Search

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD. Zinc finger protein transcription factors are developed for the selective silencing of the mutant huntingtin gene in human neurons in vitro and multiple animal models of Huntington’s disease in vivo while preserving expression of the wild-type allele.

It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.

Some camouflaged animals hide colour signals and display them only transiently. These hidden colour signals are often conspicuous and are used as a secondary defence to warn or startle predators (deimatic displays) and/or to confuse them (flash displays). The hidden signals used in these displays frequently resemble typical aposematic signals, so it is possible that prey with hidden signals have evolved to employ colour patterns of a form that predators have previously learned to associate with unprofitability. Here, we tested this hypothesis by conducting two experiments that examined the effect of predator avoidance learning on the efficacy of deimatic and flash displays. We found that the survival benefits of both deimatic and flash displays were substantially higher against predators that had previously learned to associate the hidden colours with unprofitability than against naive predators. These findings help explain the phenological patterns we found in 1568 macro-lepidopteran species on three continents: species with hidden signals tend to occur later in the season than species without hidden signals.

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein–protein interactions, modifying protein–DNA interactions is more difficult. This may be related to the structural features of protein–DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein–DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

Hawksbill turtle (Eretmochelys imbricata) populations have experienced global decline because of a history of intense commercial exploitation for shell and stuffed taxidermied whole animals, and harvest for eggs and meat. Improved understanding of genetic diversity and phylogeography is needed to aid conservation. In this study, we analyzed the most geographically comprehensive sample of hawksbill turtles from the Indo-Pacific Ocean, sequencing 766 bp of the mitochondrial control region from 13 locations (plus Aldabra, n = 4) spanning over 13500 km. Our analysis of 492 samples revealed 52 haplotypes distributed in 5 divergent clades. Diversification times differed between the Indo-Pacific and Atlantic lineages and appear to be related to the sea-level changes that occurred during the Last Glacial Maximum. We found signals of demographic expansion only for turtles from the Persian Gulf region, which can be tied to a more recent colonization event. Our analyses revealed evidence of transoceanic migration, including connections between feeding grounds from the Atlantic Ocean and Indo-Pacific rookeries. Hawksbill turtles appear to have a complex pattern of phylogeography, showing a weak isolation by distance and evidence of multiple colonization events. Our novel dataset will allow mixed-stock analyses of hawksbill turtle feeding grounds in the Indo-Pacific by providing baseline data needed for conservation efforts in the region. Eight management units are proposed in our study for the Indo-Pacific region that can be incorporated in conservation plans of this critically endangered species.

Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a pest of small fruits and cherries, and has also been noted to infest a variety of wild, ornamental, and uncultivated hosts. Identify- ing alternative hosts is critical for pest management. Research objectives were to: 1) survey fruits in the field for natural infestation of D. suzukii, 2) determine the susceptibility of fruits in laboratory no-choice studies, and 3) evaluate short-range preference between simultaneously ripe alternative hosts and culti- vated fruits in laboratory choice studies. Field surveys identified new hosts or confirmed previously re- ported hosts including: Berberis aquifolium Pursh, Oregon grape; Cornus spp., dogwood; Cotoneaster lacteus W.W. Smith, milkflower cotoneaster; Elaeagnus umbellata Thunberg, Autumn olive; Frangula purshiana (de Candolle) A. Gray, cascara buckthorn; Lindera benzoin (L.) Blume, spicebush; Lonicera caerulea L., blue honeysuckle; Morus sp., mulberry; Phytolacca americana L., pokeweed; Prunus avium (L.) L., wild cherry;Prunus laurocerasus L., cherry laurel;Prunus lusitanica L., Portuguese laurel;Rubus armeniacus Focke, Himalaya blackberry; Rubus spectabilis Pursh, salmonberry; Sambucus nigra L., black elderberry; Sarcococca confusa Sealy, sweet box; Solanum dulcamara L., bittersweet nightshade; andSymphoricarpos albus (L.) S.F. Blake, snowberry. High fruit infestations were observed in S. confusa during April-May and Lonicera spp. in June before most commercial fruits ripen. From both field and laboratory studies, there was no evidence of susceptibility during the estimated ripe period Crataegus L. 'Autumn Glory,' hawthorn; Ilex crenata Thunberg, Japanese holly; Nandina domestica Thunberg, sacred bamboo; Rhaphiolepis umbellata (Thunberg) Makino, yeddo hawthorne; Rosa acicularis Lindley, prickly rose; Skimmia japonica Thunberg, Japanese skimmia; and Viburnum davidii Franchet, David's vibur- num. Lastly, laboratory choice tests identified that several fall-ripening alternative hosts were more sus- ceptible than 'Pinot noir' or 'Pinot gris' wine grapes. By understanding host use, growers can identify high-risk areas where coordinated action may reduce infestation ofD. suzukii in crops.

