Treating human genetic conditions requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with therapies and with delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body.
View Article and Find Full Text PDFThe precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here, we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a).
View Article and Find Full Text PDFSmall Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size.
View Article and Find Full Text PDFIntroduction: This first-time-in-humans study assessed the safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of GSK2879552 in patients with relapsed or refractory SCLC.
Methods: This phase I, multicenter, open-label study (NCT02034123) enrolled patients (≥18 years old) with relapsed or refractory SCLC (after ≥1 platinum-containing chemotherapy or refusal of standard therapy). Part 1 was a dose-escalation study; Part 2 was a dose-expansion study.
Tapinarof cream is a novel topical nonsteroidal agent that represents a unique class of anti-inflammatory molecules targeting the aryl hydrocarbon receptor. Study 201851 was an open-label, 2-cohort sequential study that assessed the systemic pharmacokinetics, safety, and efficacy of tapinarof in adults with moderate to severe atopic dermatitis. A total of 11 participants were enrolled: 5 received 2% cream, and 6 received 1% cream.
View Article and Find Full Text PDFZinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.
View Article and Find Full Text PDFIgG1 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.
View Article and Find Full Text PDFMammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem.
View Article and Find Full Text PDFAnoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis.
View Article and Find Full Text PDFExit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown.
View Article and Find Full Text PDFTargeted 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.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
April 2008
Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs).
View Article and Find Full Text PDFTULA, a recently identified UBA- and SH3-containing protein, has previously been shown to regulate cell signaling through protein tyrosine kinases. In order to search for novel functions of TULA, we identified, using mass spectrometry, proteins associated with TULA. ABCE-1 also known as RLI and HP68, a host factor of HIV-1 assembly, was found among TULA-associated proteins in these experiments.
View Article and Find Full Text PDFThe lymphoid protein T-cell ubiquitin ligand (TULA)/suppressor of T-cell receptor signaling (Sts)-2 is associated with c-Cbl and ubiquitylated proteins and has been implicated in the regulation of signaling mediated by protein-tyrosine kinases. The results presented in this report indicate that TULA facilitates T-cell apoptosis independent of either T-cell receptor/CD3-mediated signaling or caspase activity. Mass spectrometry-based analysis of protein-protein interactions of TULA demonstrates that TULA binds to the apoptosis-inducing protein AIF, which has previously been shown to function as a key factor of caspase-independent apoptosis.
View Article and Find Full Text PDFJ Phys Act Health
January 2007
Background: There is little data on hiking patterns in national parks to support hiking behavior as a vehicle to meet the joint YMCA, CDC, and National Park Service initiatives to encourage physical activity through public land use.
Methods: The YMCA of the Rockies hiking program provided data from Hike Report forms completed after 343 supervised hikes for one summer season in Rocky Mountain National Park (ROMO) to assess visitor hiking patterns.
Results: Of the total hikes, 64.
Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings.
View Article and Find Full Text PDFMultisite protein phosphorylation appears to be quite common. Nevertheless our understanding of how multiple phosphorylation events regulate the function of a protein is limited in many cases. The ability to measure temporal changes in the site-specific phosphorylation profile of a protein in response to a given stimulus or cellular activity would provide an immediate indication of the functional significance of any phosphorylation site to a given process.
View Article and Find Full Text PDFTo determine functional differences between the two splice variants of PPARgamma (gamma1 and gamma2), we sought to selectively repress gamma2 expression by targeting engineered zinc finger repressor proteins (ZFPs) to the gamma2-specific promoter, P2. In 3T3-L1 cells, expression of ZFP55 resulted in >50% reduction in gamma2 expression but had no effect on gamma1, whereas adipogenesis was similarly reduced by 50%. However, ZFP54 virtually abolished both gamma2 and gamma1 expression, and completely blocked adipogenesis.
View Article and Find Full Text PDFThe chromatin architecture of a promoter is an important determinant of its transcriptional response. For most target genes, the thyroid hormone receptor (TR) activates gene expression in response to thyroid hormone (T(3)). In contrast, the thyroid-stimulating hormone alpha-subunit (TSH alpha) gene promoter is down-regulated by TR in the presence of T(3).
View Article and Find Full Text PDFProgram evaluation data from school and community applications of a physical fitness drug prevention program is presented. A train-the-trainer methodology was applied to install the program in twenty-two settings within the state of Illinois. The physical training program consisted of exercise and educational modules delivered over a twelve-week time period that focused on learning values and life skills through exercise.
View Article and Find Full Text PDFTranscriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. We used Xenopus oocytes to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v-ErbA, on partly and fully chromatinized TR-responsive templates in vivo. Repression by v-ErbA was not as efficient as that mediated by TR, was significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v-ErbA required mature chromatin to effect repression.
View Article and Find Full Text PDFThe nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) promotes adipocyte differentiation, exerts atherogenic and anti-inflammatory effects in monocyte/macrophages, and is believed to mediate the insulin-sensitizing action of antidiabetic thiazolidinedione ligands. As no complete PPARgamma antagonists have been described hitherto, we have constructed a dominant-negative mutant receptor to inhibit wild-type PPARgamma action. Highly conserved hydrophobic and charged residues (Leu(468) and Glu(471)) in helix 12 of the ligand-binding domain were mutated to alanine.
View Article and Find Full Text PDFThe thyroid hormone receptor and the highly related viral oncoprotein v-erbA are found exclusively in the nucleus as stable constituents of chromatin. Unlike most transcriptional regulators, the thyroid hormone receptor binds with comparable affinity to naked and nucleosomal DNA. In vitro reconstitution experiments and in vivo genomic footprinting have delineated the chromatin structural features that facilitate association with the receptor.
View Article and Find Full Text PDFA contemporary view of hormone action at the transcriptional level requires knowledge of the transcription factors including the hormone receptor that may bind to promoters or enhancers, together with the chromosomal context within which these regulatory proteins function. Nuclear receptors provide the best examples of transcriptional control through the targeted recruitment of large protein complexes that modify chromosomal components and reversibly stabilize or destabilize chromatin. Ligand-dependent recruitment of transcriptional coactivators destabilizes chromatin by mechanisms including histone acetylation and contacts with the basal transcriptional machinery.
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