Retroviral Insertion Sites Research Articles

InCliniGene enables high-throughput andcomprehensive in vivo clonal tracking toward clinical genomics data integration.

High-throughput clonal tracking in patients under hematopoietic stem cell gene therapy with integrating vector is instrumental in assessing bio-safety and efficacy. Monitoring the fate of millions of transplanted clones and their progeny across differentiation and proliferation over time leverages the identification of the vector integration sites, used as surrogates of clonal identity. Although γ-tracking retroviral insertion sites (γ-TRIS) is the state-of-the-art algorithm for clonal identification, the computational drawbacks in the tracking algorithm, based on a combinatorial all-versus-all strategy, limit its use in clinical studies with several thousands of samples per patient. We developed the first clonal tracking graph database, InCliniGene (https://github.com/calabrialab/InCliniGene), that imports the output files of γ-TRIS and generates the graph of clones (nodes) connected by arches if two nodes share common genomic features as defined by the γ-TRIS rules. Embedding both clonal data and their connections in the graph, InCliniGene can track all clones longitudinally over samples through data queries that fully explore the graph. This approach resulted in being highly accurate and scalable. We validated InCliniGene using an in vitro dataset, specifically designed to mimic clinical cases, and tested the accuracy and precision. InCliniGene allows extensive use of γ-TRIS in large gene therapy clinical applications and naturally realizes the full data integration of molecular and genomics data, clinical and treatment measurements and genomic annotations. Further extensions of InCliniGene with data federation and with application programming interface will support data mining toward precision, personalized and predictive medicine in gene therapy. Database URL: https://github.com/calabrialab/InCliniGene.

Characterization of an Acute Myeloid Leukemia Murine Model Driven By MLL/AF9: Effect of Retroviral Insertion Sites and Somatic Mutations on Gene Expression

The MLL/AF9 fusion is found in approximately 30% of MLL-rearranged leukemias and has an intermediate prognosis. Genomically well-characterized murine leukemia models enable us to understand leukemogenesis.We generated a retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) using the MLL/AF9 fusion gene. Fifteen of 20 mice transplanted with syngeneic bone marrow transduced with a MLL/AF9 carrying retrovirus developed leukemia after a median latency of 149 days. Half a million leukemic bone marrow (LBM) cells from two of these primary leukemias, MA03-P and MA86-P, were transplanted into irradiated recipient mice to establish secondary leukemias, MA03-S (n=3) and MA86-S (n=4). Half a million LBM cells from these secondary leukemias were further transplanted into irradiated recipient mice to generate tertiary leukemias, MA03-T (n=3) and MA86-T (n=4). The latency of the leukemias shortened from 141 days in MA03-P to 18 and 22 days in MA03-S and MA03-T, respectively. Similarly, MA86-P had a latency of 98 days, and the latency was reduced to about 28 days in MA986-S and MA986-T.We used retroviral insertion sites (RISs) to track leukemia clones during