Halyomorpha halys Stal, the brown marmorated stink bug (Hemiptera: Pentatomidae), is an invasive pest with established populations in Oregon. The generalist feeding habits of H. halys suggest it has the potential to be a pest of many specialty crops grown in Oregon, including hazelnuts, Corylus avellana L. The objectives of this study were to: 1) characterize the damage to developing hazelnut kernels resulting from feeding by H. halys adults, 2) determine how the timing of feeding during kernel development influences damage to kernels, and 3) determine if hazelnut shell thickness has an effect on feeding frequency on kernels. Adult brown marmorated stink bugs were allowed to feed on developing nuts for 1-wk periods from initial kernel development (spring) until harvest (fall). Developing nuts not exposed to feeding by H. halys served as a control treatment. The degree of damage and diagnostic symptoms corresponded with the hazelnut kernels' physiological development. Our results demonstrated that when H. halys fed on hazelnuts before kernel expansion, development of the kernels could cease, resulting in empty shells. When stink bugs fed during kernel expansion, kernels appeared malformed. When stink bugs fed on mature nuts the kernels exhibited corky, necrotic areas. Although significant differences in shell thickness were observed among the cultivars, no significant differences occurred in the proportions of damaged kernels based on field tests and laboratory choice tests. The results of these studies demonstrated that commercial hazelnuts are susceptible to damage caused by the feeding of H. halys throughout the entire period of kernel development.

Little is known about roost-site characteristics of the pallid bat (Antrozous pallidus) in the Red Hills region of Kansas and Oklahoma. The species is listed as a Species in Need of Conservation within the state of Kansas. We investigated diurnal roost characteristics of the pallid bat during two summers in the Red Hills region of Kansas and Oklahoma. Mist nets were used to capture bats within three 314-ha study sites. Radio transmitters were attached to 10 pallid bats and bats were tracked daily. Physical characteristics of 11 pallid bat roosts were recorded. Two of the 10 pallid bats radioed were females, one of which was tracked to a suspected maternity roost. The largest roost observed was a bachelor colony of 10 individuals. Most roosts (n = 8) were in vertical crevices, but pallid bats also used vertical or horizontal crevices or exfoliated cliff faces. Pallid bats roosted on cliff faces with a westerly aspect (mean = 285° ± 51.3° s). Lengths of roosting crevices ranged from 0.3 m to 3.2 m and appro...

The present study aims to establish a long-term intercontinental collaboration based on a sampling protocol using standardized repeated measures at permanent sites to document macromoth species richness and abundance through time and across the landscape. We pooled the data from two continental regions providing a total of 12 trap sites: Mt. Jirisan National Park in South Korea (2005–2007) and HJ Andrews Experimental Forest in Oregon, USA. (2004–2006). A synthesis of our data indicated that: (i) noctuids (43–52%) and geometrids (33–39%) dominated the measures of species richness; (ii) using our sampling protocols more than three years would be needed to obtain a value of 90% of empirical species richness relative to Chao-1 estimated species richness; (iii) temperature alone could not explain the peak pattern in moth abundance and species richness; (iv) the highest/ lowest proportion of species richness and abundance were present in similar elevation and forest sites. These observations established a foundation for developing a network-oriented database for assessing biotic impact of environmental and contributed to identifying species at high risk to environmental change based on empirical measures of temporal and spatial breadth.

Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity., Zinc finger nucleases have an established role in genome editing. Here, the authors report a strategy for identifying zinc finger nucleases that discriminate between desired targets and provide genome-wide specificity.

SummaryChromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts, in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.PaperFlick

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.

