Artificial Zinc Finger Proteins Research Articles

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.

Enhanced Cleavage of Double-Stranded DNA by Artificial Zinc-Finger Nuclease Sandwiched between Two Zinc-Finger Proteins

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.

Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding. In gel shift assays, we confirmed that the designed AZP has a higher affinity to the replication origin than that of Rep and that the AZP effectively inhibited the Rep binding to its replication origin in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. We will discuss properties of the AZP-transgenic plants against TYLCV infection.

Inhibition of human papillomavirus replication by using artificial zinc-finger nucleases

Recently, we have designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined a new approach to inhibition of DNA virus replication by using an AZP-nuclease fusion. In transient replication assays for HPV-18, the gene-delivered AZP-nuclease fusion reduced the viral DNA replication rate significantly. Moreover, it was demonstrated by ligation-mediated PCR that viral DNA regions close to the AZP-binding site were cleaved in the cells by the AZP-nuclease. Thus, our results demonstrate that AZP-nucleases have potentials to inhibit replication of any DNA viruses whose replication mechanisms remain unsolved.

Cell-permeable artificial zinc-finger proteins as potent antiviral drugs for human papillomaviruses

Human papillomavirus (HPV) is one of the important pharmaceutical targets because infection of the high-risk types causes invasive cervical cancer. However, effective antiviral drugs for HPV have not been developed so far. In the present study, we constructed cell-permeable artificial zinc-finger proteins (AZPs) by fusing an AZP previously generated for inhibition of HPV-18 DNA replication with a cell-penetrating peptide (CPP) as candidates for new antiviral drugs against HPV. We confirmed that these CPP-AZP fusions reduced the replication rate in transient replication assays when added to the culture medium. In particular, 250 nM CPP-AZP (designated AZP-R9) containing a 9-mer of arginine as the CPP reduced HPV-18 DNA replication to 3% of that of a control, and the 50% effective concentration (EC50) was <31 nM. Furthermore, a cytotoxicity assay revealed that the 50% inhibitory concentration (IC50) of AZP-R9 was >10 microM. Therefore, the selectivity index, defined as IC50/EC50, was >300, which is better than that of the antiviral cidofovir for HPVs. Thus, our results demonstrate that cell-permeable AZPs could serve as potent protein-based antiviral drugs.

Novel Cancer Antiangiotherapy Using the VEGF Promoter-targeted Artificial Zinc-finger Protein and Oncolytic Adenovirus

Inhibition of tumor angiogenesis through modulation of vascular endothelial growth factor (VEGF) and its signaling pathway has been clinically validated as a viable therapeutic modality in the treatment of cancer. The use of artificial transcription factors based on Cys2-His2 zinc-finger proteins (ZFPs) targeting the VEGF promoter offers a novel strategy for modulating VEGF levels in tumors. In order to demonstrate the utility of VEGF-targeted ZFPs as therapeutic agents, we generated adenoviruses (Ads) expressing VEGF promoter-targeted transcriptional repressor ZFP, F435-KOX. A replication-incompetent Ad expressing F435-KO X, namely, Ad-DeltaE1-KOX, significantly reduced VEGF expression and functionally led to inhibition of angiogenesis. In vivo, an oncolytic Ad expressing F435-KOX, namely, Ad-DeltaB7-KOX, elicited a pronounced antitumor effect against a human glioblastoma xenograft model, U87MG. Further, consistent with its expected mechanism of action, Ad-DeltaB7-KOX was shown to greatly reduce the level of VEGF and vessel density in tumor tissue and increase terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive apoptotic cells in tumors. Survival rates were also significantly increased in Ad-DeltaB7-KOX-treated mice. Taken together, the findings from this study identify F435-KOX as a novel and potent ZFP transcription factor that can inhibit VEGF-A-mediated angiogenesis and offer a novel therapeutic modality in the treatment of cancer.

