Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

Dual-locus, dual-HDR editing permits efficient generation of antigen-specific regulatory T cells with robust suppressive activity.

Adoptive regulatory T (Treg) cell therapy is predicted to modulate immune tolerance in autoimmune diseases, including type 1 diabetes (T1D). However, the requirement for antigen (ag) specificity to optimally orchestrate tissue-specific, Treg cell-mediated tolerance limits effective clinical application. To address this challenge, we present a single-step, combinatorial gene editing strategy utilizing dual-locus, dual-homology-directed repair (HDR) to generate and specifically expand ag-specific engineered Treg (EngTreg) cells derived from donor CD4+ T cells. Concurrent delivery of CRISPR nucleases and recombinant (r)AAV homology donor templates targeting FOXP3 and TRAC was used to achieve three parallel goals: enforced, stable expression of FOXP3; replacement of the endogenous T cell receptor (TCR) with an islet-specific TCR; and selective enrichment of dual-edited cells. Each HDR donor template contained an alternative component of a heterodimeric chemically inducible signaling complex (CISC), designed to activate interleukin-2 (IL-2) signaling in response to rapamycin, promoting expansion of only dual-edited EngTreg cells. Using this approach, we generated purified, islet-specific EngTreg cells that mediated robust direct and bystander suppression of effector T (Teff) cells recognizing the same or a different islet antigen peptide, respectively. This platform is broadly adaptable for use with alternative TCRs or other targeting moieties for application in tissue-specific autoimmune or inflammatory diseases.

A chemically inducible IL-2 receptor signaling complex allows for effective in vitro and in vivo selection of engineered CD4+ T cells.

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.

Radical rubrics: implementing the critical and creative thinking general capability through an ecological approach.

This article details how and why we have developed a flexible and responsive process-based rubric exemplar for teaching, learning, and assessing critical and creative thinking. We hope to contribute to global discussions of and efforts toward instrumentalising the challenge of assessing, but not standardising, creativity in compulsory education. Here, we respond to the key ideas of the four interrelated elements in the critical and creative thinking general capability in the Australian Curriculum learning continuum: inquiring; generating ideas, possibilities, actions; reflecting on thinking processes; and analysing, synthesising and evaluating reasoning and procedures. The rubrics, radical because they privilege process over outcome, have been designed to be used alongside the current NAPLAN tests in Years 5, 7 and 9 to build an Australian-based national creativity measure. We do so to argue the need for local and global measures of creativity in education as the first round of testing and results of the PISA Assessment of Creative Thinking approach and to contribute to the recognition of creative thinking (and doing) as a core twenty-first century literacy alongside literacy and numeracy.

Pancreatic islet-specific engineered Tregs exhibit robust antigen-specific and bystander immune suppression in type 1 diabetes models.

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in type 1 diabetes (T1D) mouse models. Tregs that are specific for pancreatic islets are more potent than polyclonal Tregs in preventing disease. However, the frequency of antigen-specific natural Tregs is extremely low, and ex vivo expansion may destabilize Tregs, leading to an effector phenotype. Here, we generated durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4+ T cells isolated from patients with T1D, we generated islet-specific EngTregs that suppressed effector T cell (Teff) proliferation and cytokine production. EngTregs suppressed Teffs recognizing the same islet antigen in addition to bystander Teffs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teffs or diabetogenic polyclonal Teffs in recipient mice. These data demonstrate the potential of antigen-specific EngTregs as a targeted therapy for preventing T1D.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Effect of Lure Combination on Fruit Fly Surveillance Sensitivity.