Zinc-finger nucleases (ZFNs) are chimeric endonucleases created by fusing the cleavage domain from the type IIS restriction enzyme FokI to a designed zinc-finger protein (ZFP). In order to generate a DNA double strand break (DSB) at a predetermined genomic site, two engineered ZFNs must be designed to bind DNA with the appropriate orientation and spacing (i.e., on opposite sides of the DNA with 5 or 6 bp of sequence separating them) (Carroll 2008; Cathomen and Joung 2008). This architecture (analogous to that depicted in Fig. 1A) permits the two FokI cleavage domains to heterodimerize—a prerequisite for subsequent cleavage of the targeted DNA (Bitinaite et al. 1998). Figure 1. Mutation of the catalytic domain of a ZFN monomer generates a ZFN heterodimer with nicking activity. (A) Illustration of the ZFN heterodimer architecture for single-stranded break (SSB) generation. The FokI catalytic domain of ZFN (left, blue) is mutated ... Repair of the ZFN-induced site-specific DSB provides the molecular basis for gene disruption, gene correction, or gene addition—the different potential outcomes of ZFN-mediated genome editing (for review, see Urnov et al. 2010). Specifically, a given DSB may be repaired via either the nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways (Kowalczykowski 2000; Sung and Klein 2006; Wyman and Kanaar 2006; Brugmans et al. 2007). HDR utilizes a homologous donor sequence as template for the conservative repair of the DNA break. Provision of a suitably designed donor DNA molecule can, therefore, specify gene addition or gene correction at the ZFN-induced DSB. NHEJ, on the other hand, simply catalyzes the rejoining of the two DNA ends—a process that can result in the deletion or insertion of nucleotides at the repair junction. DNA repair via NHEJ is, therefore, mutagenic. To date, ZFN-induced DNA repair via HDR or NHEJ has been utilized to target modifications to the genomes of numerous species (Le Provost et al. 2010) including Caenorhabditis elegans (Morton et al. 2006), Drosophila melanogaster (Bibikova et al. 2002), silkworms (Takasu et al. 2010), zebrafish (Doyon et al. 2008; Meng et al. 2008), sea urchins (Ochiai et al. 2010), Arabidopsis thaliana (Osakabe et al. 2010; Zhang et al. 2010), tobacco (Maeder et al. 2008; Cai et al. 2009; Townsend et al. 2009), corn (Shukla et al. 2009), mice (Carbery et al. 2010; Meyer et al. 2010), and rats (Geurts et al. 2009). ZFNs have also been used to efficiently engineer a diverse range of mammalian cell types (Urnov et al. 2010), including human stem cells, with subsequent retention of full potency and normal growth characteristics (Hockemeyer et al. 2009; Zou et al. 2009; Holt et al. 2010). Moreover, the therapeutic potential of ZFNs is currently under evaluation in clinical trials (clinicaltrials.gov identifiers NCT00842634, NCT01252641, NCT01044654). Given the markedly different genome editing outcomes mediated by NHEJ and HDR, it would be desirable to have control over the choice of pathway used and thus specify the outcome of repair. In practice, for gene disruption this can already be achieved, since NHEJ is the predominant pathway in mammalian cells (Lieber 2008) and codelivery of donor DNA is unnecessary. For gene correction and addition, however, NHEJ events represent competition to the desired homology-directed outcome at the target locus. One potential strategy for enforcing DNA repair pathway bias would be the targeted introduction of a DNA single strand break (SSB) or nick. Like the DSB, a DNA SSB/nick can theoretically stimulate homology-directed repair pathways (Holliday 1964; Meselson and Radding 1975; Radding 1982). For example, SSBs induced by the bacteriophage fd gene II protein (gIIp) stimulated HDR in yeast (Galli and Schiestl 1998), while nicks generated by specific mutants of the RAG proteins—critical components of the V(D)J recombination system—are repaired via initiation of homologous recombination (Lee et al. 2004). Moreover, experiments using a nick-inducing variant of the I-Anil LAGLIDADG homing endonuclease showed that the SSB/nicks induced by these engineered enzymes are also repaired via HDR (Smith et al. 2009). Importantly, and in contrast to a DSB, a DNA nick or SSB is not a bona fide substrate for repair via the NHEJ pathway. Thus, a targeted SSB has the potential to restrict repair to the homology-directed pathway. Here, we describe the engineering of a zinc finger nickase (ZFNickase) by mutation of a residue critical for FokI cleavage activity in one monomer of the ZFN heterodimer, thus allowing the ZFNs to heterodimerize on DNA but restricting cleavage to a single DNA strand. We show that targeting of these ZFNickases to the endogenous CCR5 locus stimulates gene correction and gene addition via HDR at frequencies of between ∼1%–8% in both transformed and primary human lines. Importantly, we show that the CCR5 ZFNickase-induced gene addition occurs without a marked increase in detectable mutagenesis by the error-prone NHEJ repair pathway. Specific outcomes of either gene addition/correction or gene disruption may, therefore, be modulated via the targeted introduction of a single- or double-strand break, respectively. Moreover, beyond the targeted event itself, elimination of DNA repair via NHEJ has the potential to eradicate mutagenesis at putative off-target cleavage sites, further improving the fidelity of genome editing.

