Carl O. Pabo

An improved zinc-finger nuclease architecture for highly specific genome editing

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

Drug discovery with engineered zinc-finger proteins

Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9–18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.

Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo

Covalent modifications of chromatin have emerged as key determinants of the genomes transcriptional competence. Histone H3 lysine 9 (H3K9) methylation is an epigenetic signal that is recognized by HP1 and correlates with gene silencing in a variety of organisms. Discovery of the enzymes that catalyze H3K9 methylation has identified a second gene-specific function for this modification in transcriptional repression. Whether H3K9 methylation is causative in the initiation and establishment of gene repression or is a byproduct of the process leading to the repressed state remains unknown. To investigate the role of HMTs and specifically H3K9 methylation in gene repression, we have employed engineered zinc-finger transcription factors (ZFPs) to target HMT activity to a specific endogenous gene. By utilizing ZFPs that recognize the promoter of the endogenous VEGF-A gene, and thus employing this chromosomal locus as an in vivo reporter, we show that ZFPs linked to a minimal catalytic HMT domain affect local methylation of histone H3K9 and the consequent repression of target gene expression. Furthermore, amino acid substitutions within the HMT that ablate its catalytic activity effectively eliminate the ability of the ZFP fusions to repress transcription. Thus, H3K9 methylation is a primary signal that is sufficient for initiating a gene repression pathway in vivo.

Engineered zinc finger proteins for controlling stem cell fate.

Stem cells are functionally defined as progenitor cells that can self‐renew and differentiate. Critical transitions in these cells are controlled via signaling pathways and subsequent transcriptional regulation. Technologies capable of modulating the levels of gene expression, especially those of transcription factors, represent powerful tools for research and could potentially be used in therapeutic applications. In this study, we evaluated the ability of synthetic zinc finger protein transcription factors (ZFP‐TFs) to cause the differentiation of embryonic stem (ES) cells. We constructed ZFP‐TFs that target the mouse Oct‐4 gene (which is a major regulator of ES cell pluripotency and self‐renewal). These designed transcription factors were able to regulate the transcription of Oct‐4, affecting the expression of downstream genes and thus regulating ES cell differentiation.

Cell Lines for Drug Discovery: Elevating Target-Protein Levels Using Engineered Transcription Factors:

Drug discovery requires high-quality, high-throughput bioassays for lead identification and optimization. These assays are usually based on immortalized cell lines, which express the selected drug target either naturally or as a consequence of transfection with the cDNA encoding the target. Natural untransfected cell lines often fail to achieve the levels of expression required to provide assays of sufficient quality with a high enough signal-to-noise ratio. Unfortunately, the use of cDNA is increasingly restricted, as the sequences for more and more genes become subject to patent restrictions. To overcome these limitations, the authors demonstrate that engineered transcription factors with Cys2-His2 zinc finger DNA-binding domains can be used to effectively activate an endogenous gene of interest without the use of isolated cDNA of the target gene. Using this approach, the authors have generated a cell line that provides a high-quality and pharmacologically validated G-protein-coupled receptor bioassay. In principle, this technology is applicable to any gene of pharmaceutical importance in any cell type. (Journal of Biomolecular Screening 2004:44-51)

Pharmacological analysis of CCK2 receptors up-regulated using engineered transcription factors.

Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.

51. A Novel Approach to Stem Cell Gene Therapy: Controlling Stem Cell ifferentiation with Engineered Zinc-Finger Protein Transcription Factors

Stem cells hold tremendous promise for gene therapy, tissue engineering, and the treatment of a diverse range of injuries and disease. However, exploiting the full potential of these pluripotent progenitor cells requires the establishment of a new set of tools capable of controlling the molecular decisions that determine whether, and how these cells differentiate. Critical transitions in stem cells are controlled via signaling pathways and subsequent transcriptional regulation. In this regard, we have previously shown that designed zinc-finger protein transcription factors (ZFP TFs) are capable of regulating the expression of targeted endogenous genes with singular specificity. In the present study, we evaluated the ability of such designed ZFP TFs to control the differentiation of embryonic stem (ES) cells. To this end, we constructed ZFP TFs that target the mouse OCT4 gene, a major regulator of ES cell pluripotency and self-renewal. We show here that introduction of an activator version of this ZFP TF resulted in increased OCT4 mRNA, and the expected concomitant effects on the expression of OCT4 responsive downstream target genes. Conversely, a repressor version of the OCT4-specific ZFP TF caused a robust reduction in OCT4 expression and the reciprocal downstream effects. Significantly, ZFP TF-dependent morphological differentiation of ES cells was also observed in a manner specific to the effector domain employed. Thus ZFP TFs are shown to be capable of controlling ES cell differentiation, raising the potential for their broad application to stem cell-based therapies.