serial transplantation. We identified 5 RISs in MA03-P. One RIS, RIS#1-03 at chromosome 7:4602500-4609499 accounted for 52.5% of the total RIS-related reads in MA03-P, while the other four RISs were each represented by fewer than 5% of the reads. Only RIS#1-03 was detected in all of the MA03 secondary and tertiary leukemias , indicating that the cells with RIS#1-03 were the dominant clone in MA03 leukemias. Two RISs were detected in MA86-P: RIS#1-86 at chromosome 19:41338500-41341999 and RIS#2-86 at chromosome 10:127106000-127109499 at 46.7% and 2.5%, respectively . RIS#1-986 was contained in the dominant clone as only this RIS was subsequently detected in the secondary and tertiary MA86 leukemias.The relatively long latency to leukemia development in our MLL/AF9 model was most likely due to the requirement of cooperating somatic mutations. We performed whole exome sequencing on DNA from LBM (n=15) and DNA from their corresponding germline (n=2). An average of 4.5 of single nucleotide variants (SNVs) and 11.4 indels affecting protein coding sequences were found in the MA03 family of leukemias (n=7) which, among others, mutated genes involved in tyrosine kinase pathways such as Epha5 and Pik3r1. We identified an average of 14.8 (SNVs) and 0.5 indels per exome in the MA86 leukemias (n=8). Transcription regulator (Brd1) and tumor suppressor genes (Stk11 and Trp53) were affected by somatic changes in the MA86 family.RNA sequencing was performed on LBM (n=15) and healthy bone marrow (HBM) (n=8). Principal component analysis (PCA) on the expression profiles showed that LBM samples clustered together. Differential gene expression analysis identified genes such as Six1, Eya1 and Bcor which had been reported in previous studies to be essential for leukemogenesis in MLL/AF9 murine model. We also observed downregulation of genes such as Gata2, Btg1, Ifitm1, which had been implicated in other types of leukemias.We next investigated the effect of the RISs and somatic mutations on gene expression. RIS#1-903 was in intron 1 of Ppp6r1. A reduction in fragments per kilobase of transcript per Million mapped reads (FPKM) of Ppp6r1 was observed in MA03 family leukemias compared to leukemias of the MA86 family which did not have RIS#1-03 and showed no difference to HBM samples (MA03: 87.71±1.5; MA86: 132.1±5.1; HBM: 77.56±1.7, p< 0.001). We then determined the expression of Tm9sf3 as it is located 600bp away from RIS#1-986. The FPKM of Tm9sf3 was significantly higher in LBM (both of MA903 and MA986 leukemias) than in HBM (LBM: 146.0±12.7; HBM: 64.66±2.8, p<0.001). In MA86 leukemias which all have RIS#1-86, the FPKM of Tm9sf was two fold higher than in MA03 leukemias without RIS#1-86 (MA86: 189.3±4.4; MA03:97.59±1.7, p < 0.001). In contrast, none of the somatic mutations had a significant effect on the expression of any of the mutated genes.In conclusion, we have established a MBMTLM driven by the MLL/AF9 fusion gene. This well-characterized model provides insights to further understand leukemia development and drug testing. Moreover, we demonstrated that RISs can have an impact on gene expression. Future work on whether Ppp6r1 and Tm9sf3 identified by our RIS analysis are drivers in MLL/AF9 leukemias is warranted. DisclosuresBrowett: MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