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR+ T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

Identification of residues critical for dimerization of the Fok1 nuclease domain of zinc-finger nucleases permits rational design of enzymes with improved cleavage activity and retained obligate heterodimerization.

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily alpha1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a alpha1,6-fucosyltransferase. FUT8(-/-) CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8(-/-) cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8(-/-) CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8(-/-) cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN-derived FUT8(-/-) cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8(-/-) cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8(-/-) cell lines was fourfold higher than in the best-producing clones of FUT8(-/-) cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8- phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies.

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Human embryonic stem cells and induced pluripotent stem cells (hESCs and hiPSCs) are powerful tools for biomedical research. Realizing the full potential of these cells requires efficient genetic modification. However, techniques to generate cell type specific lineage reporters as well as reliable tools to disrupt, repair or overexpress genes by gene targeting are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc finger nuclease (ZFN) mediated genome editing. First, using ZFNs specific for the OCT4 locus we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Secondly, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg.

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rgamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

The temporal and spatial distribution and abundance of 15 rare to uncommon species of plusiine moths were compared across a watershed landscape dominated by a coniferous forest located on the western slope of the Cascade Mountains in Oregon. The 5-yr study assessed the species in the context of functional roles related to caterpillar host plants categorized into three guilds: conifer, hardwood tree and shrub, and herbaceous-feeding species. Also, the landscape was considered in the context of five geographic zones based on elevation and habitat type. Species richness and abundance were highest within the herb-feeding guild, seven (47%) species and 74 (47%) individuals. The conifer-feeding guild consisted of four species (27%) and 64 (40%) individuals, whereas the hardwood tree and shrub guild consisted of four species (27%) and 21 (13%) individuals. In combination, zones II, III, and IV, high elevation sites with extensive subalpine meadow habitat, exhibited a species richness of 14 (93%) and 119...

Regrowth and old-growth Douglas-fir stands were sampled for ground dwelling beetles using pitfall traps in 1982, 1983 and 1999. Species richness and abundance was compared within and among years, plots, and age of forest using multivariate techniques. Representatives of 49 families and 224 taxa were collected for a total of 11,191 specimens. The abundance and species richness of ground dwelling beetles suggested four notable relationships. First, different ages of forest had significantly different beetle communities. For instance, the herb stage regrowth stands supported a number of seral specialists including: Microlestes nigrinus Mannerheim and Trachypachus holmbergi Mannerheim. Second, specifically defined plant communities were associated with unique beetle communities. Ordination analysis showed that the grouping of stands, based on beetle communities, was correlated with differences in vegetative characteristics. Third, over a 17-year-period, beetle communities in old-growth stands remained relatively stable while beetle communities in regrowth stands exhibited a high degree of change. Ordination analysis grouped old-growth stands regardless of year sampled, while young seral stands changed significantly in regards to their ordination position depending on sampling year. Fourth, beetle communities in regrowth stands changed in a manner that paralleled the predictable transformation of young plant communities into mature forest stands. Ordination analyses found that beetle communities in the herb stage regrowth stands of 1982 had changed significantly and exhibited characteristics of beetle communities in tree stage regrowth stands.

Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9-18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

Insect natural enemies selected for specific traits such as faster developmental times, higher survivorship, increased fecundity, and larger body size might be considered during production of biological control agents in certain situations. For instance, traits related to developmental rates may be selected for if genetically based variation exists within temperature-dependent responses. We compared preimaginal development, mortality, and size of Hippodamia convergens Guerin-Meneville at 4 constant temperatures (18, 22, 26, and 30°C). Total preimaginal development ranged from 51 d at 18°C to 14 d at 30°C. Developmental times were significantly different among isofemale lines of field-collected H. convergens from Oregon at all temperatures tested (18, 22, 26, and 30°C). Variation within female lines was significant only at 26°C. The lower developmental thresholds for egg, larval, and pupal stages were 9.9, 13.7, and 14.5°C, respectively. Degree-day (DD) requirements for development were 58.8, 166.7, and 52.6 for eggs, larvae, and pupae, respectively. Total preimaginal development required 231 DD above a temperature threshold of 13.6°C. Degree-day requirements for 14 field-collected female lines varied from 223 to 273, whereas the lower threshold for development ranged from 13.1 to 13.8°C. Mortality decreased with increasing temperatures. At the lower temperature (18°C), higher mortality occurred during the 4th instar and pupa. Pupal and adult weights differed across temperatures. Heavier pupae and adult beetles were reared at 22°C. Also, wings were larger among beetles reared at 22°C. The development, mortality, and size measurements provide data for establishing suitable rearing conditions for future studies on the ecology of H. convergens.

