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610. An Integrated Approach To Discover New Liver Cancer Genes and Test the Genotoxicity of Vectors for Liver Gene Therapy
RNA VIRUS VECTORS and Gas6, its human homologue. Gas6 enhances native infectivity for pseudotypes of multiple viral envelope proteins, including the wild-type Sindbis virus, Ross river virus, and baculovirus envelope proteins. Gas6 mediates binding of the virus to target cells by bridging virion envelope phosphatidylserine to Axl, a TAM receptor tyrosine kinase present on target cells. Phosphatidlyserine is a surface marker of apoptotic cells, and Gas6 was originally known to mediate clearance of apoptotic cells by phagocytes by bridging exposed phosphatidylserine on apoptotic cells to Axl on phagocytes. Our results demonstrated that envelope viruses and vectors can enter target cells using an apoptotic mimicry pathway, which is a novel alternative molecular mechanism of viral entry that can broaden host ranges and enhance infectivity of enveloped viruses and viral vectors. Our results also provided insight into the role of envelope lipid in binding and tropism of envelope viruses and vectors.
610. An Integrated Approach To Discover New Liver Cancer Genes and Test the Genotoxicity of Vectors for Liver Gene Therapy
Marco Ranzani,1 Alessio Cantore,1 Daniela Cesana,1 Cynthia Bartholomae,2 Francesca Sanvito,3 Mauro Pala,4 Fabrizio Benedicenti,1 Monica Volpin,1 Pierangela Gallina,1 Lucia Sergi Sergi,1 Alessandro Bulfone,4 Claudio Doglioni,3 Christof von Kalle,2 Manfred Schmidt,2 Giovanni Tonon,5 Luigi Naldini,1 Eugenio Montini.1 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 National Center for Tumor Diseases, Heidelberg, Germany; 3 Mouse Pathology San Raffaele Hospital, Milan, Italy; 4Bio)) flag Ltd, Pula, Italy; 5Functional Genomic of Cancer Unit, San Raffaele Scientific Institute, Milan, Italy.
Efficient liver gene transfer and long term transgene expression may allow the treatment of several hepatic and systemic diseases. However, vector integration may occasionally lead to transformation of hepatocytes, as reported for AAV vectors in mice. Therefore, sensitive preclinical models to assess the genotoxicity of vector integration in liver are needed. We devised and validated sensitive genotoxicity assays based on Cdkn2a or Pten deficient and wild type (WT) mouse strains. As a positive control of insertional mutagenesis in hepatocytes, a lentiviral vector (LV) carrying Enhanced Transthyretin enhancer/promoter (ET) in the Long Terminal Repeats (LV.ET.LTR) was generated and used to induce hepatocellular carcinoma (HCC) in mice. Systemic LV.ET.LTR injection induced HCCs in 30% of Cdkn2a-/- mice (p<0.01), 25% of Pten liver-null mice (p<0.05) and 75% of WT mice in combination with CCl4 treatment (p<0.01). By the retrieval of vector integrations from LV-induced HCCs, we identified 4 putative liver cancer genes: the well characterized Braf and Sos1 from Ras pathway, Fign whose functions are poorly understood, and the Dlk1-Dio3 imprinted region. Forced expression of the newly identified cancer genes in the mouse liver induced HCCs, thus validating their causative role in hepatocarcinogenesis. Gene expression analysis and data-mining of human cancer “omics” databases showed that these loci are frequently upregulated and/or amplified in human HCCs and that their activation leads to specific expression signatures that can distinguish different human tumor stages. Therefore, the newly identified cancer genes play a relevant role also in human hepatocarcinogenesis and the experimental models recapitulate molecular features of human HCCs. We exploited these mouse models to test the safety of self-inactivating (SIN) LVs developed for the therapy of hemophilia B. A SINLV that express factor IX transcript under the control of ET was injected in Cdkn2a-/(N=39) or WT mice (N=24). The HCC incidence was monitored till 1 year after treatment. The therapeutic vector did not induce HCCs as assessed by histopathological analysis neither in tumor prone nor WT mice + CCl4. Conversely, the mice injected with matched doses of LV.ET.LTR developed HCCs at frequency similar to previous S234
experiments. Analysis of integration to compare SINLVs and LV.ET. LTR integration pattern are currently ongoing. This study provides encouraging results on the biosafety profile of SINLVs-based liver gene therapy and uncovers the role in hepatocarcinogensis of some previously known and other completely new oncogenes that altogether may be novel candidate therapeutic targets and diagnostic markers for human HCCs.
