TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

Trinucleotide expansions cause disease by both protein-and RNAmediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.T ranslation of mRNA into protein is an exquisitely regulated, almost error-free process. Well-established rules of translational initiation have been used as a cornerstone in biology to understand gene expression and to predict the consequences of disease-causing mutations (1). For microsatellite expansion disorders, mutations within or outside ATG-initiated ORFs are thought to cause disease either by protein gain-of-function, protein loss-of-function, or RNA gain-of-function mechanisms (2, 3).Microsatellite expansion mutations that express polyglutamine (polyGln) expansion proteins include Huntington disease (Huntingtin, HD), spinal bulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17. Since the discovery of these CAG·CTG expansion mutations, efforts to understand disease mechanisms have focused on elucidating the molecular effects of the polyGln proteins expressed from these loci. Although these polyGln expansion proteins bear no similarity to each other apart from the polyGln tract, a hallmark of these diseases is protein accumulation and aggregation in nuclear or cytoplasmic inclusions. Surprisingly, although the polyGln expansion proteins are widely expressed in the CNS and other tissues, only restricted populations of neurons are vulnerable in each disease (3).Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are the best-characterized examples of RNA-mediated expansion disorders (2). The mutation causing DM1 is a CTG-repeat expansion located in the 3′ UTR of the dystrophia myotonica-protein kinase (DMPK) gene. Although DM1 can be clinically more severe than DM2, the discovery of the DM2 mutation and several mouse models provide strong support that many features of these diseases result from RNA gain-of-function effects in which the dysregulation of RNA-binding proteins is mediated by the expression of CUG and CCUG transcripts (4). Additionally, RNA gain-of-function effects have been reported for CGG and CAG expansion RNAs (5, 6).Both RNA and protein mechanisms appear to operate...

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The art and science of gene therapy has received much attention of late. The tragic death of 18-year-old Jesse Gelsinger, a volunteer in a Phase I clinical trial, has overshadowed the successful treatment of three children suffering from a rare but fatal immunological disease. In the light of the success and tragedy, it is timely to consider the challenges faced by gene therapy--a novel form of molecular medicine that may be poised to have an important impact on human health in the new millennium.

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Programmed cell death regulates a number of biological phenomena, and the apoptotic signal must itself be tightly controlled to avoid inappropriate cell death. We established a genetic screen to search for molecules that inhibit the apoptotic signal from the Fas receptor. Here we report the isolation of a gene, LFG, that protects cells uniquely from Fas but not from the mechanistically related tumor necrosis factor ␣ death signal. LFG is widely distributed, but remarkably is highly expressed in the hippocampus. LFG can bind to the Fas receptor, but does not regulate Fas expression or interfere with binding of an agonist antibody. Furthermore LFG does not inhibit binding of FADD to Fas.

We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli j3-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.