We describe here a fundamentally novel way to approach the development of a therapeutic: use of data from genome-wide association studies (GWAS) as a guide to create, in a targeted fashion, a disease-ameliorating genotype in the patient's own cells. We exemplify this by focusing on the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD): in both cases elevated levels of fetal hemoglobin (HbF) have been shown to lessen or eliminate disease symptoms. Thus, reversing HbF silencing in patients is an attractive strategy for the development of therapies. To this end, GWAS data pointed to loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, as causative for HbF elevation. While such regulatory elements are challenging to affect in a clinical setting via a conventional small molecule approach, the development of genome editing with engineered nucleases allows, in principle, a targeted intervention at the DNA level to disable enhancer function. Here, we reduce this notion to practice.The BCL11A enhancer consists of three separate cis-regulatory elements spanning a total of~1,500 bp that together drive erythroid-specific expression of BCL11A and lead to a silencing of fetal hemoglobin post-birth. Guided in part by fine-scale in vivo protein-DNA interaction data, we performed a reverse-genetic analysis of the enhancer at its endogenous location in the physiologically relevant cell type, primary human erythroid cells, using zinc finger nucleases (ZFNs). We developed highly optimized ZFNs that yield 60-80% target locus editing in human mobilized peripheral blood CD34 cells. Remarkably, we find that the ZFN-driven ablation of a single 5 bp element, GATAA, resident in the enhancer reproducibly elevates fetal globin in erythroid progeny of these CD34 cells to levels indistinguishable from those resulting from an essentially complete ZFN-driven coding knockout of BCL11A itself. We demonstrate high-efficiency ZFN-driven marking at the enhancer in peripheral blood mobilized CD34 cells at clinical scale production in a GMP-compliant setting, observe the successful engraftment and differentiation of genome-edited cells in an immunodeficient mouse model, and use an unbiased deep-sequencing based assay to comprehensively characterize the nucleus-wide specificity profile of ZFN action.Together these data illustrate the feasibility of using findings from genome-wide association studies to pursue novel therapeutic approaches in monogenic disease. In particular, our work supports the further development of fetal globin elevation via the targeted genome editing of the BCL11A erythroid-specific enhancer in both peripheral blood- and bone marrow-derived CD34 cells as a potential treatment for the hemoglobinopathies.

The multicolored Asian lady beetle, Harmonia axyridis (Pallas), an aphidophagous coccinellid, was first seen in Benton County, OR, in 1993. Coincident with the 1st occurrence of H. axyridis , we initiated a study on its temperature-dependent survival and development. Survival from 1st instar to adult emergence ranged from 83–90% between 18 and 30°C, and at 10 and 34°C was 42 and 25%, respectively. Mean time for complete development ranged from 14.8 d at 30°C to 81.1 d at l4°C. No eggs hatched at 10° or 34°C and no 1st instars transferred to 10°C survived. The mean lower threshold for egg-to-adult development of 11.2°C is typical relative to other temperate aphidophagous Coccinellidae and was not significantly different from 10°C calculated for a French population of this beetle. Conversely, the mean number of degree-clays (DD) required for complete development was higher in the Oregon population (267.3 DD) than the French population (231.3 DD), although this may have been due in part to a different diet. Times for preimaginal development and values for thermal requirements suggest that the species is a thermal generalist in western Oregon.

Lepidopterans function in the dynamics of forested ecosystems by serving as defoliators, decomposers, prey or hosts to carnivores, and pollinators. The biodiversity of Lepidoptera is thus linked into the ecosystem by influencing nutrient cycling, plant population dynamics, and predator-prey population dynamics. Two important measures of biodiversity are species richness and abundance of individuals. However, values for these measures require an ecosystem context for insightful interpretation of ecological function. We propose that such an ecosystem context is gained by an assessment of host resource requirements; in the case of Lepidoptera, this means larval host plants. The flora that contributes to the biodiversity of Lepidoptera can be grouped into 3 major vegetation types: (1) conifers, (2) hardwood trees and shrubs, and (3) herbs and grasses. We compared the macrolepidopteran biodiversity of 3 forested ecosystems: (1) western Oregon, (2) eastern Oregon, and (3) West Virginia. In respective order of the above locations, totals of 463, 385, and 475 species were found. Conifers supported 9, 10, and 1% of the species richness. By contrast, hardwoods supported 57, 45, and 61% of the species richness, whereas herbs and grasses supported 31, 42, and 31% of the species richness. The patterns in abundance of individual moths were different from species richness of moths and butterflies considered together. Comparisons of moth abundance showed conifers supported 18, 5, and 1%; hardwoods supported 69,39, and 77%; and herbs and grasses supported 11,55, and 8%. Practicesinvolved in the management offorested ecosystems are discussed in the context of how Lepidoptera may be used as an indicator taxon for the assessment of land management practices, and how biodiversity of Lepidoptera could be considered in plans for habitat restoration with a specific focus on food web relationships.

