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369. Mechanisms That Control Transformation of Polyclonal Mature T Cells
BIOLOGY OF AAV AND VECTOR DEVELOPMENT 368. Gammaretroviral Vector Integration Occurs Overwhelmingly at Boarders of DNase Hypersensitive Sites
David W. Emery, Mingdong Liu, Michael O. Dorschner, John Stamatoyannopoulos. Departments of Medicine and Genome Sciences, University of Washington, Seattle, WA. Concerns surrounding the oncogenic potential of recombinant gammaretroviral vectors has spurred a great deal of interest into vector integration site (VIS) preferences. Although gammaretroviral vectors exhibiting a modest preference for integration near the promoters of transcriptionally active genes, such associations only account for about a third of all VISs. Early studies of murine leukemia virustransformed cell clones, and more recent studies with recombinant gammaretroviral vectors, also indicated a correlation between VISs and DNase hypersensitive sites (DHSs), which mark open chromatin regions and are often associated with cis-regulatory elements such as enhancers and promoters. Although compelling, these previous studies are difficult to interpret; the older studies involved the use of functionally selected clones, while the latter studies compared VISs in one cell type to DHSs from a different cell type with a potentially very different DHS pattern. In order to study this issue more directly, we looked for correlations between gammaretroviral VISs identified in the absence of selection, and DNase hypersensitive sites mapped within the same cell line (the human fibrosarcoma HT1080) using a method called digital DNase mapping. As described elsewhere [Sabo et al., PNAS 101:16837, 2004], this approach involves sequencing individual DNaseI cleavage sites using massively parallel sequencing technologies. We compared this DHS map to 165 experimental VISs identified independently by conventional methods, as well as to 100 simulated random VISs as a negative control. These studies revealed that gammaretroviral vectors have an overwhelming preference for integrations near, but not within, DHSs. The median distance between individual VISs and their nearest DHS was only 536 bp for the experimental data set, compared to over 3 kb for the random control dataset, with a cumulative distribution that was significantly different (P<0.001, KS test). Over 75% of all VIS were within 1 kb of their nearest DHS, while approximately 89% were within 2 kb (P<0.01 in both cases). This preference was independent of the association between individual VISs and promoters (P=0.42), and is far greater then the ∼30% preference seen for promoters and active genes. As such, we believe the preference for promoters is independent of the promoters per se, and instead reflects the prevalence of DHSs at active gene promoters. The prevalence of VISs at DHSs located at even long distances from promoters, combined with the proposition that such DHSs play an important role in the regulation of distally located genes, suggest that such VISs may nevertheless represent a potential for cellular gene dysregulation. Our results also suggest that gammaretroviral vectors are either targeting the boarders of these sites because they are physically more accessible, or because they are specifically bound by proteins that mediate vector targeting. Future studies will be needed to distinguish between these two options.
369. Mechanisms That Control Transformation of Polyclonal Mature T Cells
Sebastian Newrzela,1 Zhixiong Li,2 Kerstin Cornils,3 Boris Fehse,3 Dorothee von Laer.1 1 Georg-Speyer-Haus, Frankfurt am Main, Germany; 2Hannover Medical School, Hannover, Germany; 3University Medical Center Hamburg Eppendorf, Hamburg, Germany. Background: Retroviral insertional mutagenesis in hematopoietic progenitor cells can activate neighboring proto-oncogenes and thus contribute to leukaemia development. Here, we analysed whether mature T cells bear the same risk of insertional mutagenesis. Methods: S144
To address this issue, we used the Rag-1 mouse model, which allows long-term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ∆TrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. Results: After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphoma/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes. None of the control vector-transduced mice developed malignancies. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell-transplanted animals showed no signs of leukaemia development during a follow-up of more than 1 year. Conclusions: Our results show that polyclonal mature T cells in vivo are less susceptible to transformation by known T cell proto-oncogenes than progenitor cells. The mechanisms that control transformation of mature T cells are currently under investigation. Interestingly, we found that in vitro, T cells can be immortalized by retroviral transfer of a T cell oncogene. In addition, monoclonal T lymphocytes derived from OT1 mice can be transformed with dTrkA with the same kinetics as hematopoietic stem cells in vivo, indicating that clonal competition may control leukemogenesis in mature T cell populations.
