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Key Points Targeted genetic engineering in many important model systems and in human tissue culture cells has historically been challenging. This has changed dramatically over the past 5 years with the development of zinc finger nuclease (ZFN) technology. A ZFN is an artificial endonuclease that consists of a designed zinc finger protein (ZFP) fused to the cleavage domain of the Fok I restriction enzyme. A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities. For genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. The cleavage event induced by the ZFN provokes cellular repair processes that in turn mediate efficient modification of the targeted locus. If the ZFN-induced cleavage event is resolved via non-homologous end joining, this can result in small deletions or insertions, effectively leading to gene knockout. This approach has now been used to establish facile and efficient reverse genetics (that is, reverse genetics that does not require selection) in Drosophila melanogaster , zebrafish, rats, Arabidopsis thaliana and mammalian somatic cells. If the break is resolved via a homology-based process in the presence of an investigator-provided donor, small changes or entire transgenes can be transferred, often without selection, into the chromosome; this is referred to as 'gene correction' and 'gene addition', respectively. This approach has been used to make novel alleles in D. melanogaster , mammalian cells and tobacco, and has been used to drive targeted integration in maize, tobacco and human embryonic stem and induced pluripotent stem cells. Therapeutic application of ZFN technology requires the engineering of ZFNs that are highly specific in their action. Three clinical trials with ZFNs are underway, including one in which T cells are isolated from a patient infected with HIV, treated with ZFNs that disrupt the chemokine (C-C motif) receptor type 5 ( CCR5 ) gene to make them resistant to virus infection, and transferred back to the patient. Zinc finger nucleases (ZFNs) are versatile tools for making precise modifications to genomes, and their use is now established in a range of model systems. ZFNs are also showing potential in human gene therapy, and several clinical trials are underway. Reverse genetics in model organisms such as Drosophila melanogaster , Arabidopsis thaliana , zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells — this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

TALEs (transcription activator-like effectors) are transcription factors from the plant pathogen Xanthomonas that can be readily engineered to bind new DNA sequences of interest. Miller et al . use a truncated TALE linked to a nuclease domain to edit and regulate endogenous genes in human cells. 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.

The authors selectively modify chromatin in a specific gene in vivo to examine the link between chromatin dynamics and drug- and stress-evoked responses. They report that histone methylation or acetylation at the FosB locus in nucleus accumbens is sufficient to control drug- and stress-evoked transcriptional and behavioral responses. Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo . We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

Selective inhibition of disease-related proteins underpins the majority of successful drug–target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.

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.

A pilot study in nonhuman primates was conducted, in which two Rhesus macaques received bilateral parenchymal infusions of adeno-associated virus serotype 9 encoding green fluorescent protein (AAV9-GFP) into each putamen. The post-surgical in-life was restricted to 3 weeks in order to minimize immunotoxicity expected to arise from expression of GFP in antigen-presenting cells. Three main findings emerged from this work. First, the volume over which AAV9 expression was distributed (Ve) was substantially greater than the volume of distribution of MRI signal (Vd). This stands in contrast with Ve/Vd ratio of rAAV2, which is lower under similar conditions. Second, post-mortem analysis revealed expression of GFP in thalamic and cortical neurons as well as dopaminergic neurons projecting from substantia nigra pars compacta, indicating retrograde transport of AAV9. However, fibers in the substantia nigra pars reticulata, a region that receives projections from putamen, also stained for GFP, indicating anterograde transport of AAV9 as well. Finally, one hemisphere received a 10-fold lower dose of vector compared with the contralateral hemisphere (1.5 × 10 13 vg ml −1 ) and we observed a much stronger dose effect on anterograde-linked than on retrograde-linked structures. These data suggest that AAV9 can be axonally transported bi-directionally in the primate brain. This has obvious implications to the clinical developing of therapies for neurological disorders like Huntington’s or Alzheimer’s diseases.

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.

The capacity degradation mechanism in lithium nickel-manganese-cobalt oxide （NMC） cathodes （LiNi1/3Mn1/3Co1/3O2 （NMC333） and LiNi0.4Mn0.4Co0.2O2 （NMC442）） during high-voltage （cut-off of 4.8 V） operation has been investigated. In contrast to NMC442, NMC333 exhibits rapid structural changes including severe micro-crack formation and phase transformation from a layered to a disordered rock-salt structure, as well as interfacial degradation during high-voltage cycling, leading to a rapid increase of the electrode resistance and fast capacity decline. The fundamental reason behind the poor structural and interracial stability of NMC333 was found to be correlated to its high Co content and the significant overlap between the Co3＋/4＋ t2g and O2- 2p bands, resulting in oxygen removal and consequent structural changes at high voltages. In addition, oxidation of the electrolyte solvents by the extracted oxygen species generates acidic species, which then attack the electrode surface and form highly resistive LiF. These findings highlight that both the structural and interfacial stability should be taken into account when tailoring cathode materials for high voltage battery systems.

Angiogenesis is of critical importance to tumor progression and therefore is an attractive target in the treatment of cancer. The pro-angiogenic Vascular Endothelial Growth Factor A (VEGF-A) a potent effector of angiogenesis, is over-expressed in a variety of human cancers, including highly invasive brain tumors. In this study, we have applied engineered zinc-finger protein transcription factors (ZFP TFs) to the regulation of VEGF-A expression in human cancer cell lines. We show that ZFP TFs containing either the ligand-binding domain of thyroid hormone receptor or its viral relative vErbA are potent transcriptional repressors of VEGF-A expression. The engineered ZFP chimera specifically binds its target site within the VEGF-A promoter in vitro and in vivo] and represses in a HDAC dependant manner via targeted deacetylation of promoter nucleosomes. The therapeutic relevance of ZFP-driven VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant over-expression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of this gene by >20-fold. Furthermore, the levels of VEGF-A following ZFP TF-mediated repression were comparable to those of a non-angiogenic cancer line, U251MG, suggesting that this degree of repression may be sufficient to suppress tumor angiogenesis. The use of ZFP-mediated recruitment of alternative repression domains and activities which may confer long term, epigenetic repression; such as DNA methylation, histone H3 lysine 9 methylation and HP1 recruitment in the repression of VEGF-A transcription will also be discussed.