Dataset Information

Evaluating the Safety of Retroviral Vectors Based on Insertional Oncogene Activation and Blocked Differentiation in Cultured Thymocytes.

ABSTRACT: Insertional oncogenesis due to retroviral (RV) vector integration has caused recurrent leukemia in multiple gene therapy trials, predominantly due to vector integration effects at the LMO2 locus. While currently available preclinical safety models have been used for evaluating vector safety, none have predicted or reproduced the recurrent LMO2 integrations seen in previous X-linked severe combined immunodeficiency (X-SCID) and Wiskott-Aldrich clinical gene therapy trials. We now describe a new assay for assessing vector safety that recapitulates naturally occurring insertions into Lmo2 and other T-cell proto-oncogenes leading to a preleukemic developmental arrest in primary murine thymocytes cultured in vitro. This assay was used to compare the relative oncogenic potential of a variety of gamma-RV and lentiviral vectors and to assess the risk conferred by various transcriptional elements contained in these genomes. Gamma-RV vectors that contained full viral long-terminal repeats were most prone to causing double negative 2 (DN2) arrest and led to repeated cases of Lmo2 pathway activation, while lentiviral vectors containing these same elements were significantly less prone to activate proto-oncogenes or cause DN2 arrest. This work provides a new preclinical assay that is especially relevant for assessing safety in SCID disorders and provides a new tool for designing safer RV vectors.

SUBMITTER: Zhou S

PROVIDER: S-EPMC4923324 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Evaluating the Safety of Retroviral Vectors Based on Insertional Oncogene Activation and Blocked Differentiation in Cultured Thymocytes.

Zhou Sheng S Fatima Soghra S Ma Zhijun Z Wang Yong-Dong YD Lu Taihe T Janke Laura J LJ Du Yang Y Sorrentino Brian P BP

Molecular therapy : the journal of the American Society of Gene Therapy 20160309 6

Insertional oncogenesis due to retroviral (RV) vector integration has caused recurrent leukemia in multiple gene therapy trials, predominantly due to vector integration effects at the LMO2 locus. While currently available preclinical safety models have been used for evaluating vector safety, none have predicted or reproduced the recurrent LMO2 integrations seen in previous X-linked severe combined immunodeficiency (X-SCID) and Wiskott-Aldrich clinical gene therapy trials. We now describe a new a ...[more]

PMID: 26957223

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Project description:Retroviral insertional mutagenesis screens, which identify genes involved in tumor development in mice, have yielded a substantial number of retroviral integration sites, and this number is expected to grow substantially due to the introduction of high-throughput screening techniques. The data of various retroviral insertional mutagenesis screens are compiled in the publicly available Retroviral Tagged Cancer Gene Database (RTCGD). Integrally analyzing these screens for the presence of common insertion sites (CISs, i.e., regions in the genome that have been hit by viral insertions in multiple independent tumors significantly more than expected by chance) requires an approach that corrects for the increased probability of finding false CISs as the amount of available data increases. Moreover, significance estimates of CISs should be established taking into account both the noise, arising from the random nature of the insertion process, as well as the bias, stemming from preferential insertion sites present in the genome and the data retrieval methodology. We introduce a framework, the kernel convolution (KC) framework, to find CISs in a noisy and biased environment using a predefined significance level while controlling the family-wise error (FWE) (the probability of detecting false CISs). Where previous methods use one, two, or three predetermined fixed scales, our method is capable of operating at any biologically relevant scale. This creates the possibility to analyze the CISs in a scale space by varying the width of the CISs, providing new insights in the behavior of CISs across multiple scales. Our method also features the possibility of including models for background bias. Using simulated data, we evaluate the KC framework using three kernel functions, the Gaussian, triangular, and rectangular kernel function. We applied the Gaussian KC to the data from the combined set of screens in the RTCGD and found that 53% of the CISs do not reach the significance threshold in this combined setting. Still, with the FWE under control, application of our method resulted in the discovery of eight novel CISs, which each have a probability less than 5% of being false detections.

Project description:The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system and CEBPA (encoding C/EBPalpha) is often mutated or deregulated in AML patients. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours. However, homozygous knockin mice in which wild type Cebpa has been replaced with a mutant allele (BRM2), which abolish the growth-repressing ability of C/EBPalpha, are viable, but at 8 weeks of age they display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older CebpaBRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia-like malignancy. This strongly suggests that secondary mutations in other loci must occur when developing leukemia in the CebpaBRM2/BRM2. In order to identify genes that cooperate with Cebpa mutations in the development of leukemia in CebpaBRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn CebpaBRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus, which incorporated into the genome and resulted in activation of oncogenes and leukemic progression. When leukemia was evident the mice were euthanized and analyzed. As expected the CebpaBRM2/BRM2 mice had a shorter latency than wildtype mice (186 vs. 276 days). The mice had enlarged spleen, thymus, and/or lymph nodes and were thoroughly investigated by histology, flow cytometry and southern in order to determine the leukemic phenotypes. Most of the CebpaBRM2/BRM2 mice developed an AML-like phenotype, whereas T cell leukemias were most prominent in wildtype mice, showing that mutating Cebpa specifically directs leukemic progression towards a myeloid direction. Genomic instability was analyzed by CGH. Finally, the retroviral insertion loci were identified through a splinkerette-aided PCR strategy. This led to the identification of several novel putative and previously unexplored oncogenes, which might collaborate with mutated Cebpa in the development of AML. Keywords: Disease state analysis

Dataset Information

Evaluating the Safety of Retroviral Vectors Based on Insertional Oncogene Activation and Blocked Differentiation in Cultured Thymocytes.

Publications

Evaluating the Safety of Retroviral Vectors Based on Insertional Oncogene Activation and Blocked Differentiation in Cultured Thymocytes.

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