We examined toxicity of acephate to third-instar gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), under different conditions of administration method, availability of food to larvae during bioassay, host plant, and activity of detoxifying enzymes. Larvae that had been fed field-collected foliage of white alder (Alnus rhombifolia Nutt.) were less susceptible 48 h after treatment with topically applied acephate if they were allowed to continue feeding on foliage during the bioassay period (LD 50 = 60.6 μg/g larva) than if they were not (LD 50 = 13.5 μg/g larva). All surviving larvae were replaced on their original food plant after the 48-h bioassay; of these, 14.4% of the larvae not fed during treatment died before pupation, compared with 1.3% of the larvae fed alder during treatment. The LD 50 obtained for topically treated larvae reared and treated on Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, (51.1 μg/g larva) was comparable to that obtained for larvae fed alder (60.0 μg/g larva) throughout treatment. Larvae treated orally with acephate, however, were slightly more susceptible when reared on Douglas-fir (LC 50 , 20.3 ppm) than when reared on alder (LC 50 , 27.0 ppm). Post-treatment mortality in orally treated larvae was 10.3% in those fed alder and 9.5% in those fed Douglas-fir. Higher cytochrome P-450 activities in larvae reared on Douglas-fir apparently did not enhance tolerance to acephate. Both sexes of orally treated larvae took significantly longer to pupate than did controls on both foliage types, as did topically treated males fed Douglas-fir. Pupal weight generally was slightly, but not always significantly, higher in treated than untreated larvae under all dietary and treatment regimes.

Gene flow was studied in Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), an exotic, arboreal ladybeetle predator that recently spread rapidly throughout North America. A survey of isozyme polymorphisms showed 30 of 52 resolved putative loci were polymorphic (58%), and the mean heterozygosity was 16.75 6 2.98% among all loci and 26.31 6 4.37% at only the polymorphic loci. The mean number of alleles at the 52 loci was 2.01 6 1.97. Gene frequencies were estimated in populations from Georgia, Virginia, Delaware, Rhode Island, Arkansas, Illinois, Iowa, and Oregon and differed significantly (P F 0.02) at 11 of 16 loci. Three of 16 loci (Fbp, Est-1, Tre) were not in Hardy‐Weinberg equilibrium in all populations and are not included in the F statistics. Random mating was indicated within populations (FIS 52 0.005 6 0.014) but not among populations (FST 5 0.025 6 0.005). According to Wright’s island model, FST estimates the average number of reproducing immigrants to be equivalent to ca. 10 beetles per population per generation. Thus there was a measure of genetic differentiation caused by drift, but this differentiation was small with respect to the large geographical distances among the sampled populations. The genetic data suggest that the founding North American population(s) was substantial. r1997Academic Press

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection, which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as "non-self" by the recipient. To this end, we developed designer zinc finger nucleases and employed a "hit-and-run" approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T cells, including CD19-specific T cells. The HLA-A(neg) T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by natural killer cells of cells that had lost HLA expression was circumvented by enforced expression of nonclassical HLA molecules. Furthermore, we demonstrate that zinc finger nucleases can eliminate HLA-A expression from embryonic stem cells, which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from a single donor can be administered to multiple recipients.

Development of a parasitoid, Meteorus communis (Cresson), was compared at 8 constant temperatures, while reared on a noctuid host, the variegated cutworm, Peridroma saucia (Hubner), fed a standard artificial diet. Development from oviposition to adult emergence ranged from 15.7 d at 28°C to 61.8 d at 14°C. The temperature requirement for development from egg to exit of the parasitoid larva (3rd instar) from the host was 143± 9.9 DD above a developmental threshold of 10.3 ± 0.6°C. Development from the 3rd instar to adult emergence required 132 ± 5.4 DD above a developmental threshold of 8.3 ± 0.4°C. Overall, development from egg deposition to adult emergence required 274 ± 14.5DD above a developmental threshold of 9.5 ± 0.5°C. The development of M. communis was compared with 3 populations of P. saucia for 3 temperatures.