Surveillance for invading insect pests is costly and the trapper usually finds the traps empty of the target pest. Since the successful establishment of new pests is an uncommon event, multiple lures placed into one trap might increase the efficiency of the surveillance system. We investigated the effect of the combination of the Tephritidae male lures - trimedlure, cuelure, raspberry ketone and methyl eugenol - on catch of Ceratitis capitata, Zeugodacus cucurbitae, Bactrocera tryoni, B. dorsalis, B. aquilonis and B. tenuifascia in Australia and the USA (not all species are present in each country). The increase in trap density required to offset any reduction in catch due to the presence of lures for other Tephritidae was estimated. The effect of increasing trap density to maintain surveillance sensitivity was modelled for a hypothetical population of B. tryoni males, where the effective sampling area of cuelure traps for this species has been estimated. The 3-way combination significantly reduced the catch of the methyl eugenol-responsive B. dorsalis. Unexpectedly, we found that trimedlure-baited traps that contained methyl eugenol had ×3.1 lower catch of C. capitata than in trimedlure-only-baited traps in Australia, but not in Hawaii where no difference in catch was observed, we cannot satisfactorily explain this result. Based on the data presented here and from previous research, combinations of some male lures for the early detection of tephritid flies appear compatible and where there is any reduction in surveillance sensitivity observed, this can be offset by increasing the density of traps in the area.

Cancer diagnosis and immunotherapy in the age of CRISPR.

The explosion in genome editing technologies that has occurred in the past decade has revolutionized cancer research and promises to improve cancer diagnosis and therapy. Ongoing efforts include engineering of chimeric antigen receptor-T cells using clustered regularly interspaced short palindromic repeats (CRISPR) to generate a safer, more effective therapy with improved performance in immunologically "cold" tumors, as well as clever adaptations of CRISPR enzymes to allow fast, simple, and sensitive detection of specific nucleotide sequences. While still in their infancy, CRISPR-based cancer therapeutics and diagnostics are developing at an impressive speed and it is likely they will soon impact clinical practice. Here, we summarize their history and the most recent developments.

Author Correction: Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target.

This corrects the article DOI: 10.1038/ncomms15987.

Chemical Degradation of TMR Multilure Dispensers for Fruit Fly Detection Weathered Under California Climatic Conditions.

Degradation models for multilure fruit fly trap dispensers were analyzed to determine their potential for use in large California detection programs. Solid three-component male lure TMR (trimedlure [TML], methyl eugenol [ME], raspberry ketone [RK]) dispensers impregnated with DDVP (2, 2-dichlorovinyl dimethyl phosphate) insecticide placed inside Jackson traps were weathered during summer (8 wk) and winter (12 wk) in five citrus-growing areas. Additionally, TMR wafers without DDVP, but with an insecticidal strip, were compared to TMR dispensers with DDVP. Weathered dispensers were sampled weekly and chemically analyzed. Percent loss of TML, the male lure for Ceratitis capitata (Wiedemann) Mediterranean fruit fly; ME, the male lure for Bactrocera dorsalis (Hendel), oriental fruit fly; RK, the male lure for Bactrocera cucurbitae (Coquillett), melon fly; and DDVP was measured. Based on regression analyses for the male lures, TML degraded the fastest followed by ME. Degradation of the more chemically stable RK was discontinuous, did not fit a regression model, but followed similar seasonal patterns. There were few location differences for all three male lures and DDVP. Dispensers degraded faster during summer than winter. An asymptotic regression model provided a good fit for % loss (ME, TML, and DDVP) for summer data. Degradation of DDVP in TMR dispensers was similar to degradation of DDVP in insecticidal strips. Based on these chemical analyses and prior bioassay results with wild flies, TMR dispensers could potentially be used in place of three individual male lure traps, reducing costs of fruit fly survey programs. Use of an insecticidal tape would not require TMR dispensers without DDVP to be registered with US-EPA.

Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target.

The widespread application of high-throughput sequencing methods is resulting in the identification of a rapidly growing number of novel gene fusions caused by tumour-specific chromosomal rearrangements, whose oncogenic potential remains unknown. Here we describe a strategy that builds upon recent advances in genome editing and combines ex vivo and in vivo chromosomal engineering to rapidly and effectively interrogate the oncogenic potential of genomic rearrangements identified in human brain cancers. We show that one such rearrangement, an microdeletion resulting in a fusion between Brevican (BCAN) and Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1), is a potent oncogenic driver of high-grade gliomas and confers sensitivity to the experimental TRK inhibitor entrectinib. This work demonstrates that BCAN-NTRK1 is a bona fide human glioma driver and describes a general strategy to define the oncogenic potential of novel glioma-associated genomic rearrangements and to generate accurate preclinical models of this lethal human cancer.

