brendel,2 Axel Schambach,3 Manuel Grez,2 Janine Reichenbach,1 Reinhard A. Seger,1 Ulrich Siler.1 1 Immunology/Hematology/BMT, University Children’s Hospital Zürich, Zürich, Switzerland; 2Georg-Speyer Research Institute, Frankfurt A.M., Germany; 3Experimental Hematology, Hannover Medical School, Hannover, Germany. First clinical success in gene therapy could be achieved e.g. for X-CGD and X-SCID using retroviral LTR-driven vectors. Unfortunately these results were accompanied with clonal dominance or leukemic progression as long term side effects of transactivation events. To develop a safety-optimized vector for the p47phox-decient form of CGD we cloned various myelospecic promoter candidates driving p47phox expression into a gammaretroviral self-inactivating (SIN) vector. The constructs were screened for myelospecicity and functional reconstitution in transduced murine p47phox -/- HSC upon ex vivo propagation to granulocytes. This screen demarcated the panel of promoter candidates. One of the candidates was tested in vivo in p47phox -/- mouse model and compared to SFFV driven p47phox positive control. Six weeks after reinfusion of transduced murine HSC, mice treated with the vector containing a myeloid-specic promoter driving p47phox expression showed enhanced p47phox levels in granulocytes and low/no p47phox expression in monocytes, lymphocytes and hematopoietic stem/progenitor cells in contrast to the SFFV control. Mice treated with this vector showed functional reconstitution of the NADPH-oxidase function in granulocytes as revealed by a DHR assay. p47phox CGD gene therapy using SIN vectors with a myeloid specic promoters results in tissue-specic expression of the p47phox transgene and restoration of NADPH function in granulocytes in the murine p47phox -/- CGD model.
870. Delivery of Soluble ICAM-1 in NOD Salivary Glands Can Result in B Cell Proliferation and Immunoglobulin Production
Jelle L. Vosters,1,2 Nienke Roescher,1,2 Paul P. Tak,1 John A. Chiorini.2 1 Division of Clinical Immunology and Rheumatology, Academic Medical Center, Amsterdam, Netherlands; 2Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, MD.
Objective: Sjögren’s syndrome (SS) is an autoimmune disease with unknown etiology. Accumulation and activation of lymphocytes in the salivary glands of SS patients is an important hallmark of the disease. Lymphocyte function-associated antigen-1 (LFA-1) is the receptor on lymphocytes, which binds endothelial or lymphocyte expressed Intercellular Adhesion Molecule-1 (ICAM-1), resulting in lymphocyte migration, co-stimulation and activation. Previously, a clinical trial in our department using anti-LFA-1 in SS patients resulted in exacerbated SS and induced signicant increase in local inammation and systemic autoimmunity [1]. The aim of this study is to elucidate the role of ICAM-1/LFA-1 in an autoimmune animal model by testing the effect on inammation by local expression of soluble ICAM-1. Methods and Materials: Using in vivo adeno associated viral gene transfer, we stably expressed soluble ICAM1:Fc fusion protein locally in the salivary glands in the Non-Obese Diabetic animal model of SS. Pilocarpine stimulated saliva ow was measured to address the salivary gland function and salivary glands were analyzed for focus score, composition of the inltrate, and immunoglobulins (Ig’s). Results: Local expression of ICAM1:Fc resulted in a signicant increase in B220-positive cells in the salivary glands. Also, trends towards decreased plasma cells (CD138), S336
increased CD4+ and CD8+ T cells were detected. While no change in salivary ow, focus score, or autoantibody levels was detected, statistically signicant increased levels of IgM were detected in both the salivary glands and serum. IgG and IgA also increased in salivary glands and serum, but were not statistically signicance. Conclusion: Our ndings suggest that expression of soluble ICAM-1 in the salivary gland can have a stimulatory effect on B lymphocytes. So far, this and recent clinical data suggest that these observations are induced by activated T cells. Further research will be required to understand the mechanism of ICAM-1/LFA-1 interaction blockade on lymphocyte activation and the role of ICAM-1/LFA-1 interaction in the induction of SS pathogenesis. [1] Nikolay P. Nikolov, et al. Blocking LFA-1 with Efalizumab Promotes Inammation, T Cell Activation and Autoimmunity in Primary Sjögren’s Syndrome (pSS). Arthritis Rheum., 60 suppl 10: 499 (2009).
