Vector Insertion Research Articles

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8.

Recently, we described a modified AAV2 vector-AAV2-7m8-having a capsid-displayed peptide insertion of 10 amino acids with enhanced retinal transduction properties. The insertion of the peptide referred to as 7m8 is responsible for high-level gene delivery into deep layers of the retina when virus is delivered into the eye's vitreous. Here, we further characterize AAV2-7m8 mediated gene delivery to neural tissue and investigate the mechanisms by which the inserted peptide provides better transduction away from the injection site. First, in order to understand if the peptide exerts its effect on its own or in conjunction with the neighboring amino acids, we inserted the 7m8 peptide at equivalent positions on three other AAV capsids, AAV5, AAV8, and AAV9, and evaluated its effect on their infectivity. Intravitreal delivery of these peptide insertion vectors revealed that only AAV9 benefited from 7m8 insertion in the context of the retina. We then investigated AAV2-7m8 and AAV9-7m8 properties in the brain, to better evaluate the spread and efficacy of viral transduction in view of the peptide insertion. While 7m8 insertion led to higher intensity gene expression, the spread of gene expression remained unchanged compared to the parental serotypes. Our results indicate that the 7m8 peptide insertion acts by increasing efficacy of cellular entry, with little effect on the spread of viral particles in neural tissue. The effects of peptide insertion are capsid and tissue dependent, highlighting the importance of the microenvironment in gene delivery using AAV. Biotechnol. Bioeng. 2016;113: 2712-2724. © 2016 Wiley Periodicals, Inc.

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.

BackgroundThe bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system.ResultsTo fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a “landing pad” (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %.ConclusionsIntegration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

Vecuum: identification and filtration of false somatic variants caused by recombinant vector contamination.

Advances in sequencing technologies have remarkably lowered the detection limit of somatic variants to a low frequency. However, calling mutations at this range is still confounded by many factors including environmental contamination. Vector contamination is a continuously occurring issue and is especially problematic since vector inserts are hardly distinguishable from the sample sequences. Such inserts, which may harbor polymorphisms and engineered functional mutations, can result in calling false variants at corresponding sites. Numerous vector-screening methods have been developed, but none could handle contamination from inserts because they are focusing on vector backbone sequences alone. We developed a novel method-Vecuum-that identifies vector-originated reads and resultant false variants. Since vector inserts are generally constructed from intron-less cDNAs, Vecuum identifies vector-originated reads by inspecting the clipping patterns at exon junctions. False variant calls are further detected based on the biased distribution of mutant alleles to vector-originated reads. Tests on simulated and spike-in experimental data validated that Vecuum could detect 93% of vector contaminants and could remove up to 87% of variant-like false calls with 100% precision. Application to public sequence datasets demonstrated the utility of Vecuum in detecting false variants resulting from various types of external contamination. Java-based implementation of the method is available at http://vecuum.sourceforge.net/ CONTACT: swkim@yuhs.acSupplementary information: Supplementary data are available at Bioinformatics online.

Characterizing seamless ligation cloning extract for synthetic biological applications

Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular and synthetic biology projects.

281. Enforced Expression of a Mutant HMGA2 Gene Leads to Competitive Expansion and High Level Marking of Long-Term Hematopoietic Stem Cells in Transplanted Nemestrina Macaques

An important goal in gene therapy and stem cell transplant is to expand hematopoietic stem cells (HSCs) without causing hematopoietic abnormalities. This is particularly challenging in humans and other primates, where expansion protocols that work in mouse models are often less effective in primate HSCs. One approach is to test genes that have led to clonal expansions by vector insertion site effects in human gene therapy trials. In this study, we recreated a mutant HMGA2 cDNA that was associated with a benign clonal expansion in a patient treated on a β-thalassemia gene therapy trial. A lentiviral vector was generated that expressed a cDNA for human HMGA2 with a 3’ UTR deletion that eliminates all seven let-7 microRNA binding sites and thereby avoids the known repressive effects of let-7 miRNAs on HMGA2 expression. A gamma-retroviral MSCV promoter was used to express this mutant HMGA2 cDNA along with an Ires-GFP cassette. Bone marrow CD34+ cells were collected for Nemestrina Macaques and half the cells were transduced with the lentiviral vector containing the HMGA2-GFP construct while the other half was transduced with a mCherry control vector. The transduction efficiencies were approximately the same prior to transplant. At three months post transplantation, the marking in the peripheral blood mononuclear cells was 2.9% GFP+ and 1.1% mCherry+ for animal #16, and was 2.7% GFP+ and 3.2% mCherry+ for animal #27, suggesting relatively equivalent and low levels of transduction of repopulating cells. The HMGA2-GFP marking progressively increased over 21 and 26 months in the peripheral blood leukocytes to 39% for #16 and 41% for #27 while the mCherry marked cells have decreased (Fig 1AFig 1A). Equivalent levels of marking were seen in various mature peripheral blood lineages, including circulating erythyrocytes, suggesting that expansion had occurred in pluripotent HSCs. This HSC expansion was further demonstrated by a marking analysis in bone marrow cells that showed 44.6% and 75.2% GFP marking in the CD34+CD45RA- HSC compartment for animal #16 and #27 respectively, at the latest time point. Clonality analyses using vector integration sites (VIS) showed overall oligoclonal marking in both the GFP+ and mCherry+ cells in both animals (Fig 1BFig 1B). The top most frequent VISs were present in all PB lineages indicating that expansion had occurred in multiple HSC clones. The WBC counts, lineage distribution in the peripheral blood, the percentage of CD34+ cells in the bone marrow and all the mature lineages in the peripheral blood are all within the normal range, demonstrating lack of any detectable hematopoietic abnormality. Gene expression microarray analysis of RNA from sorted BM CD34+GFP+ and CD34+mCherry+ cells showed sharp upregulation of several genes in the HMGA2-expressing cells, particularly the IGF2BP gene, a known downstream target of HMGA2. In summary, our data show that long-term HSCs from non-human primates can be progressively expanded in vivo over several years by overexpressing HMGA2. This could be useful for gene therapy, particularly for expanding and obtaining high numbers of transduced erythrocytes and granulocytes for diseases in which a naturally occurring selection advantage is not present.View Large Image | Download PowerPoint Slide

686. Forced Fetal-Globin Reactivation Mediated by the TALE-ldb1 Fusion Protein

Sickle cell disease (SCD) and thalassemia are very severe inherited disorders of the red cell and are amongst the world's most common genetic diseases. Thalassemia is caused by a serious lack of normal β-globin production in red cells whereas, sickle cell disease reflects a mutant hemoglobin that produces protein aggregates. The production of fetal-globin (HbF) may compensate for the reduction of β-globin and also it functions to inhibit sickling by participating in formation of mixed tetramers including both the γ and βS chain. Thus the induction of HbF holds tremendous promise to ameliorate the clinical symptoms of β -thalassemia and sickle cell disease (SCD). For this reason, the adult-to-fetal hemoglobin switch has been an area of long-standing interest among hematologists and basic scientists. The recent revolution in gene editing technology has provided new opportunities for high efficiency fetal-globin gene activation and provides strong potential for clinical benefit. Recently, it was reported that zinc finger artificial proteins that bind to the ? globin gene promoter could reactivate fetal-globin expression, via tethering the globin enhancer LCR to the ? globin gene promoter. We have designed Transcriptional Activator Like Effectors (TALE) targeting the ? globin gene promoters to reactivate ? globin gene expression. The EF1 short promoter was used initially to drive TALE gene expression. The TALEs were fused with a ldb1 dimerization domain, followed by a T2A GFP cassette. The various elements were then assembled into a lentiviral vector. To prevent deletions caused by repeats of TALEs during the lentivirus packing process, we changed the TALE's DNA coding sequence by using codon-optimized DNA for their expression. Both PCR and Southern blot analysis confirmed that TALEs remained as full-length intact DNA without deletion. We employed HUDEP-2 (human umbilical cord blood derived erythroid progenitor cells) to examine the function of TALEs with respect to fetal-globin gene reactivation. Quantitative RT-PCR was performed to determine the relative mRNA level of fetal-globin which was normalized to the alpha-globin mRNA content. Three of eight TALEs were found to activate fetal-globin expression in HUDEP-2 cells with increases in mRNA levels from several fold to more than 400-fold. Furthermore, we also observed a symmetrical reduction of β-globin mRNA by more than 50%. The fetal-globin level determined by HPLC was up to 40% of total globin, compared to 0.4% in control cells. To define the ability of the vector to transduce primitive CD34 cells, we performed methylcellulose culture after transduction of CD34 cells with TALE encoding lentiviral vectors at an MOI of 5. Individual red colonies were picked up to determine the vector copy number (VCN) by quantitative real time-PCR. Forty to fifty percent of the colonies were positive for vector insertion. In the future we are planning to do human CD34 cell erythroid differentiation to evaluate how the vector activates fetal-globin expression in red cells. To promote strong expression of TALE in the erythroid linage, we have replaced the EF1 short promoter with strong MND promoter or one erythroid specific ankrin promoter.

224. A Gene Therapy Clinical Study of a Patient with X-Linked Chronic Granulomatous Disease

BACKGROUND: X-linked Chronic granulomatous disease (X-CGD) is a primary immunodeficiency disease characterized by failure of the nicotinamide adenine dinucleotide phosphate enzyme system in phagocytes to produce reactive oxygen species (ROS). This disorder derives from defects in the CYBB gene encoding gp91phox and leads to recurrent infection and hyper-inflammation, occasionally represented by CGD-associated colitis (CGD colitis). Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for X-CGD, and in Europe and America, gene therapy clinical trials have been performed to patients who have no HLA-matched donor. To assess the safety and efficacy of hematopoietic stem cell gene therapy, we commenced a retroviral gene therapy clinical study using a busulfan-based preconditioning treatment regimen for CGD patients. METHODS AND PATIENT: The patient was a 27-year- old man with X-CGD. His condition was complicated by refractory subcutaneous abscess, fungal lung disease, lymphadenitis and CGD colitis, and he did not have an appropriate HLA-matched donor for HSCT. Granulocyte colony-stimulating factor -mobilized CD34+ stem cells were transduced with an MFGS-based retrovirus vector encoding the therapeutic gene of CYBB ex vivo, and infused back to the patient after busulfan-based preconditioning. Clinical and biochemical assessments were performed to evaluate the safety and efficacy of retrovirus gene therapy. RESULTS: After engraftment of 6.5×106/kg of CD34+ cells including 76% of gp91phox expressing cells, the gene-modified phagocytes appeared in peripheral blood after day +14. Dihydrorhodamine (DHR) flow cytometry assay demonstrated that the 2.3% of neutrophils in peripheral blood produced ROS, and a vector insert copy number was 2.63 per DHR+ neutrophil at day +95. His clinical symptoms of subcutaneous abscess and CGD colitis improved at almost the same time. The retrovirus insertion site of gene-modified peripheral blood cells revealed that there was no evidence of abnormal growth of clones with MDS-EVI1, SETBP1 or PRDM16 integrants at day +63. At day +182, gene marking of peripheral blood decreased to about 0.1% of neutrophils. PCR quantification of vector gene marking showed a vector insert copy number of 0.17 and 0.04 per neutrophil at day +95 and day +182, respectively. A vector insertion copy of bone marrow cells was 0.08 per marrow cell at day +183, suggesting that a reduction of gene-modified CD34+ stem cells would cause a decrease of gp91phox expressing phagocytes in peripheral blood. CONCLUSION: The therapeutic gene itself does not confer a growth advantage on the transduced cells in CGD, which would impact on the long-term gene marking and the withdrawal of clonal expansion. As other studies have reported that residual ROS production could contribute to the survival of CGD patients, the presence of gene-modified ROS-producing phagocytes led to the improvement of severe infection in our patient.

470. New Insights into rAAV Integration Mechanisms by Targeted Enrichment Sequencing

Comprehensive analysis of deep sequencing data originating from the newly introduced Targeted Enrichment Sequencing (TES) indicates so far undescribed recombinations within the Inverted Terminal Repeats of recombinant Adeno-Associated Viruses (rAAVs). For the detection of vector integration sites into the host genome we routinely apply LAM-PCR. However, TES, in which we enrich for genomic regions that include vector sequences, has major advantages over LAM-PCR, as it neither depends on the existence of a vector-specific primer binding site (often lost during rAAV integration) nor on a restriction site in the vicinity of the vector insertion site. Furthermore, regions that are captured together with the vector allow for relative quantification of vector copies per genome. As the entire vector can be sequenced by TES, mutations within the vector and transgene can be detected. In summary, we here provide new and so far unpublished information about rAAV integration patterns and show how we optimized TES to become an important complementary tool to primer-based approaches (like LAM-PCR) for mapping of vector/viral integration sites.

Abstract A01: Investigating clonal heterogeneity and transcriptional vulnerabilities in latent metastasis

Abstract Most cancer deaths are due to the poorly understood process of metastasis, which is seeded by disseminated tumor cells (DTCs). DTCs exhibit exceedingly variable latency, outgrowing immediately or lying dormant for months to years in a viable state of mass dormancy or replicative quiescence. The mechanisms enabling DTCs to survive and resist therapy are poorly understood. We have recently derived several models of latent metastasis that default to a slow-cycling state under stress, reserve the capacity for tumor-initiation, and evade immune surveillance. These DTCs resemble progenitor stem cell populations at the molecular level, actively inhibiting the pro-differentiation Wnt pathway and over-expressing Sox transcriptional factors. Tissue immunofluorescence demonstrates that latent DTCs typically manifest as solitary or clusters of infiltrating cancer cells without forming overt metastasis within several months after hematogenous dissemination in mice. This compelling observation begs the question, are these bursts truly monoclonal? Recent studies have shown that rapidly cycling DTCs occasionally migrate in groups and this improves their likelihood of establishing polyclonal metastases; however, we hypothesize that colonization in the context of delayed metastasis may in fact be monoclonal. To address this question, unique and heritable fluorescent markers were integrated into the host genome of isolated DTCs to interrogate the clonal dynamics of apparent proliferative bursts and to enable lineage tracing. A tricolor-marking strategy was employed, whereby latent derivatives were simultaneously transduced with three vectors encoding Cerulean, Venus and mCherry fluorescent proteins at equal multiplicities of infection. Transduced cells were stably marked by an extensive color palette, which was created by variations in vector copy number and insertion sites. An automated strategy for segmenting and analyzing tissue immunofluorescence was developed to measure the size and number of subclones present in indolent micro-metastases. Fluorescent lesions were represented by their angular polar hue distribution and the Shannon-Weaver diversity index (H) was used as an estimate of clonal complexity. Lineage tracing revealed latent DTCs give rise to largely monoclonal metastases, in stark contrast to their aggressive counterparts. The dynamic behavior of latent populations was analyzed according to variations in the morphology, topological growth patterns, proliferation rates and drug response of > 300 single-cell-derived subclones within latent and aggressive metastatic derivatives. Both intra- and inter-clonal heterogeneity was enriched in DTCs derived from latent populations. The most heterogeneous clones were also the most resistant to taxol. Our results support the hypothesis that DTCs undergo short bursts of proliferation followed by elimination, growth arrest and quiescence. Consequently, long-term latency may represent the sum of multiple distinct, but balanced, states. Citation Format: Ashley M. Laughney, Srinivas Malladi, Danilo Macalinao, Joan Massagué. Investigating clonal heterogeneity and transcriptional vulnerabilities in latent metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A01.

Clonal Dynamics In Vivo of Virus Integration Sites of T Cells Expressing a Safety Switch

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells.

Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4+ T Cells

The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4+ T cells with respect to a panel of 40 chromatin states. The distribution of SB transposon insertions displayed the least deviation from random, while the PB transposon and the MLV retrovirus showed unexpected parallels across all chromatin states. Both MLV and PB insertions are enriched at transcriptional start sites (TSSs) and co-localize with BRD4-associated sites. We demonstrate physical interaction between the PB transposase and bromodomain and extraterminal domain proteins (including BRD4), suggesting convergent evolution of a tethering mechanism that directs integrating genetic elements into TSSs. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. The SB transposon has the highest theoretical chance of targeting a safe harbor locus in the human genome. The data underscore the significance of vector choice to reduce the mutagenic load on cells in clinical applications.

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.

In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cell-specific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3- to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications.

The Common Genomic Locus of Evl and MiR-342 Regulates Both Lymphopoiesis and Myelopoiesis

Hematopoietic stem cell (HSC) regulation is controlled by extrinsic and intrinsic factors adapting blood cell production to the need of the organism. To search for novel HSC regulatory genes, our group has established a unique screening approach. By systematically analyzing the entire integration site (IS) repertoire of ten Wiskott-Aldrich syndrome (WAS) patients enrolled in clinical gene therapy trials, we hypothesize to identify novel key regulatory genes in hematopoiesis. By applying our screening pipeline based on the number and distance of IS to the transcription start sites (TSS) of genes, we observed a statistically significant number of therapeutic vector insertions close to 32 single genes in nine out of ten WAS patients including the Evl/miR-342 gene locus which has not been linked to hematopoiesis so far. Common insertion sites close to Evl/miR-342 accounted for up to 1.2% of relative sequencing reads within the peripheral blood (PB) of patients and clones harboring such integrations contributed to hematopoiesis for up to six years. We therefore hypothesized that the protein-coding gene Evl and/or its intronic miR-342 - which share a common genomic locus - may regulate hematopoiesis. Evl has been shown to play a pivotal role in actin cytoskeleton remodeling, and to interact with RAD51 complexes within homologous recombination. MiR-342 is a direct target of the transcription factor PU.1, which drives myeloid differentiation, and accelerates all-trans retinoic acid (ATRA)-induced differentiation of APL blasts.First of all, we investigated the candidate gene RNA expression in purified murine hematopoietic cell populations. Interestingly, we observed that Evl and miR-342 are not highly expressed in murine Lineage- Sca1+ ckit+ (LSK) cells but their expression increases profoundly with blood cell differentiation. While Evl expression was highest in lymphocytes (20-30 fold higher as compared to LSK cells), miR-342 was expressed at the highest level in macrophages (300 fold higher compared to LSK cells). To study the role of the candidates in hematopoiesis, we overexpressed Evl and miR-342 by using lentiviral vectors in murine primary LSK cells. Gene expression profiling of LSK cells overexpressing Evl revealed that 32% (62 out of 190) of the deregulated transcripts were involved in hematopoietic system development and function. Moreover, the top deregulated canonical pathways detected are essential for the development of B-cells (p=2.59*10-11). However, pathways important for myeloid cells such as immune cell trafficking and, more specifically, granulocytic adhesion and diapedesis (p=2.59*10-3) were significantly upregulated within miR-342- positive LSK cells. Functional analysis showed that Evl overexpression leads to a three- to fourfold increase of preB-cell colonies compared to control vector-transduced LSK cells. By contrast, miR-342 overexpressing cells formed a twofold higher number of myeloid colonies in semisolid medium. Next, the influence of Evl and its intronic miRNA on self-renewal and multilineage differentiation in vivo was investigated in serial bone marrow (BM) transplantation experiments. Within the PB of primary recipient mice, we detected a decrease of Evl-positive cells over time (week 4: 20.6 ± 9.6%; week 20: 4.7 ± 2.4%). Within the spleens a significantly higher donor-derived B-cell frequency was detectable (Evl: 63.6 ± 17.1%; Mock: 39.9 ± 18.3%). In line with our in vitro results, we detected a 4.3 fold higher frequency of Evl positive B-cells four weeks after secondary transplantation (Evl: 68.1 ± 9.8 %; Mock: 15.9 ± 7.5%).In summary, our data show that different hematopoietic differentiation programs are driven by one common gene locus depending on the expressed gene product. While the protein-coding gene Evl drives B-cell lymphopoiesis, its intronic miR-342 promotes myeloid differentiation. DisclosuresNo relevant conflicts of interest to declare.

127. Detection of Vector Integration Sites by Targeted Sequencing

For biosafety assessment and the understanding of viral integration mechanisms the determination of the exact position and distribution of viral integration sites in the host genome is crucial. Current methods for mapping of vector insertion sites are based on primer binding within the vector genome, its elongation into the host genome, ligation of a common adapter on the host part, and subsequent PCRs. Thus, these methods depend on the presence of the primer binding site in the viral genome and biases are introduced during restriction digest (if applied), ligation, and PCR. Here, we present results from a targeted sequencing approach (SureSelect, Agilent) in which we enriched for genomic regions including vector sequences. This approach has major advantages over primer based approaches like LAM PCR: •As less PCR cycles are needed, a more quantitative estimation of cell clonality (clones with the sample integration site) should be possible.•Genomic regions that are captured together with the vector allow for relative quantification of vector copies per genome.•Integrations of incomplete vectors (common for adeno-associated vectors) can be detected without the need of specific primers.•As the entire vector is sequenced, mutations within the vector (and transgene) can be detected.Using control samples we defined the sensitivity, background, and dynamic quantitative range of this method. As the entire vector is sequenced, mutations within the vector (and transgene) can be detected. In summary, targeted sequencing provides an important complementary tool to primer based approaches (like LAM-PCR) for mapping of vector/viral integration sites.

445. A Delectable Lentiviral Vector System for Testing Oncogene Addiction in Hematological Malignancies

Mouse models of hematological malignancies can be established by overexpressing oncogenes in hematopoietic cells followed by transplantation. It is often unclear whether the oncogene is only necessary for initiating the malignancies or if it is also required for maintenance, known as oncogene addiction. An addicted oncogene could then serve as target for therapy. We have developed a dual lentiviral vector system to distinguish these possibilities. We chose the oncogene LMO2 as our model because it is involved in a number of leukemias caused by gamma-retroviral vector insertion in gene therapy of SCID-X1 and Wiskott-Aldrich syndrome. Our previous studies showed that overexpression of LMO2 using a gamma-retroviral vector in early thymic progenitors (ETP) caused a differentiation block at the DN2 stage of development and subsequent transplant of these cells gave rise to leukemia with 100% penetrance in mice. We inserted the LoxP sequence into the 3’ U3 region of a self-inactivating CL20 lentiviral backbone and subcloned a MSCV-LMO2-i-GFP expression cassette into the vector (Fig 1A) to make a CL20-tLMO2-i-GFP vector. To deliver Cre recombinase in an inducible fashion, we constructed a second lentiviral vector in which a MSCV-CreERT2-i-mCherry expression cassette is inserted. Vectors were made by transiently transfecting 293T cells and the unconcentrated titer of these vectors were 2E6 and 3E6 TU/ml, respectively. We first tested these vectors in NIH3T3 cells. NIH3T3 cells transduced with these two vectors were subsequently sorted for GFP+mCherry+ double positive cells. We then treated the cells with various concentrations of 4-hydroxytamoxifen (4-OHT) to induce nuclear entry of Cre recombinase in order to delete the MSCV-LMO2-i-GFP expression cassette. Seven days later, 79.3% of the cells lost GFP expression in the group that was treated with 100nM 4-OHT (Fig 1B), indicating efficient cassette deletion. The loss of LMO2 protein is also confirmed by Western blot (Fig 1C). We then transduced ETP cells with the CL20-tLMO2-i-GFP vector and cultured the cells on an OP9-DL1 stroma expressing the Delta-like ligand DL1 to induce differentiation. At day 14, 90.5% of the transduced cells were blocked at the DN2 stage, demonstrating that this deletable lentiviral vector was able to express sufficient levels of LMO2 to induce the DN2 block (Fig 1D). These cells have been transplanted into sublethally irradiated Rag2-/-IL2rg-/- mice to establish the leukemia. One leukemia has been established with a GFP+ tumor, enlarged spleen of 0.534g and an erythroid phenotype as analyzed via flow cytometry. Tumor cells have been transduced with CreERT2 vector for secondary transplant and treatment with tamoxifen to establish whether LMO2 is required for tumor maintainence. In summary, we have established a dual lentiviral vector system that can be used conveniently to study oncogene addiction in hematological malignancies. View Large Image | Download PowerPoint Slide

300. AAV Vector-Mediated In Vivo Reprogramming of Various Cell Types in Adult Mice

The 2006/7 discovery that somatic cells of different tissue origins can be converted into induced pluripotent stem cells (iPSC) by mere introduction of a cocktail of “reprogramming” factors, such as Oct4, Klf4, Sox2 and c-Myc (OKSM), has revolutionized the field of regenerative medicine. A major part of this success story is due to the fact that delivery of the OKSM combination (or variants thereof) to cultured target cells can be accomplished using virtually any of the existing non-viral or viral gene transfer systems. Here, we specifically engineered Adeno-associated viral (AAV) vectors for OKSM delivery, based on their superior safety profile and their typical persistence as episomal DNA that is lost during cell division, which we considered beneficial for reprogramming. A third advantage is that AAV vectors can be pseudotyped with natural or synthetic viral capsids exhibiting high specificities and efficiencies in a given cell. Indeed, we reproducibly found that OKSM expression from four AAV vectors – each encoding a single factor under the strong and ubiquitous SFFV promoter, and pseudotyped with the potent chimeric AAV-DJ capsid – resulted in reprogramming of murine embryonic fibroblasts. Notably, the up to 0.2% reprogramming efficiency obtained with our AAV vectors is comparable to the best lentiviral system. Curiously, and defying our original prediction, we observed by PCR that the AAV vector DNA persisted in the reprogrammed cells even after extended cultivation for up to 21 passages. Using a combination of LAM-PCR and SureSelect technologies, we were then able to identify a variety of OKSM vector integration sites in the mouse genome, including a previously reported hot spot for AAV vector insertion. We speculate that the unusual high frequency of genomic AAV integration in iPSC is either related to mutational inactivation of genes that inhibit reprogramming, or merely a by-product of the relaxation of the chromatin structure triggered by OKSM expression. Based on the robust ex vivo efficiency, we next asked whether our OKSM vectors could also mediate reprogramming in vivo, and therefore applied different doses of AAV8-pseudotyped variants to adult mice via tail vein injection. Strikingly, four to eight weeks later, we found various small and large teratomas in their livers that we identified through histological analysis and isolation of residual iPSC. Moreover, by week eight, one mouse showed signs of extra-hepatic reprogramming in the pancreas and the thoracic cavity, congruent with the in vivo tropism of AAV serotype 8. These features make our novel AAV vector-mediated in vivo reprogramming strategy comparable to our previously published OKSM-transgenic mice (Nature 2013, 502:340-5), with the added benefits that AAV vectors can be applied in any mouse strain or other animals, and can be re-targeted to specific tissues, which opens up entirely novel avenues for stem cell research.

Variability of an ideal insertion vector for cochlear implantation.

Based on a three-dimensional analysis, the orientation of the basal turn of the cochlea, the disposition of the basilar membrane, and the characteristics of the hook region--all of which determine the ideal electrode insertion vector during cochlear implantation--might vary among individuals to a greater degree than previously considered. The aim of this study is to assess the variability of an ideal insertion vector among a sample of surgical candidates from a purely anatomical perspective as well as from a more intraoperative-surgical perspective. During cochlear implantation through a cochleostomy or round window approach, the angle or vector of insertion after the first entry point seems to be related to intracochlear damage, which might correlate with anatomical and functional features. Three-dimensional reconstructions of the temporal bones of 50 cochlear implant candidates (a total of 100 ears) were assessed. The spatial orientation of an ideal insertion vector for a cochleostomy and a round window approach were estimated. A difference as great as 60 degrees was observed for an ideal insertion vector among the subjects. From an intraoperative perspective, this variability involves pushing the electrodes "as near to the buttress" or "as near to the emergence of the corda tympani" as possible, depending on the case. The orientation of the basal turn and the corresponding ideal electrode insertion vector vary widely among subjects. A proper preoperative estimation on a case-to-case scenario for this feature might lead to technique adaptation during insertion, possibly contributing to minimizing electrode insertion trauma and thus optimizing the anatomical and functional results.

Multiple roles of a putative vacuolar protein sorting associated protein 74, FgVPS74, in the cereal pathogen Fusarium graminearum.

Fusarium graminearum, a member of the F. graminearum species complex, is a filamentous ascomycetous group that causes serious diseases in cereal crops. A screen of insertional mutants of F. graminearum, generated using a restriction enzyme-mediated integration method, identified a mutant designated R7048 showing pleiotropic phenotypes in several mycological traits. The vector insertion site in the R7048 genome was identified as the KpnI site within an ORF annotated as FGSG_06346 (designated FgVPS74), which showed similarity to vacuolar protein sorting-associated protein 74 in the baker yeast. Both targeted gene deletion and complementation analyses confirmed that FgVPS74 was involved in hyphal growth, conidiation, sexual development, mycotoxin production, and virulence towards host plants in F. graminearum. Electron microscopy analysis revealed no significant changes in morphology of the vacuole or other organelles, but a greater number of mitochondria were produced in the ΔFgVPS74 strain compared to the wild-type progenitor. Expression of a GFP-tagged FgVPS74 construct under its native promoter in the ΔFgVPS74 strain exhibited localization of GFP signal to putative vesicle structures, but not to the vacuolar membrane. Taken together, these findings demonstrated that a functional vacuolar protein-sorting pathway mediated by FgVPS74 is crucial for fungal growth and development in F. graminearum.

Quantitative shearing linear amplification polymerase chain reaction: an improved method for quantifying lentiviral vector insertion sites in transplanted hematopoietic cell systems.

In gene therapy trials targeting blood disorders, it is important to detect dominance of transduced hematopoietic stem cell (HSC) clones arising from vector insertion site (VIS) effects. Current methods for VIS analysis often do not have defined levels of quantitative accuracy and therefore can fail to detect early clonal dominance. We have developed a rapid and inexpensive method for measuring clone size based on random shearing of genomic DNA, minimal exponential PCR amplification, and shear site counts as a quantitative endpoint. This quantitative shearing linear amplification PCR (qsLAM PCR) assay utilizes an internal control sample containing 19 lentiviral insertion sites per cell that is mixed with polyclonal samples derived from transduced human CD34+ cells. Samples were analyzed from transplanted pigtail macaques and from a participant in our X-linked severe combined immunodeficiency (XSCID) lentiviral vector trial and yielded controlled and quantitative results in all cases. One case of early clonal dominance was detected in a monkey transplanted with limiting numbers of transduced HSCs, while the clinical samples from the XSCID trial participant showed highly diverse clonal representation. These studies demonstrate that qsLAM PCR is a facile and quantitative assay for measuring clonal repertoires in subjects enrolled in human gene therapy trials using lentiviral-transduced HSCs.

Ring Counter Based ATPG for Low Transition Test Pattern Generation.

In test mode test patterns are applied in random fashion to the circuit under circuit. This increases switching transition between the consecutive test patterns and thereby increases dynamic power dissipation. The proposed ring counter based ATPG reduces vertical switching transitions by inserting test vectors only between the less correlative test patterns. This paper presents the RC-ATPG with an external circuit. The external circuit consists of XOR gates, full adders, and multiplexers. First the total number of transitions between the consecutive test patterns is determined. If it is more, then the external circuit generates and inserts test vectors in between the two test patterns. Test vector insertion increases the correlation between the test patterns and reduces dynamic power dissipation. The results prove that the test patterns generated by the proposed ATPG have fewer transitions than the conventional ATPG. Experimental results based on ISCAS'85 and ISCAS'89 benchmark circuits show 38.5% reduction in the average power and 50% reduction in the peak power attained during testing with a small size decoding logic.