Cox-2-derived PGE2 induces Id1-dependent radiation resistance and self-renewal in experimental glioblastoma.

BACKGROUND: In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. METHODS: Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. RESULTS: In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1. CONCLUSIONS: In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients.

Weathering and Chemical Degradation of Methyl Eugenol and Raspberry Ketone Solid Dispensers for Detection, Monitoring, and Male Annihilation of Bactrocera dorsalis and Bactrocera cucurbitae (Diptera: Tephritidae) in Hawaii.

Solid male lure dispensers containing methyl eugenol (ME) and raspberry ketone (RK), or mixtures of the lures (ME + RK), and dimethyl dichloro-vinyl phosphate (DDVP) were evaluated in area-wide pest management bucket or Jackson traps in commercial papaya (Carica papaya L.) orchards where both oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett), are pests. Captures of B. dorsalis with fresh wafers in Jackson and bucket traps were significantly higher on the basis of ME concentration (Mallet ME [56%] > Mallet MR [31.2%] > Mallet MC [23.1%]). Captures of B. cucurbitae with fresh wafers in Jackson and bucket traps were not different regardless of concentration of RK (Mallet BR [20.1%] = Mallet MR [18.3%] = Mallet MC [15.9%]). Captures of B. dorsalis with fresh wafers, compared with weathered wafers, were significantly different after week 12; captures of B. cucurbitae were not significantly different after 16 wk. Chemical analyses revealed presence of RK in dispensers in constant amounts throughout the 16-wk trial. Degradation of both ME and DDVP over time was predicted with a high level of confidence by nonlinear asymptotic exponential decay curves. Results provide supportive data to deploy solid ME and RK wafers (with DDVP) in fruit fly traps for detection programs, as is the current practice with solid TML dispensers placed in Jackson traps. Wafers with ME and RK might be used in place of two separate traps for detection of both ME and RK responding fruit flies and could potentially reduce cost of materials and labor by 50%.

Electrochemical and Spectroscopic Evidence on the One-Electron Reduction of U(VI) to U(V) on Magnetite.

Reduction of U(VI) to U(IV) on mineral surfaces is often considered a one-step two-electron process. However, stabilized U(V), with no evidence of U(IV), found in recent studies indicates U(VI) can undergo a one-electron reduction to U(V) without further progression to U(IV). We investigated reduction pathways of uranium by reducing U(VI) electrochemically on a magnetite electrode at pH 3.4. Cyclic voltammetry confirms the one-electron reduction of U(VI) to U(V). Formation of nanosize uranium precipitates on the magnetite surface at reducing potentials and dissolution of the solids at oxidizing potentials are observed by in situ electrochemical atomic force microscopy. XPS analysis of the magnetite electrodes polarized in uranium solutions at voltages from -0.1 to -0.9 V (E(0)(U(VI)/U(V))= -0.135 V vs Ag/AgCl) show the presence of only U(V) and U(VI). The sample with the highest U(V)/U(VI) ratio was prepared at -0.7 V, where the longest average U-O(axial) distance of 2.05 ± 0.01 Å was evident in the same sample revealed by extended X-ray absorption fine structure analysis. The results demonstrate that the electrochemical reduction of U(VI) on magnetite only yields U(V), even at a potential of -0.9 V, which favors the one-electron reduction mechanism. U(V) does not disproportionate but stabilizes on magnetite through precipitation of mixed-valence state U(V)/U(VI) solids.

Fluorescence in situ hybridization detects increased sperm aneuploidy in men with recurrent pregnancy loss.

