Sangamo BioSciences

About: Sangamo BioSciences is a based out in . It is known for research contribution in the topics: Zinc finger & Zinc finger nuclease. The organization has 312 authors who have published 397 publications receiving 56406 citations.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

A TALE nuclease architecture for efficient genome editing

Abstract: 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.

Genome editing with engineered zinc finger nucleases

Abstract: 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.

Highly efficient endogenous human gene correction using designed zinc-finger nucleases

Abstract: 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.

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.

Abstract: Background CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe. Methods We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. Results One serious adverse event was associated with infusion of the ZFN-modified autologous C...