871. Modulation of CXCR4 Expression by Novel Growth Factor Cocktail on Stem Cells
Somayeh Shahrokhi,1 Kamran Alimoghaddam,2 Massoumeh Ebtekar,1 Zohre Shari,3 Seyed Hamidolah Ghaffari,2 Ali Akbar Pourfathollah,1 Mahboobeh Mohseni,1 Ardeshir Ghavamzadeh.2 1 Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran; 2 Hematology, Oncology and Stem Cell Transplantation Research Center of Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; 3Research Center, Department of Immunology, Iranian Blood Transfusion Organization, Tehran, Islamic Republic of Iran. Background: CXCR4 is critical adhesion molecule in homing process. Modulation of its expression on cord blood (CB) CD34+ could overcome delay following cord blood transplantation. Complicated network of growth factors including cytokines and neuropeptides in microenvironment has important role in regulation of this adhesion molecule. So, we aimed to assess role of 2 neuropeptide substance p (SP) and Calcitonin gene related peptide (CGRP) in addition to cytokine cocktail on CXCR4. Material and Methods: CD34+cells puried from CB were cultured in a serum-free liquid culture system. Different concentrations of SP and/or CGRP were used in combination with cytokine cocktail. Protein and genomic levels of CXCR4 was assessed by owcytometry and real time RT-PCR. Results: Our data show increased CXCR4+CD34+ cells cultured with SP and/or CGRP by day 7. Concentration 10-9M either SP or CGRP increased genomic expression of CXCR4 molecule, by day 11 compare to control group. Conclusions: Our experiment indicates that SP and CGRP induce CXCR4 protein expression in 7 days culture and enhance its genomic expression. Consequently, overexpression of CXCR4 improves engraftment of CB CD34+ cells.
Lung and Respiratory Disease Gene & Cell Therapy 872. Efcient Gene Delivery to Pig Airways Using Helper-Dependent Adenoviral Vectors
Huibi Cao,1 Jonathan C. Yeung,2 Cathleen Duan,1 Allan Coates,1,3 Kitty Leung,1 Shaf Keshavjee,2 Jim Hu.1,3,4 1 Physiology & Experimental Medicine, The Hospital for Sick Children, Toronto, ON, Canada; 2Thoracic Surgery Research Laboratories, Division of Thoracic Surgery, The Toronto General Hospital Research Institute, Toronto, ON, Canada; 3Departments of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 4Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Gene therapy is currently being developed for respiratory diseases such as Cystic Fibrosis. While various gene therapy vectors have been successfully used to deliver genes to mouse lungs, gene transfer to the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY airway epithelium of humans has proven to be inefcient. Because of signicant differences in anatomic and physiologic features between mouse and human lungs, development of large animal models for gene transfer is critical for the development of clinical lung gene therapy. Pigs share many features of lung biology with humans, including anatomy, histology, electrolyte transport, and immune responses. Their bronchi show similar patterns of branching and histology, possess similar abundance of submucosal glands and have similar products of secretion. Recently, a porcine CFTR-/- model has been created and the neonatal CF pigs displayed a high degree of similarity in electrophysiological and pathological changes to human neonates with CF (Rogers et al, Science, 2008). Therefore, pigs could become an important pre-clinical model for lung gene therapy. In this study, we used wild-type pigs as a model to investigate the technique of viral vector delivery, to explore the distribution and expression of foreign genes, and to analyze the host immune responses to Ad vector and transgene products in pig airways. Using an Aeroprobe catheter under exible bronchoscopic guidance, we aerosolized 5x1012 particles of HD-K18-LacZ in 5 ml of normal saline containing 0.1% L-a-lysophosphatidylcholine into the left lung. X-gal staining of reporter gene expression and histological analyses were performed 8 days post gene delivery. A robust level of reporter expression was detected by X-gal staining in tissue blocks from the left lung, but not the control right lung. The nuclear staining of the transduced cells in bronchial and alveolar epithelia was characteristic of the expression of the nuclear-targeted beta-galactosidase. More interestingly, many submucosal gland cells, the major CFTR-expressing cells in the airway, were transduced. Pigs tolerated the procedure and the vector very well. These results suggest that highly efcient delivery of HD-Ad vectors to the airway of large animals can be achieved and will provide important insights into the design of clinical studies for lung gene therapy.
873. SIV Vector Pseudotyped with SeV-F/ HN Envelope Proteins Produces Long Lasting Expression in the Murine Lung, Is Readministrable and Transfects Human Airway Models
Uta Griesenbach,1 Makoto Inoue,2 Cuixiang Meng,1 Andrea M. Brum,1 Raymond Farley,1 Nikki Newman,1 Eiji Akiba,2 Mamoru Hasegawa,2 Eric W. F. W. Alton.1 1 Department of Gene Therapy and UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, United Kingdom; 2 DNAVEC Coorporation, Tskuba, Japan.