611. Lentiviral Vector Common Integration Sites in the ALD and MLD Clinical Trials and in a Preclinical Model Reflect a Benign Integration Bias and Not Oncogenic Selection
Alessandra Biffi,1 Cynthia C. Bartolomae,2 Daniela Cesana,1 Natalie Cartier,3 Marco Ranzani,1 Martina Cesani,1 Fabrizio Benedicenti,1 Tiziana Plati,1 Merella Stefania,1 Alessia Capotondo,1 Jacopo Sgualdino,1 Patrick Aubourg,3 Gianluigi Zanetti,4 Christof von Kalle,2 Manfred Schmidt,2 Luigi Naldini,1 Eugenio Montini.1 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 National Center for Tumor Diseases, Heidelberg, Italy; 3INSERM, University Paris-Descartes, Paris, France; 4Center for Advanced Studies, Research, and Development in Sardinia, Pula, Italy. HIV-derived lentiviral vectors (LV) have shown good efficacy and safety data in preclinical models and in a recent Hematopoietic Stem Cell (HSC)-based clinical trial for X-linked adrenoleukodystrophy (ALD). However, a careful analysis of LV integration sites in ALD patients’ derived cells showed that a relevant number of Common Insertion Sites (CIS) were present. This observation raises concerns because the detection of CIS is a well established hallmark of insertional mutagenesis in mice and clinical trials. Indeed, in γretroviral-based clinical trials, different patients developed malignancies triggered by vector integrations at CIS recurrently targeting the same protooncogenes. Thus, it is possible that the occurrence of CIS in the ALD clinical trial is a still silent effect of genotoxicity caused by LV integrations that confer a selective advantage. To understand if CIS generated by LV integrations are the product of genotoxicity, we generated our own dataset of LV integrations from transduced human HSC after transplant in immunodeficient mice. This integration profile was compared to LV integrations found in the ALD clinical trial, in other gene therapy trials that reported insertional mutagenesis, as well as in retroviral and transposon-mediated oncogene tagging studies in mice. LV CIS in hematochimeras were the same found in ALD patients and clustered in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CIS found in tumor cells from clinical trials and oncogenes tagging studies in mice, do not form clusters, target always a single gene and are contained in narrow genomic intervals. Further statistical analysis show that genotoxic CIS, but not LV CIS, have a higher targeting frequency with respect neighboring genes. Moreover, integration site analysis was also performed on a patient enrolled in the recent clinical trial for the cure of metachromatic leukodystrophy (MLD) in Milan, Italy (6-months follow-up). The 5000 unique LV integration sites obtained from transduced in vitro cultured CD34 cells and in different tissues and cell types at 30, 90 and 120 days after transplant, provide a photograph of the early phases of engraftment and multilineage repopulation. A preliminary analysis of the MLD integration profile show strong similarities to our experimental model and the ALD clinical trial. CIS genes, which are largely overlapping are also clustered in the same megabase-wide chromosomal regions. These findings imply that LV CIS in our experimental model, the ALD and MLD clinical trials are produced by an integration bias towards specific genomic regions rather than by oncogenic selection.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
610. An Integrated Approach To Discover New Liver Cancer Genes and Test the Genotoxicity of Vectors for Liver Gene Therapy
Marco Ranzani,1 Alessio Cantore,1 Daniela Cesana,1 Cynthia Bartholomae,2 Francesca Sanvito,3 Mauro Pala,4 Fabrizio Benedicenti,1 Monica Volpin,1 Pierangela Gallina,1 Lucia Sergi Sergi,1 Alessandro Bulfone,4 Claudio Doglioni,3 Christof von Kalle,2 Manfred Schmidt,2 Giovanni Tonon,5 Luigi Naldini,1 Eugenio Montini.1 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 National Center for Tumor Diseases, Heidelberg, Germany; 3 Mouse Pathology San Raffaele Hospital, Milan, Italy; 4Bio)) flag Ltd, Pula, Italy; 5Functional Genomic of Cancer Unit, San Raffaele Scientific Institute, Milan, Italy.