Biology of AAV and Vector Development 370. Structural Studies of Adeno-Associated Virus Serotypes towards Improved Gene Therapy Applications
Mavis Agbandje-McKenna,1 Lakshmanan Govindasamy,1 HyunJoo Nam,1 Brittney Gurda,1 Robert Ng,1 Robert McKenna,1 Sergei Zolotukhin,1 Nicholas Muzyczka,1 Norm Olson,2 Timothy Baker,2 John A. Chiorini,3 Olga Kozyreva,4 R. Jude Samulski.4 1 University of Florida, Gainesville, FL; 2University of California, San Diego, San Diego, CA; 3GTTB, NIDCR, National Institutes of Health, Bethesda, MD; 4University of North Carolina at Chapel, Chapel Hill, NC. The development of viruses, such as the Adeno-associated viruses (AAV), to deliver corrective genes into the cells or tissues of a target organism has gained momentum over the past few years. AAVs are members of the dependovirus genus of the Parvoviridae family, requiring a helper virus to cause a productive infection. They are non-pathogenic and non-toxic, and can package, and deliver foreign DNA into cells, making them attractive vectors for gene therapy applications. Twelve distinct AAV serotypes (AAV1-12) have been sequenced, and more than 100 additional variants have been identified in human and non-human primate tissues. Molecular studies indicate that each serotype has unique cellular transduction characteristics. In addition, mutagenesis, cell binding, and transduction studies show that the AAV capsid VP amino acid sequences play a central role in the observed tissue recognition and transduction efficiency disparities. These observations and a goal to enhance the efficacy of AAV gene therapy applications, by viral capsid modifications for specific cell/tissue targeting, has generated a need for a detailed understanding of their capsid structures and interactions with cell surface ligands. Towards this goal, we are conducting structural studies of the representative members of the AAV antigenic clades and clonal isolates, alone and complexed with receptor ligands. We have determined the structures of AAV1, AAV4, AAV5, AAV7, AAV8, and AAV9 to ∼3.0 Å resolution by X-ray crystallography, the structure of AAV6 alone to ∼9.7 Å and of AAV2 complexed with heparin sulfate to ∼18 Å resolution by cryo-EM and image reconstruction. X-ray diffraction data has also been collected for AAV6 compexed with Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
David W. Emery, Mingdong Liu, Michael O. Dorschner, John Stamatoyannopoulos. Departments of Medicine and Genome Sciences, University of Washington, Seattle, WA. Concerns surrounding the oncogenic potential of recombinant gammaretroviral vectors has spurred a great deal of interest into vector integration site (VIS) preferences. Although gammaretroviral vectors exhibiting a modest preference for integration near the promoters of transcriptionally active genes, such associations only account for about a third of all VISs. Early studies of murine leukemia virustransformed cell clones, and more recent studies with recombinant gammaretroviral vectors, also indicated a correlation between VISs and DNase hypersensitive sites (DHSs), which mark open chromatin regions and are often associated with cis-regulatory elements such as enhancers and promoters. Although compelling, these previous studies are difficult to interpret; the older studies involved the use of functionally selected clones, while the latter studies compared VISs in one cell type to DHSs from a different cell type with a potentially very different DHS pattern. In order to study this issue more directly, we looked for correlations between gammaretroviral VISs identified in the absence of selection, and DNase hypersensitive sites mapped within the same cell line (the human fibrosarcoma HT1080) using a method called digital DNase mapping. As described elsewhere [Sabo et al., PNAS 101:16837, 2004], this approach involves sequencing individual DNaseI cleavage sites using massively parallel sequencing technologies. We compared this DHS map to 165 experimental VISs identified independently by conventional methods, as well as to 100 simulated random VISs as a negative control. These studies revealed that gammaretroviral vectors have an overwhelming preference for integrations near, but not within, DHSs. The median distance between individual VISs and their nearest DHS was only 536 bp for the experimental data set, compared to over 3 kb for the random control dataset, with a cumulative distribution that was significantly different (P<0.001, KS test). Over 75% of all VIS were within 1 kb of their nearest DHS, while approximately 89% were within 2 kb (P<0.01 in both cases). This preference was independent of the association between individual VISs and promoters (P=0.42), and is far greater then the ∼30% preference seen for promoters and active genes. As such, we believe the preference for promoters is independent of the promoters per se, and instead reflects the prevalence of DHSs at active gene promoters. The prevalence of VISs at DHSs located at even long distances from promoters, combined with the proposition that such DHSs play an important role in the regulation of distally located genes, suggest that such VISs may nevertheless represent a potential for cellular gene dysregulation. Our results also suggest that gammaretroviral vectors are either targeting the boarders of these sites because they are physically more accessible, or because they are specifically bound by proteins that mediate vector targeting. Future studies will be needed to distinguish between these two options.
