Showing papers by "David Baltimore published in 2003"

Chimeric Nucleases Stimulate Gene Targeting in Human Cells

Abstract: Correction of gene defects in human somatic cells by targeting as has been used in murine embryonic stem cells (1, 2) has been precluded by the low spontaneous rate of gene targeting (3). However, creation of a DNA double-stranded break (DSB) in the genomic target (DSB-GT) can stimulate homologous recombination by over 1000-fold (4). We can rapidly and quantitatively measure gene targeting by correcting a mutation in a green fluorescent protein (GFP) gene that has been stably integrated into the genome (5) (fig. S1). With an optimized GFP gene targeting system, the introduction of a DSB by I–Sce I (Sce) stimulated GT >40,000-fold and the absolute rate of gene targeting reached 3 to 5% (fig. S2). Such a system, however, depends on the prior introduction of a Sce binding site into the target gene and cannot be used for endogenous genes. Chimeric nucleases (CNs) have the potential to create sequence-specific DSBs (6). CNs—fusions between zinc finger binding DNA binding domains and the endonuclease domain of Fok I—can sitespecifically cleave naked DNA in vitro (6), extrachromosomal DNA in Xenopus oocytes (7), and chromosomal DNA in Drosophila (8). CNs work as dimers, and their efficiency depends on the spacing and orientation of the zinc finger binding sites with respect to the length of the amino acid linker between the DNA binding and endonuclease domains (7, 9). QQR is an artificial zinc finger DNA binding domain that recognizes the sequence 5′-GGGGAAGAA-3′ with nanomolar affinity (10). We modified QQR chimeric nucleases (QQR-CNs) (7, 9) (Fig. 1A) and tested whether they stimulated gene targeting (Fig. 1B). The background rate of gene targeting was 0.71 events per million transfected cells (fig. S1C). QQRL18-CN stimulated gene targeting 17-fold on target QQR6 and 260-fold on target QQR8 (Fig. 1B). QQRL0-CN did not stimulate gene targeting on target QQR8, but it was as efficient as Sce in stimulating gene targeting by over 2000-fold on target QQR6 (Fig. 1B). QQRL18-CN showed some preference for an 8–base pair (bp) spacing between binding sites whereas QQRL0-CN preferred 6-bp spacing. Thus, removing the linker between the zinc spacfinger and the nuclease domains increased the activity and specificity of the fusion protein in mammalian cells. As controls, we showed that the CNs did not stimulate gene targeting if (i) they lacked a nuclear localization signal, (ii) there was a single binding site rather than an inverted repeat binding site in the target, and (iii) the cognate binding site was changed. Thus, homodimers of CNs are potent stimulators of gene targeting in human somatic cells.

Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5

Abstract: Double-stranded RNAs ≈21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.

NF-kappa B functions in synaptic signaling and behavior.

Abstract: Ca(2+)-regulated gene transcription is essential to diverse physiological processes, including the adaptive plasticity associated with learning. We found that basal synaptic input activates the NF-kappa B transcription factor by a pathway requiring the Ca(2+)/calmodulin-dependent kinase CaMKII and local submembranous Ca(2+) elevation. The p65:p50 NF-kappa B form is selectively localized at synapses; p65-deficient mice have no detectable synaptic NF-kappa B. Activated NF-kappa B moves to the nucleus and could directly transmute synaptic signals into altered gene expression. Mice lacking p65 show a selective learning deficit in the spatial version of the radial arm maze. These observations suggest that long-term changes to adult neuronal function caused by synaptic stimulation can be regulated by NF-kappa B nuclear translocation and gene activation.

Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance

Abstract: A Cre/lox-conditional mouse line was generated to evaluate the role of ATR in checkpoint responses to ionizing radiation (IR) and stalled DNA replication. We demonstrate that after IR treatment, ATR and ATM each contribute to early delay in M-phase entry but that ATR regulates a majority of the late phase (2–9 h post-IR). Double deletion of ATR and ATM eliminates nearly all IR-induced delay, indicating that ATR and ATM cooperate in the IR-induced G2/M-phase checkpoint. In contrast to the IR-induced checkpoint, checkpoint delay in response to stalled DNA replication is intact in ATR knockout cells and ATR/ATM and ATR/p53 double-knockout cells. The DNA replication checkpoint remains intact in ATR knockout cells even though the checkpoint-stimulated inhibitory phosphorylation of Cdc2 on T14/Y15 and activating phosphorylation of the Chk1 kinase no longer occur. Thus, incomplete DNA replication in mammalian cells can prevent M-phase entry independently of ATR and inhibitory phosphorylation of Cdc2. When DNA replication inhibitors are removed, ATR knockout cells proceed to mitosis but do so with chromosome breaks, indicating that ATR provides a key genome maintenance function in S phase.