A lady beetle native to Asia,Harmonia axyridisPallas, is established in the Pacific Northwest of North America. Ten years occurred between the intentional release of beetles and the first observation of an individual on July 18, 1991, in Kings County, Washington. The beetle was extremely abundant in western Washington and Oregon throughout 1993–1994, where the species ranged from mideastern Washington, south to just north of the California/Oregon border, east to an elevation of 1371 m in the Cascade Mountains and west to the Pacific Coast. Field sampling confirmed the habits and habitats ofH. axyridisas a generalist, semiarboreal, aphidophagous predator. Our records associateH. axyridiswith 17 aphid prey species on 17 plant hosts. In western Oregon,H. axyridisco-occurs with 11 species of native and 2 species of exotic aphidophagous coccinellids on trees and shrubs. In arboreal habitats, 70% of individual lady beetles and 82% of live adult coccinellid mass were represented byH. axyridis,while constituting only 4% of the coccinellids in alfalfa, clover, and peppermint. AdultH. axyridisdispersed to feeding sites from overwintering aggregations, which formed the preceding October, during March. At least two generations, with a partial to complete third generation, occur per year. Parasitism of field-collected adult beetles byDinocampus coccinellae(Schrank) was less than 1%.

The influence of temperature on the development rate and expression of 3 elytral color patterns in the lady beetle, Calvia quatuordecimguttata (L.), was assessed at 6 constant temperatures between 10 and 34°C. The expression of the 3 elytral color patterns was discrete and independent of temperature treatment. No differences were observed among the color morphs in the real or physiological time for egg-to-adult development, or for pupal weight. However, temperature did influence survival and growth rates of the overall population. No eggs hatched at 34°C. Larval mortality was high at 10°C and moderate between 14 and 30°C. Development time from oviposition to adult was 14.6 ± 0.9 d at 30°c and 95.6 ± 9.0 d at 10°C. The total heat-unit requirement for egg to adult development was 274.1 DD above a mean development-threshold of 8.2°C. Pupal weight differed among temperature treatments and was greatest at 10-22°C, and lowest at 26 and 30°C.

Individuals of the convergent lady beetle Hippodamia convergens Guerin-Meneville, selected for a fast developmental rate (intensely selected line) through five generations, were assessed for unintended selective effects on adult weight and longevity, survivorship, food consumption, fecundity, and population growth parameters. Beetles from an intensely selected line were compared with beetles from a moderately selected line and field-collected wild types. After selection, survivorship from first instar to adult emergence at 18, 22, and 26°C was improved by 9.4, 8.5, and 22.2%, respectively, in the intensely selected line. Selection of individuals for fast development did not affect adult weight (19.6 mg), fecundity (344 eggs), longevity (53 days), nor third-fourth instar consumption of aphids (106 mg of pea aphids). Also, the intrinsic rate of increase (rm) and number of beetle generations per year were calculated for the intensely selected line, the moderately selected line, and wild-type beetles. The rm was higher for the intensely (0.099) than for the moderately selected line (0.091) and wild type (0.092). A degree-day model demonstrated that the intensely selected line may complete 3.9 and 5.3 generations in Corvallis and Hermiston, Oregon, respectively. The moderately selected line and wild-type populations would only complete 3.4 and 3.2 generations in Corvallis, respectively, and 4.6 and 4.5 generations in Hermiston, respectively.

Techniques optimal for the mass culture of a lady-beetle, Eriopis connexa Mulsant, imported into the United States for biological control of the Russian wheat aphid Diuraphis noxia (Mordvilko) were determined using three prey species and two temperature treatments. Also, survival of eggs and pupae after various periods of storage at 4°C was monitored. Conditions that favored production of large beetles (11.8 mg) were not the conditions providing the highest larval survival (91.7%), which in turn were not the conditions promoting the most rapid larval growth (6.3 days). An index based on larval developmental time, larval survival, and adult weight indicated that the most optimal rearing condition for beetle production was to use the Russian wheat aphid as prey at 34°C. The least favorable conditions involved feeding beetles the pea aphid Acyrthosiphon pisum (Harris) at 34°C. Eggs cannot be kept for more than 1 day at 4°C without incurring 30+% mortality; after 14 days mortality was 83%. Pupae were stored at 4°C for 3 weeks with no mortality but pupae stored longer than 7 weeks suffered 100% mortality.

