Siyuan Tan

A TALE nuclease architecture for efficient genome editing

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.

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.

Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

302. Engineered Zinc Finger Transcription Factors: A Platform for Therapeutic Gene Regulation with Exquisite Specificity

A challenge of modern medicine is the development of effective yet highly specific drugs. To this end, we are developing artificial zinc finger protein transcription factors (ZFP TFs) as direct therapeutic agents for the treatment of disease. Here we show that ZFP TFs can be designed to activate or repress a wide range of clinically relevant genes, including Phospholamban, a regulator of heart contractivity, Chk2, a kinase with a role in DNA damage pathway and P2X7, a ligand-gated channel. Moreover, using these examples and the Affymetrix GeneChipR platform we show that these ZFP TFs regulate the targeted gene with exceptional and often singular specificity genome wide, despite the complexity of mammalian genome. This exquisite specificity is independent of both gene target and species, while occurring in the context of maximal efficacy. This combination of potency and specificity supports the clinical potential of ZFP TF technology.