Engineered Zinc Finger Protein Transcription Research Articles

Persistent repression of tau in the brain using engineered zinc finger protein transcription factors.

Neuronal tau reduction confers resilience against β-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

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.

Transcriptional activation of Brassica napus β‐ketoacyl‐ACP synthase II with an engineered zinc finger protein transcription factor

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.

991. Engineered Zinc Finger Protein Transcription Factors as a Potential Therapy for Neuropathic Pain

991. Engineered Zinc Finger Protein Transcription Factors as a Potential Therapy for Neuropathic Pain

(108) Engineered zinc finger protein transcription factors as a potential therapy for neuropathic pain

(108) Engineered zinc finger protein transcription factors as a potential therapy for neuropathic pain

32. HSV-Mediated Delivery of a TrkA-Repressing Engineered Zinc Finger Protein Transcription Factor Reduces Nociception in a Mouse Model of Bone Cancer Pain

32. HSV-Mediated Delivery of a TrkA-Repressing Engineered Zinc Finger Protein Transcription Factor Reduces Nociception in a Mouse Model of Bone Cancer Pain

Controlling gene expression in Drosophila using engineered zinc finger protein transcription factors

Zinc finger protein transcription factors (ZFP TFs) have been designed to control the expression of endogenous genes in a variety of cells. However, thus far the use of engineered ZFP TFs in germline transgenic settings has been restricted to plants. Here we report that ZFP TFs can regulate gene expression in transgenic Drosophila. To demonstrate this, we targeted the promoter of the well-characterized fushi tarazu (ftz) gene with a ZFP TF activator using the VP16 activation domain from Herpes simplex virus, and ZFP TF repressors using the Drosophila methyl-CpG binding domain (MBD)-like Δ protein. Heat-shock-inducible expression of the ZFP TF activator and repressors resulted in reciprocal effects on ftz regulation, as deduced from changes in the staining pattern and intensity of ftz and en gene expression, and from the cuticular analysis of first instar larvae. These data demonstrate the utility of ZFP TFs as tools for controlling gene expression in the context of a metazoan organism.

Gene Transfer of an Engineered Transcription Factor Promoting Expression of VEGF-A Protects Against Experimental Diabetic Neuropathy

Peripheral neuropathy is a common, irreversible complication of diabetes. We investigated whether gene transfer of an engineered zinc finger protein transcription factor (ZFP-TF) designed to upregulate expression of the endogenous vascular endothelial growth factor (VEGF)-A gene could protect against experimental diabetic neuropathy. ZFP-TF-driven activation of the endogenous gene results in expression of all of the VEGF-A isoforms, a fact that may be of significance for recapitulation of the proper biological responses stimulated by this potent neuroprotective growth factor. We show here that this engineered ZFP-TF activates VEGF-A in appropriate cells in culture and that the secreted VEGF-A protein induced by the ZFP protects neuroblastoma cell lines from a serum starvation insult in vitro. Importantly, single and repeat intramuscular injections of formulated plasmid DNA encoding the VEGF-A-activating ZFP-TF resulted in protection of both sensory and motor nerve conduction velocities in a streptozotocin-induced rat model of diabetes. These data suggest that VEGF-A-activating ZFP-TFs may ultimately be of clinical utility in the treatment of this disease.

1000. Down-Regulating the Epithelial Glycoprotein-2 Promoter Utilizing Engineered Zinc Finger Protein Transcription Factors