We have previously shown that simian immunodeciency virus pseudotyped with F and HN protein from Sendai virus (F/HN-SIV) transduces murine nasal epithelium efciently (3-5% of respiratory epithelial cells after perfusion with 5x108 TU) and importantly can be repeatedly administered. We now show that expression in the nasal epithelium persists for the life-time of the animals (16-25 months, 9 of 9 C57Bl/6 mice). The vast majority of published studies have assessed lentiviral vectors in mouse nasal epithelium, and transduction of lung epithelium, particularly without pre-conditioning through polidocanol treatment or tissue damage, remains challenging. Here, we show that F/HN-SIV transduces lung epithelium efciently and dose-dependently. In contrast to other studies, we show that lentivirus-mediated gene expression in the lung is stable for at least 13 months (latest time-point currently analyzed) (month 2: 362660±63922photons/sec, month 13: 431454±65647photons/sec, n=5-8 mice). We also show, for the rst time, that lentivirus can be repeatedly administered to the lung (2 doses of F/HN-SIV-GFP followed by a 3rd dose of F/HN-SIV-Lux at monthly interval) without loss of activity compared to a single dose of F/HN-SIV-Lux (1 Dose: 40178±4843, 3 Doses: 39080±7490 RLU/mg protein, n=21/ group). Importantly, we also show that gene expression increases with increasing number of doses (10 doses at daily interval) (Dose 1: Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
4693± 899, Dose 10: 239212±48362 RLU/mg protein, n=8/group, p< 0.0005). Importantly, we have not observed acute or chronic toxicity in 12 months follow-up studies in mice. We have also assessed the performance of F/HN-SIV in various airway ex vivo models. (a) The virus transduces human air liquid interface cultures efciently with expression persisting for at least 4 months. (b) The virus transduces freshly obtained human nasal brushings dose-dependently. (c) The virus transduces human lung slices efciently and expression persists for the life-span of the slices. These data suggest that, F/HN-SIV may be a suitable vector for cystic brosis gene therapy.
874. Airway Lentiviral Gene Transfer In Marmosets
David Parsons,1,2,4 Richard Bright,2,4 Darren Miller,2 Don Anson.3,4 Respiratory and Sleep Medicine, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 2Women’s and Children’s Health Resarch Insitute, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 3SA Pathology, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 4Centre for Stem Cell Research, University of Adelaide, Adelaide, South Australia, Australia. 1
Preclinical studies examining effectiveness and safety in nonhuman primates (NHP) will be important in developing clinicallyappropriate gene transfer protocols to treat cystic brosis (CF) airway disease. Extended in-vivo lentiviral (LV) gene expression suited to long lasting CF airway correction has been described in mice (Stocker et al 2009, J Gene Med). We report ndings from the rst studies of LV gene transfer in lungs of the marmoset (Callithrix jacchus), a small NHP where lung anatomy and physiology is similar to humans. Methods: Pretreatment with lysophosphatidylcholine (LPC, 0.1%, 200-350 ul) was followed one hour later by LV-LacZ vector pseudotyped with vesicular stomatitis virus G (∼ 1.2 x109 TU/ ml, 350-500ul), each delivered via an ET tube into the mid-trachea of four anaesthetised animals (2 M, 2F). After 7 days trachea, lungs and other organs in two animals were examined for LacZ reporter gene expression via standard Xgal staining protocols. Regular blood samples, secretions and tissue samples were collected and examined for presence of vector particles. Results: A rapid transient O 2 desaturation was noted in some animals after LPC administration, but behavioural and physiological indices were normal post-procedure. Body weights followed usual post-anaesthesia trajectories. Patchy epithelial cell LacZ gene expression was evident en face and in cross-sections primarily in conducting airways. Discrete and limited patches of haemorrhage and neutrophil/mononuclear cell inltration were deemed unremarkable by a veterinary pathologist. No evidence of lacZ gene expression was observed in any other tissue. LV vector capsid protein levels (p24) were present in serum at Day 1 but absent from Day 2 onwards. Further histological, immunological and RTPCR analyses are in progress. Conclusions: These initial studies suggest LPC/LV dosing procedures are well-tolerated and are able to induce target-cell gene expression in a non-human primate. The remaining 2 animals are being maintained for longer-term assessment of the effects of the single-dose lentiviral lung gene transfer on lung gene expression.
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