OBJECTIVE: To investigate, in men presenting with recurrent pregnancy loss (RPL), the prevalence of sperm autosome and sex chromosome aneuploidy. DESIGN: Retrospective study. SETTING: Male infertility clinic. PATIENT(S): A total of 140 men with RPL provided semen samples, and five normozoospermic controls provided 140 semen samples for comparison. Recurrent pregnancy loss, documented in the female partners, was defined as a prior miscarriage and/or recurrent IVF/intracytoplasmic sperm injection failure. INTERVENTION(S): Fluorescence in situ hybridization (FISH) was used to detect numerical abnormalities in sex chromosomes (X, Y) and autosomes (13, 18, 21) in ejaculated sperm. MAIN OUTCOME MEASURE(S): Sperm aneuploidy in men with RPL and normozoospermic controls. RESULT(S): Men with RPL had a greater percentage of sperm aneuploidy within the sex chromosomes and chromosomes 18 and 13/21 (1.04% vs. 0.38%; 0.18% vs. 0.03%; 0.26% vs. 0.08%). In total, 40% of men with normal sperm density and motility had abnormal sperm aneuploidy in all the chromosomes analyzed. Men with abnormal sperm density and motility had a higher proportion of sperm sex chromosome aneuploidy than men with normal density/motility (62% vs. 45%). Men with normal strict morphology (>4%) had lower rates of sex chromosome and sperm aneuploidy than men with abnormal strict morphology (28% vs. 57%). There was no association between sperm DNA fragmentation and sperm aneuploidy. CONCLUSION(S): Men with RPL have increased sperm aneuploidy compared with controls. A total of 40% of men with RPL and normal sperm density/motility had abnormal sperm aneuploidy. Men with oligoasthenozoospermia and abnormal strict morphology had a greater percentage of sperm aneuploidy compared with men with normal semen parameters.

BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages.

Hair follicle stem cells (HFSCs) and their transit amplifying cell (TAC) progeny sense BMPs at defined stages of the hair cycle to control their proliferation and differentiation. Here, we exploit the distinct spatial and temporal localizations of these cells to selectively ablate BMP signaling in each compartment and examine its functional role. We find that BMP signaling is required for HFSC quiescence and to promote TAC differentiation along different lineages as the hair cycle progresses. We also combine in vivo genome-wide chromatin immunoprecipitation and deep-sequencing, transcriptional profiling, and loss-of-function genetics to define BMP-regulated genes. We show that some pSMAD1/5 targets, like Gata3, function specifically in TAC lineage-progression. Others, like Id1 and Id3, function in both HFSCs and TACs, but in distinct ways. Our study therefore illustrates the complex differential roles that a key signaling pathway can play in regulation of closely related stem/progenitor cells within the context of their overall niche.

Safe storage and effective monitoring of CO2 in depleted gas fields.

Carbon capture and storage (CCS) is vital to reduce CO(2) emissions to the atmosphere, potentially providing 20% of the needed reductions in global emissions. Research and demonstration projects are important to increase scientific understanding of CCS, and making processes and results widely available helps to reduce public concerns, which may otherwise block this technology. The Otway Project has provided verification of the underlying science of CO(2) storage in a depleted gas field, and shows that the support of all stakeholders can be earned and retained. Quantitative verification of long-term storage has been demonstrated. A direct measurement of storage efficiency has been made, confirming that CO(2) storage in depleted gas fields can be safe and effective, and that these structures could store globally significant amounts of CO(2).

Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal.

Cellular aging is linked to deficiencies in efficient repair of DNA double strand breaks and authentic genome maintenance at the chromatin level. Aging poses a significant threat to adult stem cell function by triggering persistent DNA damage and ultimately cellular senescence. Senescence is often considered to be an irreversible process. Moreover, critical genomic regions engaged in persistent DNA damage accumulation are unknown. Here we report that 65% of naturally occurring repairable DNA damage in self-renewing adult stem cells occurs within transposable elements. Upregulation of Alu retrotransposon transcription upon ex vivo aging causes nuclear cytotoxicity associated with the formation of persistent DNA damage foci and loss of efficient DNA repair in pericentric chromatin. This occurs due to a failure to recruit of condensin I and cohesin complexes. Our results demonstrate that the cytotoxicity of induced Alu repeats is functionally relevant for the human adult stem cell aging. Stable suppression of Alu transcription can reverse the senescent phenotype, reinstating the cells' self-renewing properties and increasing their plasticity by altering so-called "master" pluripotency regulators.

Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.