13 research outputs found
Pre-clinical validation of a humanized anti-EGFR variant III chimeric antigen receptor and phase I trial of CART-EGFRvIII in glioblastoma
Exome sequencing and disease-network analysis of a single family implicate a mutation in KIF1A in hereditary spastic paraparesis
Whole exome sequencing has become a pivotal methodology for rapid and cost-effective detection of pathogenic variations in Mendelian disorders. A major challenge of this approach is determining the causative mutation from a substantial number of bystander variations that do not play any role in the disease etiology. Current strategies to analyze variations have mainly relied on genetic and functional arguments such as mode of inheritance, conservation, and loss of function prediction. Here, we demonstrate that disease-network analysis provides an additional layer of information to stratify variations even in the presence of incomplete sequencing coverage, a known limitation of exome sequencing. We studied a case of Hereditary Spastic Paraparesis (HSP) in a single inbred Palestinian family. HSP is a group of neuropathological disorders that are characterized by abnormal gait and spasticity of the lower limbs. Forty-five loci have been associated with HSP and lesions in 20 genes have been documented to induce the disorder. We used whole exome sequencing and homozygosity mapping to create a list of possible candidates. After exhausting the genetic and functional arguments, we stratified the remaining candidates according to their similarity to the previously known disease genes. Our analysis implicated the causative mutation in the motor domain of KIF1A, a gene that has not yet associated with HSP, which functions in anterograde axonal transportation. Our strategy can be useful for a large class of disorders that are characterized by locus heterogeneity, particularly when studying disorders in single families
Both the genomic and antigenomic RNA strands of the arbovirus RVFV generate vsiRNAs.
<p>(A) RNA species produced during RVFV infection. (−) strand genomic segments and mRNAs are depicted in blue, (+) strand antigenomes and mRNAs in red. (B) RVFV vsiRNA size distribution (control library). (C) Distribution of 21 nt RVFV vsiRNAs across the three viral genomic segments. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) RVFV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt RVFV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) RVFV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt RVFV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p
VACV terminal repeat-derived vsiRNAs are derived from long, repeat-containing precursors.
<p>(A) RNA secondary structure prediction of one of sixty 70-mer repeats located at the genomic termini. The abundant repeat-associated VACV vsiRNA is mapped in red. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s004" target="_blank">Figure S4</a>. (B) Expression analysis of VACV terminal repeat-associated transcripts in <i>Drosophila</i> DL1 cells and mouse embryonic fibroblasts (MEFs) by RT-PCR. The forward primer (red) lies within the 70-mer repeat sequence, while the reverse primer (green) binds a unique sequence outside of the repetitive region. The banding pattern of PCR products reflects the amplification of variable numbers of 70-mer repeats, as depicted in the diagram. M = DNA ladder.</p
DCV genomic strand RNA is preferentially targeted by antiviral RNAi.
<p>(A) RNA species produced during DCV infection. (+) strand genome is depicted in blue, (−) strand antigenome in red. (B) DCV vsiRNA size distribution (control library). (C) Distribution of 21 nt DCV-derived vsiRNAs across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) DCV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt DCV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) DCV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt DCV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p
RNA transcripts produced by VACV are targeted by the <i>Drosophila</i> RNA silencing pathway.
<p>(A) The VACV genome is a dsDNA molecule with covalently closed identical termini. (B) VACV vsiRNA size distribution (control library). (C) Distribution of 21 nt VACV vsiRNAs across the viral genome. vsiRNAs mapping to the (+) strand are depicted in blue, (−) strand in red. Black arrows mark genomic termini. (D) VACV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt VACV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) VACV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt VACV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p
VSV vsiRNAs are concentrated at the 5′ genomic terminus.
<p>(A) RNA species produced during VSV infection. (−) strand genome is depicted in blue, (+) strand antigenome and mRNAs in red. (B) VSV vsiRNA size distribution (control library). (C) Distribution of 21 nt VSV vsiRNAs across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) VSV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt VSV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) VSV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt VSV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p
A putative hairpin within the RVFV S segment generates abundant vsiRNAs in <i>Drosophila</i> and mosquito cells.
<p>(A) RNA secondary structure prediction of S segment IGR. The highly abundant vsiRNAs are mapped in red. (B) Northern blot analysis of RVFV-infected <i>Drosophila</i> DL1 cells, <i>Aedes aegypti</i> Aag2 cells, and <i>Aedes albopictus</i> C6/36 cells, probed for the S segment stem loop vsiRNAs and tRNA<sup>val</sup> as a loading control.</p
FoxO3 Regulates Neural Stem Cell Homeostasis
SummaryIn the nervous system, neural stem cells (NSCs) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3−/− mice have fewer NSCs in vivo than wild-type counterparts. NSCs isolated from adult FoxO3−/− mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSCs suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSCs might have important implications for counteracting brain aging in long-lived species
Validation of a humanized anti-EGFR variant III chimeric antigen receptor for a Phase I trial of CART-EGFRvIII in glioblastoma
Chimeric antigen receptors (CARs) are synthetic molecules designed to re-direct T cells to specific antigens; CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of novel surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFR variant III) results from an in-frame deletion of a portion of the extracellular domain. In glioblastoma, the EGFRvIII mutation is oncogenic, portends a poor prognosis, and is thought to be enriched in glioblastoma stem cells. However, because the neoepitope of EGFR variant III is based on a small peptide sequence, an antibody or single-chain variable fragment (scFv) directed to this epitope must be rigorously tested to confirm lack of cross-reactivity to the ubiquitously expressed wild-type EGFR. We chose a vector backbone encoding a second generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFv’s and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low affinity scFv was chosen based on its specificity for EGFR variant III over wild type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead candidate CAR in vitro against EGFR expressing keratinocytes and in vivo in immunodeficient mice grafted with normal human skin; a cetuximab-based CAR served as a positive control. EGFRvIII-directed CAR-T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFR variant III+ glioblastoma. We have designed a phase I clinical study of CAR T cells transduced with humanized scFv directed to EGFR variant III in patients with either residual or recurrent glioblastoma (NCT02209376)