Genetic analysis of NF-κB/Rel transcription factors defines functional specificities

Abstract: The NF-κB transcription factors consist of dimeric proteins of the Rel homology family. They activate many promoters containing highly divergent κB-site sequences. We have generated cell lines lacking individual and multiple NF-κB proteins and used them to establish interactions between components of the NF-κB–IκB signaling system. Functional compensation within the family of dimers was evident in knockout cell lines. Analysis of transiently transfected genes gave an impression of promiscuity that was not borne out by analysis of endogenous genes. Using TNFα as an inducer, a panel of endogenous genes showed a wide range of subunit specificities as well as highly variable kinetics of induction. Comparing the function and subunit specificity of genes with the sequence of the κB DNA-binding site we found little correlation, indicating that NF-κB family member specificity for endogenous promoters is not solely encoded by the κB site sequence itself.

Use of chimeric nucleases to stimulate gene targeting

Abstract: Gene targeting is a technique to introduce genetic change into one or more specific locations in the genome of a cell. For example, gene targeting can introduce genetic change by modifying, repairing, attenuating or inactivating a target gene or other chromosomal DNA. In one aspect, this disclosure relates to methods and compositions for gene targeting with high efficiency in a cell. This disclosure also relates to methods of treating or preventing a genetic disease in an individual in need thereof. Further disclosed are chimeric nucleases and vectors encoding chimeric nucleases.

Essential role for STAT5 signaling in CD25+CD4+ regulatory T cell homeostasis and the maintenance of self-tolerance.

Abstract: A population of CD25(+)CD4(+) regulatory T cells (T regs) functions to maintain immunological self tolerance by inhibiting autoreactive T cell responses. CD25(+)CD4(+) T regs are present in low, but steady, numbers in the peripheral lymphoid tissues of healthy mice. Recent studies have shown that IL-2 is an essential growth factor for these cells. How this cytokine functions to regulate CD25(+)CD4(+) T reg homeostasis and prevent autoimmune disease remains unknown. In conventional CD4(+) T cells, IL-2 triggers signaling pathways that promote proliferation and survival by activating the STAT5 transcription factor and by increasing the expression of the antiapoptotic protein, Bcl-2. We show here that bcl-2 deficiency does not affect CD25(+)CD4(+) T reg homeostasis, and that ectopic expression of this molecule fails to rescue CD25(+)CD4(+) T reg numbers or to prevent the development of autoimmunity in IL-2-deficient mice. Furthermore, transient activation of STAT5 is sufficient to increase CD25(+)CD4(+) T reg numbers in IL-2-deficient mice. Our study uncovers an essential role for STAT5 in maintaining CD25(+)CD4(+) T reg homeostasis and self-tolerance.

The need for a global HIV vaccine enterprise

Abstract: A new collaborative model of research is needed to increase resources, to prioritize the RD (ii) to increase the pace, reduce the overlap, and more systematically explore the elements of and delivery systems for vaccines; (iii) to use common standards for the prompt comparative testing of vaccine candidates; (iv) to expand resources for manufacturing vaccine candidates to speed their use in human trials; and (v) to increase the capacity for international clinical trials and to focus this effort toward quickly measuring the effectiveness of vaccine protection as prototype vaccine candidates are identified.

Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.

Abstract: Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

Erratum: NF-κB functions in synaptic signaling and behavior (Nature Neuroscince (2003) 6 (1072-1078))

Abstract: Nat. Neurosci. 6, 1072–1078 (2003) Due to a typesetting error in the Fig. 3 legend, the symbol μ was mistakenly printed as the letter 'm'. This led to erroneous concentration descriptions for reagents in panels c and d. The corrected legend appears below. Figure 3 Ca2+-dependent pathway of NF-κB activation.

On Over-Weighting the Bottom Line

Abstract: Universities in the Marketplace The Commercialization of Higher Education. Derek Bok. Princeton University Press, Princeton, NJ, 2003. 247 pp. $22.95, £15.95. ISBN 0-691-11412-9. Bok discusses what he sees as the inherent dangers that arise when universities undertake money-making ventures with corporations.

Use of zinc finger nucleases to stimulate gene targeting

Abstract: The present invention relates to a vector comprising (a) a nucleic acid encoding a chimeric nuclease, wherein the chimeric nuclease comprises: (i) a DNA binding domain comprising a zinc finger domain; (ii) a Fok I cleavage domain; (iii) a nuclear localization signal (NLS); and (b) a nucleic acid comprising a repair substrate that comprises: (i) a nucleic acid sequence that is substantially identical to a region flanking a target sequence in chromosomal DNA; and (ii) a nucleic acid sequence which replaces the target sequence upon recombination between the repair substrate and the target sequence.

Utilisation de nucleases chimeres pour stimuler le ciblage de genes

Abstract: Le ciblage de genes est une technique permettant d'introduire une modification genetique au niveau d'un ou de plusieurs emplacements specifiques dans le genome d'une cellule. Par exemple, le ciblage d'un gene peut introduire une modification genetique par modification, reparation, attenuation ou inactivation d'un gene cible ou de tout autre ADN chromosomique. Dans un premier mode de realisation, l'invention se rapporte a des methodes et a des compositions pour le ciblage de genes a haute efficacite dans une cellule. L'invention se rapporte egalement a des methodes de traitement ou de prevention d'une maladie genetique chez un individu justiciable d'un tel traitement. L'invention se rapporte en outre a des nucleases chimeres et a des vecteurs codant ces nucleases chimeres.