Efficient liver gene transfer and long term transgene expression may allow the treatment of several hepatic and systemic diseases. However, vector integration may occasionally lead to transformation of hepatocytes, as reported for AAV vectors in mice. Therefore, sensitive preclinical models to assess the genotoxicity of vector integration in liver are needed. We devised and validated sensitive genotoxicity assays based on Cdkn2a or Pten deficient and wild type (WT) mouse strains. As a positive control of insertional mutagenesis in hepatocytes, a lentiviral vector (LV) carrying Enhanced Transthyretin enhancer/promoter (ET) in the Long Terminal Repeats (LV.ET.LTR) was generated and used to induce hepatocellular carcinoma (HCC) in mice. Systemic LV.ET.LTR injection induced HCCs in 30% of Cdkn2a-/- mice (p<0.01), 25% of Pten liver-null mice (p<0.05) and 75% of WT mice in combination with CCl4 treatment (p<0.01). By the retrieval of vector integrations from LV-induced HCCs, we identified 4 putative liver cancer genes: the well characterized Braf and Sos1 from Ras pathway, Fign whose functions are poorly understood, and the Dlk1-Dio3 imprinted region. Forced expression of the newly identified cancer genes in the mouse liver induced HCCs, thus validating their causative role in hepatocarcinogenesis. Gene expression analysis and data-mining of human cancer “omics” databases showed that these loci are frequently upregulated and/or amplified in human HCCs and that their activation leads to specific expression signatures that can distinguish different human tumor stages. Therefore, the newly identified cancer genes play a relevant role also in human hepatocarcinogenesis and the experimental models recapitulate molecular features of human HCCs. We exploited these mouse models to test the safety of self-inactivating (SIN) LVs developed for the therapy of hemophilia B. A SINLV that express factor IX transcript under the control of ET was injected in Cdkn2a-/(N=39) or WT mice (N=24). The HCC incidence was monitored till 1 year after treatment. The therapeutic vector did not induce HCCs as assessed by histopathological analysis neither in tumor prone nor WT mice + CCl4. Conversely, the mice injected with matched doses of LV.ET.LTR developed HCCs at frequency similar to previous S234
experiments. Analysis of integration to compare SINLVs and LV.ET. LTR integration pattern are currently ongoing. This study provides encouraging results on the biosafety profile of SINLVs-based liver gene therapy and uncovers the role in hepatocarcinogensis of some previously known and other completely new oncogenes that altogether may be novel candidate therapeutic targets and diagnostic markers for human HCCs.
611. Lentiviral Vector Common Integration Sites in the ALD and MLD Clinical Trials and in a Preclinical Model Reflect a Benign Integration Bias and Not Oncogenic Selection
Alessandra Biffi,1 Cynthia C. Bartolomae,2 Daniela Cesana,1 Natalie Cartier,3 Marco Ranzani,1 Martina Cesani,1 Fabrizio Benedicenti,1 Tiziana Plati,1 Merella Stefania,1 Alessia Capotondo,1 Jacopo Sgualdino,1 Patrick Aubourg,3 Gianluigi Zanetti,4 Christof von Kalle,2 Manfred Schmidt,2 Luigi Naldini,1 Eugenio Montini.1 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 National Center for Tumor Diseases, Heidelberg, Italy; 3INSERM, University Paris-Descartes, Paris, France; 4Center for Advanced Studies, Research, and Development in Sardinia, Pula, Italy. HIV-derived lentiviral vectors (LV) have shown good efficacy and safety data in preclinical models and in a recent Hematopoietic Stem Cell (HSC)-based clinical trial for X-linked adrenoleukodystrophy (ALD). However, a careful analysis of LV integration sites in ALD patients’ derived cells showed that a relevant number of Common Insertion Sites (CIS) were present. This observation raises concerns because the detection of CIS is a well established hallmark of insertional mutagenesis in mice and clinical trials. Indeed, in γretroviral-based clinical trials, different patients developed malignancies triggered by vector integrations at CIS recurrently targeting the same protooncogenes. Thus, it is possible that the occurrence of CIS in the ALD clinical trial is a still silent effect of genotoxicity caused by LV integrations that confer a selective advantage. To understand if CIS generated by LV integrations are the product of genotoxicity, we generated our own dataset of LV integrations from transduced human HSC after transplant in immunodeficient mice. This integration profile was compared to LV integrations found in the ALD clinical trial, in other gene therapy trials that reported insertional mutagenesis, as well as in retroviral and transposon-mediated oncogene tagging studies in mice. LV CIS in hematochimeras were the same found in ALD patients and clustered in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CIS found in tumor cells from clinical trials and oncogenes tagging studies in mice, do not form clusters, target always a single gene and are contained in narrow genomic intervals. Further statistical analysis show that genotoxic CIS, but not LV CIS, have a higher targeting frequency with respect neighboring genes. Moreover, integration site analysis was also performed on a patient enrolled in the recent clinical trial for the cure of metachromatic leukodystrophy (MLD) in Milan, Italy (6-months follow-up). The 5000 unique LV integration sites obtained from transduced in vitro cultured CD34 cells and in different tissues and cell types at 30, 90 and 120 days after transplant, provide a photograph of the early phases of engraftment and multilineage repopulation. A preliminary analysis of the MLD integration profile show strong similarities to our experimental model and the ALD clinical trial. CIS genes, which are largely overlapping are also clustered in the same megabase-wide chromosomal regions. These findings imply that LV CIS in our experimental model, the ALD and MLD clinical trials are produced by an integration bias towards specific genomic regions rather than by oncogenic selection.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy