Assessment and improvement of the Plasmodium yoelii yoelii genome annotation through comparative analysis

Motivation: The sequencing of the Plasmodium yoelii genome, a model rodent malaria parasite, has greatly facilitated research for the development of new drug and vaccine candidates against malaria. Unfortunately, only preliminary gene models were annotated on the partially sequenced genome, mostly by in silico gene prediction, and there has been no major improvement of the annotation since 2002

Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Background: There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5 delta 32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation. Methods and Findings: We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student's t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic. Conclusions: Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy

Whole Genome DNA Methylation Analysis of Active Pulmonary Tuberculosis Disease Identifies Novel Epigenotypes: PARP9/miR-505/RASGRP4/GNG12 Gene Methylation and Clinical Phenotypes

We hypothesized that DNA methylation patterns may contribute to the development of active pulmonary tuberculosis (TB). Illumina&rsquo;s DNA methylation 450 K assay was used to identify differentially methylated loci (DML) in a discovery cohort of 12 active pulmonary TB patients and 6 healthy subjects (HS). DNA methylation levels were validated in an independent cohort of 64 TB patients and 24 HS. Microarray analysis identified 1028 DMLs in TB patients versus HS, and 3747 DMLs in TB patients after versus before anti-TB treatment, while autophagy was the most enriched signaling pathway. In the validation cohort, PARP9 and miR505 genes were hypomethylated in the TB patients versus HS, while RASGRP4 and GNG12 genes were hypermethylated, with the former two further hypomethylated in those with delayed sputum conversion, systemic symptoms, or far advanced lesions. MRPS18B and RPTOR genes were hypomethylated in TB patients with pleural involvement. RASGRP4 gene hypermethylation and RPTOR gene down-regulation were associated with high mycobacterial burden. TB patients with WIPI2/GNG12 hypermethylation or MRPS18B/FOXO3 hypomethylation had lower one-year survival. In vitro ESAT6 and CFP10 stimuli of THP-1 cells resulted in DNA de-methylation changes of the PARP9, RASGRP4, WIPI2, and FOXO3 genes. In conclusions, aberrant DNA methylation over the PARP9/miR505/RASGRP4/GNG12 genes may contribute to the development of active pulmonary TB disease and its clinical phenotypes, while aberrant DNA methylation over the WIPI2/GNG12/MARPS18B/FOXO3 genes may constitute a determinant of long-term outcomes

Production of <i>tat/rev</i> shRNA-containing lentiviral vectors at high titer using a modified helper plasmid.

<p>(<b>A</b>) Three synonymous mutations indicated by an underline were introduced into <i>rev</i> in the lentiviral helper plasmid pCMVRdelta8.74 which expresses Gag-Pol and Rev to create pCMVRdelta8.74-sIM, which contains three mismatches at the <i>tat/rev</i> site I shRNA target sequence. (<b>B</b>) Lentiviral vectors were prepared by transient transfection using either the lentiviral helper plasmid pCMVRdelta8.74, which contains the wild-type <i>rev</i> sequence (wt-<i>rev</i>), or the pCMVRdelta8.74-sIM helper, which contains the mutated (sIM-<i>rev</i>) and titered using the EGFP reporter. The RSC-UsI-SC46-IMPGW vector containing the tat/rev shRNA is produced at low titer using the wt <i>rev</i> helper, but is produced at 38-fold higher titer using the sIM-<i>rev</i> helper.</p

Lentiviral anti-HIV vectors.

<p>The lentiviral vector RSC-SMPGW contains an SFFV promoter (S) driving expression of the P140K mutant of MGMT (M) and also contains a human PGK promoter (P) driving expression of EGFP (G). This vector also contains a safety modified woodchuck hepatitis virus post transcriptional regulatory element (W). The following anti-HIV transgenes were added to this vector; the transmembrane-localized C46 HIV envelope fusion inhibitor expressed from the SFFV promoter with an IRES expressing MGMT (RSC-SC46-IMPGW), and the U6 driven <i>tat/rev</i> site I shRNA combined with the C46-expressing vector (RSC-UsI-SC46-IMPGW). The long terminal repeats (LTR) and lentiviral <i>cis</i>-acting region (CAR) are also indicated.</p

Lentiviral transduction and engraftment of pigtailed macaque CD34⁺ cells.

*<p>Monkey and weight at time of transplantation in brackets.</p>†<p>Multiplicity of infection based on titer determined by transduction of HT1080 cells.</p>‡<p>Percentage of fluorescence-positive cells assessed by flow cytometry for EGFP or EYFP in liquid cultures on day 11 (M05270) or 6 (M05189) after transduction.</p

Flow-cytometric analysis of transgene-expressing cells in peripheral blood subpopulations and bone marrow CD34⁺ cells.

<p>(<b>A</b>) Displayed is the percentage of transgene positive cells in different leukocyte subpopulations in the peripheral blood of monkey M05189 that received the C46 membrane fusion inhibitor and EGFP-expressing vector. In this macaque EGFP-expressing cells were found at significant percentages in all lineages examined. (<b>B</b>) Lineage-specific staining for macaques M05189 and M05270. Macaque M05189 and the control EYFP arm of M05270 had significant percentages of transgene-expressing cells in all lineages. For the experimental EGFP-expressing arm of M05270 that received the vector with the C46 fusion inhibitor and the U6-driven site I shRNA, the percentage of transgene-expressing cells was low in all lineages examined.</p

Transgene expression levels in peripheral blood cells of macaques and MGMT-mediated in vivo selection using O6BG and BCNU.

<p>The percentages of EGFP and EYFP-expressing leukocytes detected by flow-cytometry are shown for monkeys M05189 (<b>A</b>) and M05270 (<b>B</b>). Granulocytes and lymphocytes were gated based on forward and side-scatter properties and the percentage of EGFP or EYFP-expressing gated cells is shown. Administration of O6BG and BCNU to macaque M05189 is indicated by an arrow. For animal M05270 the control arm vector transduced cells are shown with back diamonds connected by a solid line (granulocytes) or dotted line (lymphocytes), and anti-HIV vector RSC-UsI-SC46-IMPGW transduced cells are shown by open boxes connected by a solid line (granulocytes) or dotted line (lymphocytes).</p