Multiple Genes Surrounding Bcl-xL, a Common Retroviral Insertion Site, Can Influence Hematopoiesis Individually or in Concert

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS) Bcl-xL, which revealed seven genes located within ±150 kb from the RVIS for our study. We confirmed that Bcl-xL enhanced the competitiveness of HSPCs, whereas the Bcl-xL neighbor Id1 hindered HSPC long-term repopulation. This negative influence of Id1 could be counteracted by co-expressing Bcl-xL. Interestingly, >90% of early reconstituted myeloid cells were found to originate from transduced HSPCs upon simultaneous overexpression of Bcl-xL and Id1, which implies that Bcl-xL and Id1 can collaborate to rapidly replenish the myeloid compartment under stress conditions. To directly compare the competitiveness of HSPCs conveyed by multiple transgenes, we developed a multiple competitor competitive repopulation (MCCR) assay to simultaneously screen effects on HSPC repopulating capacity in a single mouse. The MCCR assay revealed that multiple genes within a CIS can have positive or negative impact on hematopoiesis. Furthermore, these data highlight the importance of studying multiple genes located within the proximity of an insertion site to understand complex biological effects, especially as the number of gene therapy patients increases.

VISPA2: a scalable pipeline for high-throughput identification and annotation of vector integration sites

BackgroundBioinformatics tools designed to identify lentiviral or retroviral vector insertion sites in the genome of host cells are used to address the safety and long-term efficacy of hematopoietic stem cell gene therapy applications and to study the clonal dynamics of hematopoietic reconstitution. The increasing number of gene therapy clinical trials combined with the increasing amount of Next Generation Sequencing data, aimed at identifying integration sites, require both highly accurate and efficient computational software able to correctly process “big data” in a reasonable computational time.ResultsHere we present VISPA2 (Vector Integration Site Parallel Analysis, version 2), the latest optimized computational pipeline for integration site identification and analysis with the following features: (1) the sequence analysis for the integration site processing is fully compliant with paired-end reads and includes a sequence quality filter before and after the alignment on the target genome; (2) an heuristic algorithm to reduce false positive integration sites at nucleotide level to reduce the impact of Polymerase Chain Reaction or trimming/alignment artifacts; (3) a classification and annotation module for integration sites; (4) a user friendly web interface as researcher front-end to perform integration site analyses without computational skills; (5) the time speedup of all steps through parallelization (Hadoop free).ConclusionsWe tested VISPA2 performances using simulated and real datasets of lentiviral vector integration sites, previously obtained from patients enrolled in a hematopoietic stem cell gene therapy clinical trial and compared the results with other preexisting tools for integration site analysis. On the computational side, VISPA2 showed a > 6-fold speedup and improved precision and recall metrics (1 and 0.97 respectively) compared to previously developed computational pipelines. These performances indicate that VISPA2 is a fast, reliable and user-friendly tool for integration site analysis, which allows gene therapy integration data to be handled in a cost and time effective fashion. Moreover, the web access of VISPA2 (http://openserver.itb.cnr.it/vispa/) ensures accessibility and ease of usage to researches of a complex analytical tool. We released the source code of VISPA2 in a public repository (https://bitbucket.org/andreacalabria/vispa2).

An association between type A porcine endogenous retrovirus copy number and hematological parameters and gender in miniature pigs

Pig is the most promising species for transplantation of organs and cells into humans, although implementation of xenotransplantation in clinical practice has been hindered by the risk of infecting the recipient with zoonotic infectious diseases. Porcine endogenous retroviruses (PERV) are capable of incorporating copies of DNA into the genome of a host cell. Based on the nucleotide sequence of the envelope gene (env), three main types of pig retrovirus, PERV-A, PERV-B and PERV-C, have been recognized, with PERV-A and PERV-B having the capability of infecting human cell lines in vitro. Selection for animals with low copy number of retroviruses in the genome using simple phenotypic indications is required for the widespread implementation of xenotransplantation. The objective of this study was to evaluate the correlation between PERV-A env gene copy number and hematological parameters, gender and coat color in miniature pigs of the Institute of Cytology and Genetics (ICG) SB RAS. Reference values for eighteen blood parameters of miniature pigs were determined, including white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), absolute (LYM#) and relative (LYM%) lymphocyte counts, absolute (MID#) and relative (MID%) monocyte, basophil and eosinophil counts, absolute (GRA#) and relative (GRA%) granulocyte counts, hematocrit (HCT) and thrombocrit (PCT), mean cell volume (MCV) and mean platelet volume (MPV). Males had significantly higher reference values for WBC, MID#, GRA# and red cell distribution width (RDW-CV) as compared to females. The mean corpuscular hemoglobin concentration (MCHC) and platelet distribution width (PDW-CV) were significantly higher in female animals. No correlation between PERV-A env gene copy number and the coat color of animals was detected, suggesting that retroviral insertion sites and genes that determine the coat color of miniature pigs, namely KIT (chromosome 8) and MC1R (chromosome 6), are either located far apart on same chromosome or on different chromosomes. The copy number of PERV-A env gene in males was lower than in females. Presence of multiple copies of PERV-A on the X-chromosome is the most probable cause of such gender-related differences in miniature pigs. Thus, male miniature pigs of ICG SB RAS should be the source of material for xenotransplantation.

High-Throughput Mapping and Clonal Quantification of Retroviral Integration Sites.

We describe here a method to identify the position of retroviral insertion sites and simultaneously to quantify the absolute abundance of each clone, i.e., the number of cells having the provirus inserted at a given place in the host genome. The method is based on random shearing of the host cell DNA, followed by a linker-mediated PCR to amplify the genomic regions flanking the proviruses, and high-throughput sequencing of the amplicons. The quantification of the abundance of each infected clone allowed us to develop two new metrics: i. the oligoclonality index, which quantifies the nonuniformity of the distribution of clone abundance, and ii. an estimator of the total number of clones in the body of the host. These new tools are valuable for the study of retroviral infections and can also be adapted for the tracking of gene-edited cells.

The LORE1 insertion mutant resource.

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity canbe used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes.

The NCI Retrovirus Integration Database is a MySql-based relational database created for storing and retrieving comprehensive information about retroviral integration sites, primarily, but not exclusively, HIV-1. The database is accessible to the public for submission or extraction of data originating from experiments aimed at collecting information related to retroviral integration sites including: the site of integration into the host genome, the virus family and subtype, the origin of the sample, gene exons/introns associated with integration, and proviral orientation. Information about the references from which the data were collected is also stored in the database. Tools are built into the website that can be used to map the integration sites to UCSC genome browser, to plot the integration site patterns on a chromosome, and to display provirus LTRs in their inserted genome sequence. The website is robust, user friendly, and allows users to query the database and analyze the data dynamically. Availability: https://rid.ncifcrf.gov; or http://home.ncifcrf.gov/hivdrp/resources.htm.

523. Human Umbilical Vein Endothelial Cell, HUVEC, Co-Culture Promotes Robust Expansion and Maintains Phenotypic Integrity of Rhesus Hematopoietic Stem and Progenitor Cells, HSPC, Prior to Autologous Tansplantation

The development of ex-vivo HSPC expansion techniques is particulary relevant for improving cord blood transplantation and gene therapy. Despite successful long-term, multilineage reconstitution of expanded human cord blood HSPC in immunodeficient mice, early phase clinical trials have failed to demonstrate improved outcomes. Thus, it is critical to develop a robust pre-clinical model to study ex-vivo expansion strategies, particularly function of long term HSPC and engrafment of all lineages, difficult in xenograft models. We have previously used retroviral insertion site retrieval and autologous competitive transplantation in rhesus macaques to track hematopoietic engraftment and ontogeny at a single HSPC level, in addition to comparing ex-vivo expanded and unexpanded HSPC (Gomes et al, Mol Ther). More recently we have developed retrieval of 31bp diverse barcodes as a more robust and quantitative in vivo HSPC tracking approach (Wu et al, Cell Stem Cell). Modified human endothelial cells (HUVEC) with the ability to be maintained in serum free media for prolonged periods have shown to support and expand human HSPC in murine Butler et al, Blood). We have developed a barcoded rhesus autologous transplant model to evaluate expansion of HPSC on HUVEC versus unexpanded or cytokine expanded rhesus HSPC. We first tested the feasibility of rhesus CD34+ cell expansion on HUVEC versus cytokines/fibronectin for 8 days. We also evaluated for the diversity of expanded HSPC by retrieving transduced viral barcode DNA tags from individual cells by low cycle PCR followed by Illumina sequencing. On average, rhesus CD34+ cells expanded 76 fold (± 53) on HUVEC versus 13 fold (±7) in cytokines (n=4; p=0.03). By morphologic assessment, the HUVEC expanded cell fraction contained higher numbers of progenitors and differentiating cells, with up to 97% CD34+CD45+ cells in the HUVEC expanded fraction and 65% in the cytokine expanded fraction. The HUVEC expanded rhesus CD34+ cells were capable of multi-lineage colony formation after 8 days of expansion. Within the HUVEC expanded cell fractions, fold expansion was similar whether CD34+ cells were frozen after (C1) or before (C2) transduction-expansion. However, viability and percentages of CD34+CD45+ cells were higher in C2 compared to C1 (55% versus 26%). GFP transduction efficiency was also higher in C2 compared to C1 (43% vs. 28%). We demonstrated a slightly lower percentage but higher absolute number of CD34+CD38-CD45RA-CD90+CD49f+Rho-low putative long-term HSC after expansion on HUVEC compared to in cytokines over 26 days in culture (0.018% vs. 0.016% pre and post expansion, respectively). Retrieved barcode analysis and quantitation revealed that in the HUVEC expanded fraction, the top 20 clones constituted up 15% of the total valid reads by day 8, in contrast to only 3.9% of total clones in the cytokine expanded fraction. Cells have been transplanted into autologous macaques, in a competitive model comparing non-expanded, cytokine-expanded and HUVEC-expanded conditions, and in vivo expansion and barcode analysis will be presented. Our data thus far show that peripheral blood CD34+ cells from rhesus macaques expand more robustly in the presence of HUVEC and cytokines as compared to expansion in cytokines alone, and barcode analysis suggests that the HUVEC fraction selectively expanded a subset of highly proliferative cells, which may represent true HSCs within the CD34+ cell fraction.

18F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility

Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75(+)-selected donor T cells (1.0-13 × 10(5))/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-((18)F)fluoro-3-hydroxymethyl-butyl]guanine ([(18)F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [(18)F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [(18)F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation.

532. Foamy Viral Vector Integration Sites in SCID-Repopulating Cells After MGMTP140K-Mediated In Vivo Selection

Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector transduced human repopulating cells is needed. Human CD34+ human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT) P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rgnull (NSG) mice, and selected in vivo for gene-modified cells. The retroviral insertion site (RIS) profile of repopulating clones was examined using modified genomic sequencing PCR (MGS-PCR). We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus (SFFV) promoter known to be genotoxic. However we did observe that highly captured sites were found more often near proto-oncogenes than less frequently captured sites (Figure 1Figure 1). Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy, but also suggests additional safety features should be developed and evaluated.Figure 1(a) Proximity of RIS to TSS of proto-oncogenes in Network of Cancer Genes 4.0 (NCG 4.0) cancer gene list (n = 2048). (b) Proportion of RIS within NCG 4.0 proto-oncogene transcripts or within 50 kb of a proto-oncogene TSS. In vivo low and in vivo high represent the lowest and highest thirds of RIS ranked by recovery frequency. Abbreviations: ns, not significant; RIS, retroviral insertion site; TSS, transcription start site.View Large Image | Download PowerPoint Slide

Foamy viral vector integration sites in SCID-repopulating cells after MGMTP140K-mediated in vivo selection.

Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector-transduced human repopulating cells is needed. Human CD34(+) human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT)P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rγ(null) mice, and selected in vivo for gene-modified cells. The retroviral insertion site profile of repopulating clones was examined using modified genomic sequencing PCR. We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus promoter known to be genotoxic. Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy but also suggests additional safety features should be developed and evaluated.

Haploid genetic dissection of immune molecule trafficking (TECH1P.836)

Abstract To perform their expected functions, immune molecules must be targeted to specific cellular compartments such as the plasma membrane. Improper protein targeting can lead to major forms of immune disease such as immunodeficiency and persistent inflammation. Our group developed a novel haploid genetics approach in order to unravel protein targeting on a genome-wide scale. We take advantage of human myeloid cell lines (e.g. KBM7 and HAP1) that exhibit a haploid karyotype. Retroviral insertional mutagenesis of these cells offers an efficient method to generate a library of mutant cells with complete disruption of specific genes. After selections of desired phenotypes (e.g., mislocalization of a protein), the retroviral insertion sites of the mutant clones can be mapped by deep sequencing. We have used this haploid genetics approach to characterize the plasma membrane protein CD59, a key player in the complement system. We identified ~20 genes involved in the biosynthesis, maturation, and trafficking of CD59. These genes are involved in GPI lipid synthesis, oligosaccharide modification, as well as protein trafficking at both the early and late stages of the secretory pathway. This haploid genetic screen provides the first genome-wide map of a complement protein. Many factors identified in the screen represent novel components of the complement system, and may serve as therapeutic targets for treating immune disorders.

Retroviral insertional mutagenesis as a platform to study proto-oncogenes and their collaborating partners

Retroviral insertional mutagenesis (RIM) is a well known adverse effect in gene therapy trials which can lead to leukemia or preleukemic conditions. However, it can also serve as a powerful tool to study cancer and stem cell biology by studying genes adjacent to insertion sites. This study is to demonstrate the capacity of RIM to study proto-oncogenes in stem cell regulation and their potential collaborating partners in leukemogenesis. By recording retroviral insertion sites from hematopoietic dominant clones in murine bone marrow transplantation (BMT) model, our group has developed the Insertional Dominance Database (IDDb).

Abstract PR03: Genome-wide screens for Wnt signaling in human haploid cells

Abstract Wnt signaling is at the core of human development and stem cell biology. Defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer. Using HAP1 cells, a human haploid cell line, we conducted a set of genome-wide forward genetic screens to identify new Wnt pathway components. We constructed a HAP1 line harboring a GFP reporter driven by the Wnt-responsive TCF promoter, we mutagenized the cells by insertion of a gene-trap retrovirus throughout the haploid genome, and we sorted the mutagenized population for cells exhibiting phenotypes of interest using FACS. In order to find 1. negative regulators, 2. positive regulators and 3. feedback modulators of the pathway, we sorted for 1. cells expressing GFP in the absence of Wnt, 2. cells with reduced GFP expression after strong Wnt stimulation and 3. cells with enhanced GFP expression after mild Wnt stimulation, respectively. Deep sequencing of amplified genomic DNA flanking the retroviral insertion sites in the sorted cell populations revealed genes whose disruption may have led to the phenotype of interest. Among the most frequently disrupted genes (some with over 150 unique retroviral insertions) we found many of the known components of the canonical Wnt pathway, including LRP6, casein kinase I, APC, β-catenin, DOT1-L, TCF4 and the recently discovered negative feedback regulator ZNRF3. Unexpectedly, the screen for positive regulators revealed retroviral insertions in AXIN2 (a scaffold protein found in the β-catenin destruction complex) predicted to yield a C-terminally truncated form that appears to act as a dominant negative. We are currently assessing hits not previously known to be involved in Wnt signaling. This abstract is also presented as Poster A40. Citation Format: Andres Lebensohn, Casey Hughes, Caleb Marceau, Rajat Rohatgi, Jan Carette. Genome-wide screens for Wnt signaling in human haploid cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR03.

A novel haploid genetics approach to dissect immune pathways (P3235)

Abstract Unbiased loss-of-function genetic screens in model organisms have been major driving forces for modern biological research. However, many biological functions of humans such as adaptive immunity are not found in model organisms amenable to classic genetic screens. Despite tremendous efforts, human somatic cell genetics remains unattainable due to the inability to efficiently induce homozygous mutations in diploid human cells. Our group developed a novel haploid genetics approach to dissect immune pathways in human cells. We take advantage of human myeloid cells lines (e.g. KBM7) that exhibit a haploid karyotype. Retroviral insertional mutagenesis of the haploid cells generates a library of mutant cells with individual genes completely disrupted. First, we validated the haploid mutant library by screening for mutants resistant to the cytotoxicity of diphtheria toxin. After selection, the retroviral insertion sites of the resistant clones were mapped by deep sequencing. We identified HBEGF, the known cell surface receptor for diphtheria toxin. We also isolated multiple endocytic membrane proteins, which likely mediate the internalization of diphtheria toxin. Next, we demonstrated that the haploid mutant library is also suitable for studying complex immune pathways such as the elimination of tumor cells by cytotoxic T lymphocytes. This haploid genetics approach provides a novel and powerful platform for dissecting immune pathways on a genome-wide scale.

P051 Mapping the TNF-induced necroptosis pathway by experimental test-tube genetics in human haploid cells

Introduction TNF signaling can result in NF-kB and MAPK activation, apoptosis or necrosis depending on cell type and cell state. Until recently necrosis has been seen as an uncontrolled mode of cell death when compared to the programmed apoptosis. The identification of RIP3 as a crucial mediator of TNF-induced necrosis clearly demonstrates that in many cases necrosis is controlled by a dedicated signaling cascade, leading to the definition of necroptosis [3] , [4] , [6] . Increasing evidences demonstrate that RIP3-mediated necroptosis participates in inflammation and in both innate and adaptive immunity. Despite this, the signaling events downstream of RIP3 kinase activation remain largely obscure. Methods Here we describe the design of a haploid genetic screen in human KBM7 cells that aims to identify new components of TNF-induced RIP3-dependent necroptosis signaling pathways. The KBM7 chronic myeloid leukemia cell line is haploid for all chromosomes except chromosome 8. This allows gene inactivation through random insertional mutagenesis using a retroviral gene-trap vector and subsequent insertion site mapping [1] , [2] . Results Our data demonstrate that the TNF signaling pathway is functional in KBM7 cells resulting in NF-kB and MAPK activation, apoptosis or necroptosis depending on defined experimental settings. Under necroptosis inducing conditions, cell death occurs without detectable caspase activation and is suppressed by the RIP1 kinase inhibitor necrostatin-1. Based on these results, mutagenized KBM7 cells will be submitted to TNF-induced necroptosis and resistant clones mapped to identify genes required for necroptotic cell death. Mixed lineage kinase domain-like protein (MLKL) has been recently shown to participate in TNF-induced necroptosis [5] . An MLKL-deficient KBM7 cell clone is completely resistant to TNF-induced necroptosis while retaining sensitivity to TNF-induced apoptosis. This finding confirms by gene inactivation in human cells that MLKL is crucial for TNF-necroptosis and, importantly, validates our screening approach. Conclusion We expect the screen to genetically define the essential molecular elements required for necroptosis. Identified components will then be characterized using affinity-purification coupled to mass spectrometry. Our study should thus contribute significantly to the molecular understanding of necroptosis, a process whose relevance in inflammation, innate and adaptive immunity has just began to be fully appreciated.