Top of pageAbstract Engineered zinc finger protein transcription factors (ZFP-TFs) are emerging as novel powerful tools to regulate endogenous gene expression. Zinc finger proteins consist of modular building blocks, with each domain recognizing 3-4 base pairs. Stitching these modular domains together results in the recognition of an extended DNA sequence, increasing the specificity of the target sequence. Specific amino acid positions within each domain recognize the DNA. Altering the amino acid residues at these positions therefore results in the recognition of different base pairs. A wide variety of effector domains can be attached to the DNA-binding zinc finger protein, for example to activate or inhibit gene transcription. In this study we utilized engineered ZFP-TFs to specifically inhibit the expression of the epithelial glycoprotein-2 (EGP-2). EGP-2 is expressed in the most prevalent types of carcinomas, including breast, ovarian, and colon cancer. Recently, others have demonstrated that down-regulation of EGP-2 using siRNA or ODNs resulted in a decrease in the migration, invasion, and proliferation potential of tumor cells. Therefore, inhibiting the expression of EGP-2 can provide a novel anti-cancer treatment for a broad range of tumor types. Down-regulating EGP-2 expression by targeting the mRNA requires the targeting of many molecules. Direct down-regulation of the EGP-2 expression at the DNA level using engineered ZFP-TFs can therefore be more effective. We engineered a zinc finger protein that specifically bound to a 9 base-pair target sequence in the EGP-2 promoter sequence. After attaching a gene-expression inhibition domain to the DNA-binding ZFP, co-transfection of EGP-2-positive cells with a plasmid expressing the engineered ZFP-TF and a plasmid containing the luciferase gene under the control of the EGP-2 promoter resulted in 60% inhibition of luciferase expression. To further increase the specificity for binding to the EGP-2 promoter, we engineered three different ZFP-TFs targeting 18 base pair sequences within the EGP-2 promoter. Co-transfection experiments demonstrated that the engineered ZFP-TFs caused up to 80% down-regulation of the EGP-2 promoter while no down-regulation of a non-target promoter was observed. Combining inhibition at the DNA level with inhibition at the RNA level using siRNA might even further enhance the down-regulation of EGP-2 promoter activity. In this study we have demonstrated that ZFP-TFs can be engineered to down-regulate the EGP-2 promoter, generating a novel therapeutic for the treatment of a broad variety of tumor types.

788. Engineered Zinc Finger Protein Transcription Factors as a Potential Therapy for Choroidal Neovascularization

Choroidal neovascularization (CNV) is the most common cause of severe vision loss in patients with age-related macular degeneration (AMD). Anti-angiogenesis therapies, especially those that target Vascular Endothelial Growth Factor (VEGF), have shown promise in treating neovascular AMD. However, targeting VEGF alone maynot yield maximal therapeutic effects. Zinc-finger protein transcription factors (ZFP TF) can be engineered to regulate the expression of virtually any endogenous gene. We sought to test the effects in vitro and in vivo ZFPs designed to repress the transcription of VEGF-A and those designed to activate the transcription of Pigment Epithelium Derived Factor (PEDF), a potent anti-angiogenic factor.

1073. Down-Regulation of Epithelial Glycoprotein-2 and VEGF-D by Engineered Zinc Finger Protein Transcription Factors

Engineered zinc finger protein (ZFP) transcription factors are emerging as powerful tools to regulate endogenous gene expression as they are capable of selectively down- or upregulate all (splice) variants of a gene (family), in contrast to siRNA. Zinc finger proteins consist of modular building blocks recognizing 3|[ndash]|4 base pairs each. Stitching these fingers together results in the recognition of an extended and therefore unique DNA sequence. The small size of these transcription factors allows easy manipulation for efficient delivery and multiple transcription factors can be delivered utilizing one vector. As the target DNA sequence does not have to be palindromic, it is feasible to specifically target virtually any DNA sequence in the human genome.

Gene regulation in planta by plant-derived engineered zinc finger protein transcription factors

The ability to modify plant traits is of great commercial potential in agricultural biotechnology. To this end we have engineered plant-based zinc finger protein transcription factors (ZFP TFs) that minimize the use of non-plant DNA sequences. This novel architecture supports the use of tandem arrays of zinc-finger DNA recognition domains such that the ZFP TF binds a contiguous DNA target site - thus emulating the design of ZFP TFs described previously for mammalian gene regulation. We show that this plant-based ZFP TF architecture supports high affinity DNA binding while allowing the specificity of the DNA-protein interaction to be determined by the amino acid sequences of the recognition helices. This plant-based backbone thus supports the use of previously characterized DNA recognition helices originally identified in a mammalian ZFP context without using mammalian DNA sequences. Moreover, we show that plant-based ZFP TFs employing this new architecture can up-regulate endogenous ADH activity by > 20-fold in transgenic Arabidopsis. Thus plant-based ZFP TFs are shown to be potent regulators of gene expression in vivo.