369. Mechanisms That Control Transformation of Polyclonal Mature T Cells
Sebastian Newrzela,1 Zhixiong Li,2 Kerstin Cornils,3 Boris Fehse,3 Dorothee von Laer.1 1 Georg-Speyer-Haus, Frankfurt am Main, Germany; 2Hannover Medical School, Hannover, Germany; 3University Medical Center Hamburg Eppendorf, Hamburg, Germany. Background: Retroviral insertional mutagenesis in hematopoietic progenitor cells can activate neighboring proto-oncogenes and thus contribute to leukaemia development. Here, we analysed whether mature T cells bear the same risk of insertional mutagenesis. Methods: S144
To address this issue, we used the Rag-1 mouse model, which allows long-term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ∆TrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. Results: After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphoma/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes. None of the control vector-transduced mice developed malignancies. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell-transplanted animals showed no signs of leukaemia development during a follow-up of more than 1 year. Conclusions: Our results show that polyclonal mature T cells in vivo are less susceptible to transformation by known T cell proto-oncogenes than progenitor cells. The mechanisms that control transformation of mature T cells are currently under investigation. Interestingly, we found that in vitro, T cells can be immortalized by retroviral transfer of a T cell oncogene. In addition, monoclonal T lymphocytes derived from OT1 mice can be transformed with dTrkA with the same kinetics as hematopoietic stem cells in vivo, indicating that clonal competition may control leukemogenesis in mature T cell populations.
Biology of AAV and Vector Development 370. Structural Studies of Adeno-Associated Virus Serotypes towards Improved Gene Therapy Applications
Mavis Agbandje-McKenna,1 Lakshmanan Govindasamy,1 HyunJoo Nam,1 Brittney Gurda,1 Robert Ng,1 Robert McKenna,1 Sergei Zolotukhin,1 Nicholas Muzyczka,1 Norm Olson,2 Timothy Baker,2 John A. Chiorini,3 Olga Kozyreva,4 R. Jude Samulski.4 1 University of Florida, Gainesville, FL; 2University of California, San Diego, San Diego, CA; 3GTTB, NIDCR, National Institutes of Health, Bethesda, MD; 4University of North Carolina at Chapel, Chapel Hill, NC. The development of viruses, such as the Adeno-associated viruses (AAV), to deliver corrective genes into the cells or tissues of a target organism has gained momentum over the past few years. AAVs are members of the dependovirus genus of the Parvoviridae family, requiring a helper virus to cause a productive infection. They are non-pathogenic and non-toxic, and can package, and deliver foreign DNA into cells, making them attractive vectors for gene therapy applications. Twelve distinct AAV serotypes (AAV1-12) have been sequenced, and more than 100 additional variants have been identified in human and non-human primate tissues. Molecular studies indicate that each serotype has unique cellular transduction characteristics. In addition, mutagenesis, cell binding, and transduction studies show that the AAV capsid VP amino acid sequences play a central role in the observed tissue recognition and transduction efficiency disparities. These observations and a goal to enhance the efficacy of AAV gene therapy applications, by viral capsid modifications for specific cell/tissue targeting, has generated a need for a detailed understanding of their capsid structures and interactions with cell surface ligands. Towards this goal, we are conducting structural studies of the representative members of the AAV antigenic clades and clonal isolates, alone and complexed with receptor ligands. We have determined the structures of AAV1, AAV4, AAV5, AAV7, AAV8, and AAV9 to ∼3.0 Å resolution by X-ray crystallography, the structure of AAV6 alone to ∼9.7 Å and of AAV2 complexed with heparin sulfate to ∼18 Å resolution by cryo-EM and image reconstruction. X-ray diffraction data has also been collected for AAV6 compexed with Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy