Vector Insertion Research Articles

Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown.In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/- mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays.Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant.The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/- recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/- bone marrow and spleen were retained in the blood stream.In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal.The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation.In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Homologous recombinatorial cloning without the creation of single-stranded ends: exonuclease and ligation-independent cloning (ELIC).

We describe a new type of molecular cloning that complements the available strategies for homologous recombinatorial cloning. Purified, linear double-stranded DNA molecules with homologous ends are simply mixed in water and they transform readily into E. coli. Insert and linear vector need as few as ten base pairs of homologous sequence at their ends and essentially no incubation or enzyme treatments are needed for creating recombinants from linear fragments. Our method outcompetes most existing cloning methods in simplicity and affordability and is well-suited for high-throughput applications.

Tracking vector insertion sites to explore the biology of transduced cells in vivo

Tracking vector insertion sites to explore the biology of transduced cells in vivo

Safety and immunogenicity of a candidate tuberculosis vaccine MVA85A delivered by aerosol in BCG-vaccinated healthy adults: a phase 1, double-blind, randomised controlled trial

SummaryBackgroundIntradermal MVA85A, a candidate vaccine against tuberculosis, induces high amounts of Ag85A-specific CD4 T cells in adults who have already received the BCG vaccine, but aerosol delivery of this vaccine might offer immunological and logistical advantages. We did a phase 1 double-blind trial to compare the safety and immunogenicity of aerosol-administered and intradermally administered MVA85AMethodsIn this phase 1, double-blind, proof-of-concept trial, 24 eligible BCG-vaccinated healthy UK adults were randomly allocated (1:1) by sequentially numbered, sealed, opaque envelopes into two groups: aerosol MVA85A and intradermal saline placebo or intradermal MVA85A and aerosol saline placebo. Participants, the bronchoscopist, and immunologists were masked to treatment assignment. The primary outcome was safety, assessed by the frequency and severity of vaccine-related local and systemic adverse events. The secondary outcome was immunogenicity assessed with laboratory markers of cell-mediated immunity in blood and bronchoalveolar lavage samples. Safety and immunogenicity were assessed for 24 weeks after vaccination. Immunogenicity to both insert Ag85A and vector modified vaccinia virus Ankara (MVA) was assessed by ex-vivo interferon-γ ELISpot and serum ELISAs. Since all participants were randomised and vaccinated according to protocol, our analyses were per protocol. This trial is registered with ClinicalTrials.gov, number NCT01497769.FindingsBoth administration routes were well tolerated and immunogenic. Respiratory adverse events were rare and mild. Intradermal MVA85A was associated with expected mild local injection-site reactions. Systemic adverse events did not differ significantly between the two groups. Three participants in each group had no vaccine-related systemic adverse events; fatigue (11/24 [46%]) and headache (10/24 [42%]) were the most frequently reported symptoms. Ag85A-specific systemic responses were similar across groups. Ag85A-specific CD4 T cells were detected in bronchoalveolar lavage cells from both groups and responses were higher in the aerosol group than in the intradermal group. MVA-specific cellular responses were detected in both groups, whereas serum antibodies to MVA were only detectable after intradermal administration of the vaccine.InterpretationFurther clinical trials assessing the aerosol route of vaccine delivery are merited for tuberculosis and other respiratory pathogens.FundingThe Wellcome Trust and Oxford Radcliffe Hospitals Biomedical Research Centre.

Development and Evaluation of Quality Metrics for Bioinformatics Analysis of Viral Insertion Site Data Generated Using High Throughput Sequencing.

Integration of viral vectors into a host genome is associated with insertional mutagenesis and subjects in clinical gene therapy trials must be monitored for this adverse event. Several PCR based methods such as ligase-mediated (LM) PCR, linear-amplification-mediated (LAM) PCR and non-restrictive (nr) LAM PCR were developed to identify sites of vector integration. Coupling the power of next-generation sequencing technologies with various PCR approaches will provide a comprehensive and genome-wide profiling of insertion sites and increase throughput. In this bioinformatics study, we aimed to develop and apply quality metrics to viral insertion data obtained using next-generation sequencing. We developed five simple metrics for assessing next-generation sequencing data from different PCR products and showed how the metrics can be used to objectively compare runs performed with the same methodology as well as data generated using different PCR techniques. The results will help researchers troubleshoot complex methodologies, understand the quality of sequencing data, and provide a starting point for developing standardization of vector insertion site data analysis.

542. A Novel Non-Restrictive, Semi-Quantitative Method for Vector Insertion Site Analysis Based on Random Shearing of Genomic DNA

542. A Novel Non-Restrictive, Semi-Quantitative Method for Vector Insertion Site Analysis Based on Random Shearing of Genomic DNA

Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni.

Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.

Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation

Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

A modified version of the digestion–ligation cloning method for more efficient molecular cloning

Here we describe a modified version of the digestion–ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.

A Role for H/ACA and C/D Small Nucleolar RNAs in Viral Replication

We have employed gene-trap insertional mutagenesis to identify candidate genes whose disruption confer phenotypic resistance to lytic infection, in independent studies using 12 distinct viruses and several different cell lines. Analysis of >2,000 virus-resistant clones revealed >1,000 candidate host genes, approximately 20 % of which were disrupted in clones surviving separate infections with 2–6 viruses. Interestingly, there were 83 instances in which the insertional mutagenesis vector disrupted transcripts encoding H/ACA-class and C/D-class small nucleolar RNAs (SNORAs and SNORDs, respectively). Of these, 79 SNORAs and SNORDs reside within introns of 29 genes (predominantly protein-coding), while 4 appear to be independent transcription units. siRNA studies targeting candidate SNORA/Ds provided independent confirmation of their roles in infection when tested against cowpox virus, Dengue Fever virus, influenza A virus, human rhinovirus 16, herpes simplex virus 2, or respiratory syncytial virus. Significantly, eight of the nine SNORA/Ds targeted with siRNAs enhanced cellular resistance to multiple viruses suggesting widespread involvement of SNORA/Ds in virus–host interactions and/or virus-induced cell death.

Restriction Enzyme-Mediated Integration 방법으로 확보한 Fusarium oxysporum 형질전환체의 후자리산 생성능 분석

후자린산(FA)는 Fusarium 속 균이 생성하는 독소로서 다른 곰팡이독소보다 독성은 낮으나 다른 독소와 중복 오염시 전체 독성을 증진시키는 것으로 알려졌다. 현재까지 FA 생합성 관련 효소나 유전자가 Fusarium oxysporum에서 밝혀지지 않았기 때문에 본 연구에서는 관련 생합성 유전자의 발굴을 위해 제한효소를 통한 무작위 삽입 형질전환방법인 REMI를 이용하여 FA 생성 F. oxysporum 균주의 생합성유전자의 결손을 시도하였다. F. oxysporum 균주 2주를 대상으로 REMI를 시도한 결과, 평균 3.2주 (<TEX>$1{\mu}g$</TEX> DNA 당)의 효율로 7,100주 이상의 형질전환체를 육성하였다. FA 미생성 형질전환체를 스크리닝 하기 위해 FA가 함유된 배양액에서 다양한 식물종자의 발아여부를 조사한 결과, 11종의 종자 중 가장 감수성인 배추종자를 선발하였다. 각 형질전환체는 Czapek-Dox broth에서 3주간 배양한 후 배양여액을 배추종자의 발아여부 검정에 사용하였다. 검정결과 총 5,000여 주의 REMI 형질전환체 중 53주의 배양여액에서 종자가 발아하지 않아, 이들을 FA 미(저) 생성 추정 형질전환체로 선발하였다. 이중 26주의 FA 생성량을 HPLC로 분석한 결과, 2주의 형질전환체에서 모균주 생성량의 1% 이하의 FA가 검출되었다. 이 중 형질전환체 1주로부터 REMI 벡터 삽입 부위 게놈 DNA의 염기서열(252 bp)을 확보하였으며, 이 부위는 F. fujikuroi의 미동정 게놈부위와 93% 유사성이 있음을 확인하였다. 이 부위의 FA생성 관련성 증명을 위해서는 추후 연구가 필요하다. Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/<TEX>${\mu}g$</TEX> DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at <TEX>$25^{\circ}C$</TEX> then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production.

A simple and highly efficient method to identify the integration site of a transgene in the animal genome

Because genetic manipulation occasionally disrupts the expression of the neighboring genes, the chromosomal locus where the transgene has been integrated should be identified in the use of transgenic organisms. By using a new blend of thermostable DNA polymerase, we established a highly efficient method of inverse polymerase chain reaction for this purpose. By using this protocol, we successfully determined the vector integration sites of 2 mouse lines, NSE-tTA and tetO-Cre, the combination of which is a useful tool in neuroscience research. On the basis of this information, we quantified the relative expression amount of the chromosomal genes adjacent to these transgenes and found that the insertion of the tetO-Cre vector significantly altered the mRNA level of one of the examined genes. Considering the potential risk of the insertion effect, we recommend that the vector integration sites of any transgenic lines should be determined routinely by using this method, and that the expression levels of their neighboring genes should be determined.

Safety Of a Gamma Globin Expressing Lentivirus Vector In a Non-Human Primate Model For Gene Therapy Of Sickle Cell Disease

Strategies for human gene therapy trials targeting hematopoietic stem cells (HSCs) are complicated by studies in murine models due to differences in stem cell behavior, short life-span and limited HSCs that could be transduced and transplanted when studying safety of viral vectors. Recent reports on adverse genotoxic events with integrating viral vectors in clinical trials utilizing autologous gene corrected HSCs underscores the need for safer gene transfer vectors. Non-human primates are relevant models due to similarities in the behavior of hematopoietic stem/progenitor cells, global gene expression profile, ability to assess long-term engraftment of transduced cells and safety of gene-modified HSCs, and thus could relatively accurately predict risk of vector genotoxicity. As a preclinical step towards globin gene therapy for hemoglobinopathies, we used pigtailed macaque HSC transplantation (HSCT) model to ascertain long-term safety and stable transgene expression from sGbG, a lentiviral vector (LV) encoding human γ-globin coding sequences from a β-globin promoter and locus control region (LCR). We observed upregulation of endogenous macaque fetal hemoglobin post-HSCT, which decreased to minimal levels by two years post-HSCT, a well-documented phenomenon following HSCT in humans. However, fetal hemoglobin (HbF) (comprised of macaque α and human γ-globin) expression remained steady at 12-15% even after 700 days post-HSCT. At 2.5 years post-HSCT, the HbF expression in a macaque transplanted with HSCs gene-modified with sGbG was stable in the range of 13% vs. 0.1% for control macaque; the average vector copy ranged between 0.13 and 0.28 with stable gene marking during the analysis period. In order to evaluate the LV integration site clonal population in sGbG transduced macaque repopulating cells, modified genome sequencing PCR was performed on genomic DNA from white blood cells and PCR products were sequenced. The junction sequences were mapped to the rhesus macaque genome assembly. A total of 177 unique vector insertions were retrieved at 6 months post-HSCT (early) and 102 vector insertions at 2.5 years (late) post-HSCT respectively. The relative distribution of vector insertions into chromosomes revealed a slight over-representation into Chromosome 16, both at early and late time points. Analysis of distribution of LV integrations of with respect to transcription start sites (TSS) revealed no insertions within the 2.5kb region of TSS. The frequency of insertions was concentrated near the 10-50kb window of TSS both upstream (18.6%) and downstream (15.6%) respectively. Interestingly, among the retrieved insertion sites, only 10% (17 insertions) were common at both time points, while 90% of insertions were unique at each time point, suggesting clonal fluctuations, with multiple HSC clones contributing to hematopoiesis at an early time point, and unique, HSC clones emerged at a later time point. Comparison of the top ten most frequently detected insertion sites at both time points revealed one insertion at Chromosome 16 mapping to an intron of KIAA0195 (an uncharacterized protein expressed ubiquitously), retrieved at both time points contributed to 3.27% and 9.23% of gene modified cells at early and late time points, respectively. No insertions were near MDS/EVI1, PRDM16 or HMGA2 loci. Other oncogenes and cancer associated genes were in the vicinity of some integrants; however, there was no significant clustering of insertions in gene regions. To assess the effect of insertions on flanking gene expression or putative cancer associated genes, we performed mRNAseq on whole blood RNA from sGbG macaque and two control macaques. A comparative analysis of transcript levels of >30,000 genes revealed no difference in global gene expression profile, gene insertions and genes within 300kb region of the LV insertion sites. Importantly, transcript levels of the most abundant clone observed (KIAA0195, Chr16: 70791901) and flanking genes, the tRNA splicing endonuclease subunit SEN54 and CASK interacting protein 2 differed from two control macaques analyzed by <2 fold. In summary, long-term follow-up data from a macaque that received cells gene-modified with a human γ-globin LV reveal polyclonal reconstitution of transduced cells, HSC clonal fluctuation, and a normal transcriptional profile, suggesting low risk of genotoxicity from this vector.Arumugam P, Burtner C: Equal Contribution Disclosures:No relevant conflicts of interest to declare.

Clonal Diversity Analysis Of Integrating Vector Transduced Hematopoietic Cells Using LAM-PCR Followed By Illumina-Based Next-Generation Sequencing Is Affected By False Positivity That Arises From Both Reaction Biochemistry and Bioinformatic Analysis

We have previously reported that LAM-PCR libraries derived from non-vector transduced NSG repopulating cells, even those proven negative by high-resolution gel analysis, subsequently prepared following standard Illumina library preparation and sequencing protocol, yielded thousands of reads that contained full length LTR (30nt) fragments, met all quality control filters, were mappable to loci within the human genome, and were thus indistinguishable from true reads. We observed 430,385 of these reads in two mocks, with one having 106 identified vector insertion sites (VIS) and the other 27. To determine the source of this mock positivity, we modified our LAM biochemistry to include a 6nt barcode sequence in the linker fragment that could provide a second identity verification in addition to the Illumina adaptor index (double-indexing). Now when we performed LAM-NGS we were able to filter out any read where the linker barcode did not match with the identified Illumina index, which resulted in the elimination of all but 2 reads, neither of which could be mapped. As the sequencing libraries generated from the vector treated NSG repopulating cell LAM-PCR products were also double-indexed, we could identify any read that had been assigned to the incorrect source (mis-indexing). This occurred at a rate of approximately 0.1-1% in polyclonally reconstituted animals, but could be as high as 100% in mice with one or two identified VIS. This risk of mis-indexing appears constant, such that reads derived from vector insertion sites with the highest read counts are most likely to result in mock positivity, therefore the simple subtraction of the VIS found in mock will result in the loss of true signal and cannot be recommended.For any assay to be successfully translated to the clinic, one must have an understanding of its performance characteristics, so we sought to determine the sensitivity and specificity of the LAM-NGS assay. To this end, we developed a set of controls consisting of genomic DNA purified from mixtures of Jurkat cell clones that contained known VIS to derive LAM-NGS libraries of predictable complexity (a 15 VIS and a 32 VIS library). Using these controls with standard LAM-PCR/ Illumina biochemistry followed by standard bioinformatic analysis, even with the incorporation of the double indexing strategy to remove all mis-indexed reads, numerous predicted VIS still remain as false positive calls. Our 15 VIS control had a sensitivity of 100% but a specificity 21% (55 false positive of 70 total VIS identified), and our 32 VIS control performed even worse with a sensitivity/specificity of 53%/13% (119 false positive of 136 total VIS identified). Several factors were common to the non mis-indexed false positive reads: 1. False positive VIS usually arise from shorter reads (<90nt) which, due to the highly repetitive nature of the human genome, can be mapped to multiple locations or map better to different locations depending on how long the read is and 2. False positive VIS are more prevalent in reads with lower read counts (<100, with many of them =1) where a read is likely a synthesis artifact where regions of low complexity such as a run of poly-T will result in indels or other PCR induced artifacts such as chimera formation that alter the best mapping location. Neither of these criteria was sufficient as filters and many true VIS became undetectable when arbitrary limits were placed on read length or total read count. Using different mapping algorithms (we tested BLAT, BWA, BFast, and GSNAP), while altering the absolute number and where many of these false positive reads mapped, none could eliminate this problem and all showed some degree of both success and failure.By using a set of defined controls and double indexing biochemistry, we have been able to assess the sensitivity and specificity of the LAM-NGS assay and to elucidate the mechanism underlying much of the assay's false positivity. By using this double indexing strategy, we have eliminated mis-indexed reads, but false positivity from real reads mapped to incorrect locations as well as low numbers of reads arising from still unclear sources are present in the LAM-NGS assay. As much of these false positive reads are due to PCR artifact we are currently testing low PCR cycle approaches and as read length is also an issue, controlling LAM fragment size using sonication instead of restriction endonucleases is also being evaluated. Disclosures:No relevant conflicts of interest to declare.

Modeling and Targeting MLL-PTD/RUNX1 Related MDS/AML In Mouse

Myelodysplastic syndromes (MDS) are heterogeneous disorders in which the hematopoietic stem cells (HSCs) in the bone marrow are defective, resulting in insufficient normal blood cells. MDS progress to secondary acute myeloid leukemia (sAML) in about one third of patients, as additional genetic abnormalities are acquired. Because of the similar molecular mechanisms under these two related disease categories, MDS with increased blasts (>5%) and AML with multilineage dysplasia and/or antecedent MDS, are also defined as MDS/AML. MLL and RUNX1/CBFb regulate normal hematopoiesis, and we have shown that they form a regulatory complex to regulate downstream target genes. Mutations of MLL1 (in-frame partial tandem duplication, MLL-PTD, or MLL translocations) or RUNX1 are found in about 28% of MDS, particularly in high-risk MDS or therapy-related MDS. sAML frequently contains both MLL-PTD and RUNX1 mutations, arguing for cooperative leukemogenic synergy between these two molecular lesions. However, Mll-PTD knock-in mice or Runx1Δ/Δ mice do not develop spontaneous MDS or AML. RUNX1 mutations can cause mouse MDS/AML in murine retroviral transduction mediated overexpression and BMT, however, the latency is long (8-14 months) and retroviral vector insertion mutagenesis at Evi1 or Mn1loci seems critical for MDS/AML development in this model. Indeed RUNX1 mutations cooperate with Evi1 upregulation in both murine MDS/AML model and human AML. Thus, we hypothesize that combining RUNX1 mutations with MLL-PTD may facilitate its transformation toward MDS and/or sAML.To understand the impact of RUNX1 mutation cooperativity with MLL-PTD, we first expressed MDS relevant patient-derived RUNX1 mutants (D171N and 291fsX300) in the context of Mll-PTD knock-in mouse bone marrow cells and performed BMT and in vitro CFU replating assay. RUNX1 mutations (D171N and 291fsX300) could not transform WT BM cells. However, they could transform MLL-PTD BM cells and undergo serial replating. Interestingly, D171N and 291fsX300 transformed MLL-PTD cells form different type of clones: MLL-PTD/D171N clones are bigger and diffuse, while MLL-PTD/291fsX300 clones are smaller but denser. In BMT assay, the MLL-PTD/D171N and MLL-PTD/291fsX300 BMT mice developed MDS and MDS/AML (2-10 months) after BMT. The MLL-PTD/D171N BMT mice developed anemia, neutropenia with leukodysplasia and left-shifted differential counts, and a hypo-cellular marrow with excess blasts, while MLL-PTD/291fsX300 BMT mice developed rather similar trilineage dysplasia features but present hyper-cellular marrow with high percent of blasts, some of the mice were diagnosed as MDS/AML. Interestingly, the MLL-PTD/291fsX300 BMT mice also develop myelo-fibrosis (MF) in the BM. We further generated Mll-PTD/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia one month after pI-pC injection, and developed pancytopenia 2-4 months later. The CBC exhibited increased MCV, RDW and severe anemia. All these Mll-PTD/Runx1Δ/Δ mice died of MDS induced complications within 8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspiration. Similar but accelerated lethal MDS were found in recipients transplanted with PTD/Runx1Δ/Δ BM cells compared with controls (median survival: 68 days VS undefined). Low dose decitabine (DAC 0.3 mg/kg, twice a week, subcutaneous injection) were used to treat these recipients, and we found significantly longer median survival in DAC treated recipients than controls (median survival: 94.5±6.4 VS 53.5±3.5 days, p<0.001). Neither Mll-PTD nor Runx1Δ/Δ BM cells could replate more than 4 times with M3434 methaltheloase, however, PTD/Runx1Δ/Δ BM cells could be replated more than 6 months in vitro. We also treated these cells in vitro with DAC (0.5 uM). Fewer colony numbers and increased differentiated cells (Gr1+/Mac1+) were found in DAC treated cells than PBS treated controls (CFU numbers/1x105seeded cells: 34±7.7 vs 619±30.5, p<0.001).In conclusion, our study demonstrates that: 1) RUNX1 mutations and complete deletions cause MDS or MDS/AML phenotypes in Mll-PTD background; 2) Decitabine is a promising drug to treat MLL-PTD/RUNX1 related MDS/AML. These exciting new models allow us to identify and analyze MDS/AML-initiating cells (MIC) and major targets that are critical for clonal evolution and pathogenesis of MDS/AML and therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Gene Therapy Model of X-linked Severe Combined Immunodeficiency Using a Modified Foamy Virus Vector

X-linked severe combined immunodeficiency (SCID-X1) is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc) gene, and characterized by a complete defect of T and natural killer (NK) cells. Gene therapy for SCID-X1 using conventional retroviral (RV) vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV) vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO) mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.

Membrane-bound SIV envelope trimers are immunogenic in ferrets after intranasal vaccination with a replication-competent canine distemper virus vector

We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination.

Role of HMGA2 in human hematopoietic stem and progenitor cells

Hematopoietic stem cells maintain life long blood generation during homeostasis and stress. Factors controlling the key characteristics of these cells such as self-renewal, quiescence and the potential to form various blood lineages are poorly understood. We reasoned that genes found to be located near common sites of vector insertion in clinical gene therapy studies might have important functions in this regard. The DNA binding protein, high mobility Group AT hook 2 (HMGA2) is one such factor. HMGA2 activation coupled with a myeloid biased clonal dominance of stem/progenitor cells was observed in a clinical gene therapy trial for β-thalassemia.

Hepatic lentiviral gene transfer is associated with clonal selection, but not with tumor formation in serially transplanted rodents

Lentiviral (LV) vectors are promising tools for long-term genetic correction of hereditary diseases. In hematopoietic stem cell gene therapies adverse events in patients due to vector integration-associated genotoxicity have been observed. Only a few studies have explored the potential risks of LV gene therapy targeting the liver. To analyze hepatic genotoxicity in vivo, we transferred the fumarylacetoacetate hydrolase (FAH) gene by LV vectors into FAH((-/-)) mice (n = 97) and performed serial hepatocyte transplantations (four generations). The integration profile (4,349 mapped insertions) of the LV vectors was assessed by ligation-mediated polymerase chain reaction and deep sequencing. We tested whether the polyclonality of vector insertions was maintained in serially transplanted mice, linked the integration sites to global hepatocyte gene expression, and investigated the effects of LV liver gene therapy on the survival of the animals. The lifespan of in vivo gene-corrected mice was increased compared to 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) control animals and unchanged in serially transplanted animals. The integration profile (4,349 mapped insertions) remained polyclonal through all mouse generations with only mild clonal expansion. Genes close to the integration sites of expanding clones may be associated with enhanced hepatocyte proliferation capacity. We did not find evidence for vector-induced tumors. LV hepatic gene therapy showed a favorable risk profile for stable and long-term therapeutic gene expression. Polyclonality of hepatocyte regeneration was maintained even in an environment of enforced proliferation.

INTEGRATION OF LIDAR DATA INTO A MUNICIPAL GIS TO STUDY SOLAR RADIATION

Abstract. Identifying the right roofs to install solar panels inside a urban area is crucial for both private citizens and the whole local population. The aim is not easy because a lot of consideration must be made: insolation, orientation of the surface, size of the surface, shading due to topography, shading due to taller buildings next the surface, shading due to taller vegetation and other possible problems typical of urban areas like the presence of chimneys. Accuracy of data related to the analyzed surfaces is indeed fundamental, and also the detail of geometric models used to represent buildings and their roofs. The complexity that these roofs can reach is elevated. This work uses LiDAR data to obtain, with a semi-automatic technique, the full geometry of each roof part complementing the pre-existing building data in the municipal cartography. With this data is possible to evaluate the placement of solar panels on roofs of a whole city analyzing the solar potential of each building in detail. Other traditional techniques, like photogrammetry, need strong manual editing effort in order to identify slopes and insert vector on surfaces at the right height. Regarding LiDAR data, in order to perform accurate modelling, it is necessary to obtain an high density point cloud. The method proposed can also be used as a fast and linear workflow process for an area where LiDAR data are available and a municipal cartography already exist: LiDAR data can be furthermore successfully used to cross-check errors in pre-existent digital cartography that can remain otherwise hidden.