There are approximately 7000 known rare and orphan diseases, over a third of which affect the central nervous system, virtually all do not have adequate treatment options. Shire is committed to developing innovative medicines to treat the fundamental biochemical abnormalities that result in pathologies caused by lysosomal storage disorders and other rare neurological diseases by selecting the right biological target based on extensive knowledge of disease pathophysiology and the right therapeutic modality from our array of technology platforms that includes antibodies, modified RNA, small molecules, gene therapy and protein therapeutics. This approach is particularly relevant for Huntington’s disease (HD), a rare and fatal neurodegenerative disease caused by a CAG trinucleotide repeat expansion in exon 1 of one copy of the Huntingtin (Htt) gene, resulting in expression of an aggregation-prone mutant protein. As this mutant protein is believed to be a primary cause of the pathophysiology in HD, Htt-lowering approaches are being explored using various technologies. Here, we will describe the use of an engineered zinc-finger protein transcription factor (ZFP TF) that preferentially down-regulates expression from the disease-causing copy of the Htt gene relative to the normal, unexpanded copy of the gene in both in vitro and in vivo HD models. Results presented here support the further development of allele-specific ZFP TFs as a potential therapy for HD.

Temperature-dependent development ofCoccinella trifasciata LeConte from Corvallis, Oregon, was assessed for the general population and compared among a subset of isofemale lines. All eggs died at 10 and 34°C. Survival ranged between 63.3–96.7% from 18–34°C. Development from oviposition to adult ranged from 44.2 days at 18°C to 11.1 days at 34°C. Mean adult weight did not differ among temperatures, and was 15.2 mg overall. For the species, heat-unit requirements for development from egg to adult were 227 degree-days above a developmental threshold of 12.7°C. Values for the developmental threshold differed among isofemale lines, and ranged from 11.4–14.5°C with heat-unit requirements ranging from 186–260 degree-days above their respective threshold. The isofemale line producing the fastest rate of immature development at 18°C was characterized by a heat-unit requirement of 260 degree-days above a developmental threshold of 11.4°C. The isofemale line producing the slowest rate of immature development at 18°C was characterized by a heat-unit requirement of 186 degree-days above a developmental threshold of 14.5°C. Implications of using isofemale lines in culturing biological control agents are discussed.

While adoptive transfer of T-cells modified to express a chimeric antigen receptor or tumor antigen specific T-cell receptor (TCR) has shown great promise for the treatment of malignant cancers, most current clinical applications are limited by the use of autologous T-cell products. Targeting of the TCR and HLA Class I genes in primary T-cells thus represent attractive targets for genome editing in order to produce universal T cells from allogeneic donors. Elimination of the native TCR and HLA class I proteins on T-cells would, respectively, reduce the risk of graft-versus-host disease and host-versus-graft clearance mediated by the adaptive immune system. We have developed clinical grade zinc finger nuclease (ZFN) reagents that can efficiently target the T-cell receptor alpha constant (TRAC) and beta-2-microglobulin (B2M) loci. ZFN encoding mRNAs were introduced into purified T-cells by electroporation. Without selection, T-cells treated with TRAC specific ZFNs showed modification of up to 89% of alleles as gauged by deep sequencing. Flow analysis showed 91% of treated cells were negative for CD3 expression. Results from ZFN mRNA dose titration studies showed that the level of TRAC gene modification by deep sequencing was highly correlated with the percentage of cells that stain negative for CD3 expression by FACS (Spearman rho = 0.96; p < 0.0001). B2M is a subunit in all HLA class I molecules, and represents a conserved target for eliminating HLA class I presentation in cells from different donors. Analysis of T-cells transfected with mRNAs encoding B2M specific ZFNs showed up to 94% of alleles were modified, as determined by deep sequencing. FACS analysis showed that 89% of treated cells were negative for expression of HLA-A, B, C. Similar to TRAC modified cells, a marked correlation was observed between the percentage of B2M alleles modified and percentage of cells lacking surface expression of HLA-A,B, and C (Spearman rho = 0.57; p < 0.0001). ZFN mediated genome editing was well tolerated. T-cells treated with TRAC or B2M specific ZFNs showed similar viability and growth characteristics as mock transfected cells. Together, these highly efficient ZFN reagents permit the highly efficient double knockout of TCR and HLA class I surface expression in primary human T-cells for potential use in the development of allogeneic cell therapies.

Temperature-dependent development of a Czechoslovakian population of the parasitoid Aphidius matricariae Haliday was measured at eight constant temperatures between 11 and 31°C using the Russian wheat aphid, Diuraphis noxia (Mordvilko), as a host. Development rate from oviposition to emergence of adult wasps increased linearly with increasing temperature between 11 and 26°C. No adult wasps emerged from parasitized aphids at 31°C. Requirements for development from oviposition to adult eclosionwere 273.1 ± 5.9 degree-days above a threshold of 4.5 ± 0.4°C. These results are compared with previously reported data on temperature-dependent development of other populations of A. matricariae and the host, D. noxia .