MiRNA Lentiviral Vector Integration and Gene Targeting Efficacy in Cardiac Progenitors

Stem and progenitor cardiac cells are challenging for possible cell therapy application. Several research laboratories are exploiting the feasibility of autologous cell therapy approach to get rid of immunosuppressive treatments responsible for undesirable side effects. Recently, we showed that cardiac progenitors isolated from Sgcb null mice, animal model of limb-girdle muscular dystrophy type 2E, undergo an aberrant differentiation in vitro and in vivo due to the dysregulation of miR669. This miRNA family is able to inhibit the skeletal myogenic program directly targeting MyoD 3’ UTR. Using lentiviral technology we provided evidence that it is possible to rescue the dystrophic aberrant phenotype by miRNA669overexpression without gene correction. However, how the viruses carrying the miRNAs were positioned in the genome upon transduction and how their localization site could influence the rescue potential was not analysed. Here we investigate the integration profile of lentiviral vector carrying the pre-miR669 in infected polyclonal and clonal populations derived from Sgcb cardiac progenitors. Our study reveals that the retroviral insertion sites (RIS) are largely restricted to coding genes (65%). Although with the limitation of our analysis, we found no hits for cancer-related genes and several sequenced RIS brought to light genes mainly involved in muscle function. Thus our data show that lentiviral vector insertional profile is cell-specific, however, the chromatin state of target cells positively influences the viral integrations.

Assessing Potential Genotoxicity of Sleeping Beauty Transposition Events in T-Cell Immunotherapy by Supercomputer-Based High Throughput Profiling,

Abstract 4174The first-in-human clinical trial using Sleeping Beauty (SB) transposition for T-cell immunotherapy is under way at the University of Texas MD Anderson Cancer Center. To understand potential genotoxic risks involved in our non-viral SB transposition-based immunotherapy, we employed high throughput sequencing (Illumina) and profiling (BlueBioU supercomputer) of the SB insertion sites in T cells. Our SB clinical vector inserted 99.999% of the time at expected TA dinucleotide sites, with 44% of the insertions localizing to intragenic loci and the remainder 56% intergenic. The vast majority (96.5%) of intragenic insertions are intronic while the majority (>60%) of intergenic transpositions fall within non-coding repeat regions. By linking microarray gene expression profile to the vector insertion profile, we find that the number of transcriptional start sites (TSS) hit is proportional to the expected ‘open’ conformational loci for the starting population of quiescent T cells. Compared to immunotherapy infusing T cells genetically modified with retrovirus, the integration profiles for SB-modified T cells are favored as: 1) there are more insertions within intergenic regions (56% SB vs. 44% in retroviral), 2) insertions concentrated at TA sites which have a different genomic distribution profile than retroviral insertion sites, 3) TSS associated with quiescent T cells were favored reflecting the electro-transfer of SB DNA plasmids into non-proliferating T cells vs. retroviral protocols that require transduction of activated T cells. Scanning for potential danger loci (e.g. oncogene, tumor suppressor, miRNA etc.), SB integration profiles compare favorably with retroviral insertions. In addition, we identified potential “safe harbor” genetic loci for future targeted modifications. Opportunities in immunotherapy and other personalized medicine modalities will be fully realized when the actual and perceived risks are understood and perhaps mitigated. In the absence of targeted insertions by clinical vectors, the next best option is random integration with an evenly distributed insertion profile across the whole genome to minimize hot spot and clonal expansion of therapeutic T cells. We now seek to proactively profile the T-cell products prior to infusion and follow up sampling to help clinicians in data-driven decision making in a time-sensitive manner. Our methods and findings are consistent with the human application of SB system and the harnessing high throughput supercomputing resources to help clinicians mitigate potential risks. Disclosures:No relevant conflicts of interest to declare.