107. Use of Adeno-Associated Viral Vectors for Effective Delivery of Engineered Zinc Finger Protein Transcription Factors

Zinc finger protein transcription factors (ZFP TFs) can be designed to regulate the expression of any endogenous gene, and thus provide a powerful platform for drug target validation and human therapeutics development. To deliver ZFP TFs into target cells that are difficult to transfect and for long-term expression of therapeutic ZFP TFs in mammalian tissues, we produced adeno-associated viral (AAV) vectors that express ZFP TFs using the triple plasmid transfection method. Crude AAV lysates were used to infect a range of cell types including a human osteosarcoma cell line (U2OS), human primary uterine smooth muscle cells (UtSMC) and rat primary cardiomyoctes. We show here that AAV vectors are capable of efficiently delivering ZFP TFs that cause the down-regulation of the checkpoint kinase 2 (CHK2) or phospholamban gene in these cells. Our results indicate that AAV vectors are effective vehicles for delivering therapeutic ZFP TFs and provide a convenient tool for functional screening of ZFP TFs in hard-to-transfect cells. The AAV vector that expresses a phospholamban-repressing ZFP TF validated here is currently being tested in pre-clinical animal models of congestive heart failure.

325. Identification of Mechanistically Distinct Repression Domains at Endogenous Genes Using Engineered Zinc Finger Proteins

Engineered zinc finger protein transcription factors (ZFP TFs) are capable of controlling the expression of targeted endogenous genes, and thus provide potential therapeutics for the treatment of disease. The functionality of ZFP TFs results from the modularity of the DNA binding and effector domains allowing such ZFP-fusion proteins to address a given domain to a specific chromosomal gene in vivo. This technology thus allows novel effector domains to be assayed for function directly at an endogenous gene. Since this system requires no insertion of binding sites or other foreign DNA into the region of interest, the efficacy of a given ZFP-domain fusion can be determined in the context of truly native chromatin. This is of particular significance to transcriptional regulation since promoter specific modification of histones plays a dynamic role in the process of gene control. We show here that a ZFP targeted to an endogenous reporter gene can be used to successfully identify new effector domains capable of repressing gene expression in vivo. Furthermore we show that the requirements for gene repression at an endogenous gene are not always recapitulated by more standard plasmid-based reporter assays. Finally we use this system to identify different effector domains that employ alternative repression pathways, and show that a domain from the TGF-beta inducible early gene is capable of driving gene repression in an HDAC and DNA methylation independent manner. The ability to screen for, and employ alternative effector domains with defined sensitivity toward known drugs opens the potential for the combinatorial use of such compounds with ZFP TF therapeutic interventions to both accentuate, or limit their function.

324. The Degron Motif: A Protein Destruction Module with Potent Transcriptional Activation Properties for ZFP-Targeted Gene Regulation

Engineered zinc finger protein transcription factors (ZFP TFs) can be designed to regulate any intended gene target, with single gene specificity within the genome. This mode of gene up-regulation outperforms that of the cDNA since (i) very low levels of expression of the ZFP TF activator are required for a dramatic effect on the gene target; and (ii) the ZFP TF also upregulates all of the splice variants, a fact which may, in many instances, be critical to therapeutic efficacy. To fully exploit the remarkably low steady-state levels of engineered ZFP TFs necessary for in vivo function we describe here a ZFP TF construct that has been modified to induce proteasome mediated self-destruction following transcriptional activation of the target gene. To do this, we have made use of the motif, a protein degradation module present in the most potent of natural transcription factors, including VP16. We show that incorporation of the degron motif into an engineered ZFP TF resulted in the proteasome-dependent degradation of ZFP TF, leading to an almost undetectable level of ZFP TF protein at steady state. Importantly, the low protein levels observed for these degron-containing ZFP TFs did not impair their ability to activate gene expression. Indeed, for the genes tested, equivalent or even increased activation of the target gene was obtained by the degron-constructs. Furthermore, we show that a degron containing ZFP TF may be combined with a regulatable switch such as the progesterone receptor ligand-binding domain, resulting in mifpristone-dependent up-regulation of the target gene. The degron motif thus represents a potent, self-limiting activation domain that permits maximal efficacy of the ZFP TF while simultaneously restricting ZFP TF protein levels. The specificity and potency of ZFP TFs can thus be controlled even within the context of gene therapy.

780. Repression of Endogenous VEGF-A Gene with Engineered Zinc Finger Protein Transcription Factors

780. Repression of Endogenous VEGF-A Gene with Engineered Zinc Finger Protein Transcription Factors