New Redesigned Zinc‐Finger Proteins: Design Strategy and Its Application

The design of DNA-binding proteins for the specific control of the gene expression is one of the big challenges for several research laboratories in the post-genomic era. An artificial transcription factor with the desired DNA binding specificity could work as a powerful tool and drug to regulate the target gene. The zinc-finger proteins, which typically contain many fingers linked in a tandem fashion, are some of the most intensively studied DNA-binding proteins. In particular, the Cys(2)His(2)-type zinc finger is one of the most common DNA-binding motifs in eukaryotes. A simple mode of DNA recognition by the Cys(2)His(2)-type zinc-finger domain provides an ideal framework for designing proteins with new functions. Our laboratory has utilized several design strategies to create new zinc-finger peptides/proteins by redesigning the Cys(2)His(2)-type zinc-finger motif. This review focuses on the aspects of design strategies, mainly from our recent results, for the creation of artificial zinc-finger proteins, and discusses the possible application of zinc-finger technology for gene regulation and gene therapy.

Development of protein-based antiviral drugs for human papillomaviruses

Recently, we designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined another approach to delivery of AZPs. We constructed cell-permeable AZPs by fusing an AZP previously generated for inhibition of HPV-18 DNA replication with a cell-penetrating peptide (CPP), and confirmed that these CPP-AZP fusions reduced the replication rate in transient replication assays when added to the culture medium. In particular, 250 nM CPP-AZP reduced HPV-18 DNA replication to 3% of that of a control experiment. Western blot analysis detected 7% of the CPP-AZP added to the culture medium in the cell lysates, and demonstrated that greater internalization of CPP-AZP into mammalian cells causes greater inhibition of viral DNA replication. Furthermore, CPP-AZP did not show any significant cytotoxixity in MTT assays. Thus, our results demonstrate that cell-permeable AZPs could serve as potent protein-based antiviral drugs.

Inhibition of tomato yellow leaf curl virus replication by artificial zinc-finger proteins

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed an AZP binding to the direct repeat to block the TYLCV Rep binding and confirmed in gel shift assays that the designed AZP has a higher affinity to the replication origin than that of Rep. Furthermore, we demonstrated in competitive binding assays that the AZP effectively inhibited the Rep binding in vitro. We discuss properties of the AZP for inhibition of TYLCV replication in detail.

Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins

Several techniques are available to study chromosomes or chromosomal domains in nuclei of chemically fixed or living cells. Current methods to detect DNA sequences in vivo are limited to trans interactions between a DNA sequence and a transcription factor from natural systems. Here, we expand live cell imaging tools using a novel approach based on zinc finger-DNA recognition codes. We constructed several polydactyl zinc finger (PZF) DNA-binding domains aimed to recognize specific DNA sequences in Arabidopsis and mouse and fused these with GFP. Plants and mouse cells expressing PZF:GFP proteins were subsequently analyzed by confocal microscopy. For Arabidopsis, we designed a PZF:GFP protein aimed to specifically recognize a 9-bp sequence within centromeric 180-bp repeat and monitored centromeres in living roots. Similarly, in mouse cells a PZF:GFP protein was targeted to a 9-bp sequence in the major satellite repeat. Both PZF:GFP proteins localized in chromocenters which represent heterochromatin domains containing centromere and other tandem repeats. The number of PZF:GFP molecules per centromere in Arabidopsis, quantified with near single-molecule precision, approximated the number of expected binding sites. Our data demonstrate that live cell imaging of specific DNA sequences can be achieved with artificial zinc finger proteins in different organisms.

287. An Omega-Based Bacterial Selection System for Engineering Artificial Zinc Finger Proteins

287. An Omega-Based Bacterial Selection System for Engineering Artificial Zinc Finger Proteins

Application of artificial zinc-finger proteins to inhibition of DNA replication of human papillomavirus

Recently, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to inhibition of replication of a mammalian DNA virus, human papillomavirus (HPV) type 18. Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed to block binding of the HPV-18 E2 replication protein to the replication origin. Both the designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited the viral DNA replication: AZP(HPV)-2, especially, reduced the replication level to approximately 10%. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses, but also to mammalian DNA viruses.

Regulation of cancer-related growth factor expression by artificial zinc-finger proteins

One attractive approach to anticancer therapy is repression of expression of vascular endothelial growth factor (VEGF) gene, which is a potent target for prevention of tumor growth. To achieve this, artificial transcription factors (ATF) designed for VEGF gene regulation were fused to cell-penetrating peptides (CPPs). We demonstrated ATFs fused to CPPs, designated CPP-ATFs or designed regulatory proteins (DRPs), could penetrate into mammalian cells and transiently repress expression of a reporter gene, which was under control of the VEGF promoter/5'-UTR. We discuss gene-regulatory properties of CPP-ATFs in detail.

Inhibition of DNA Replication of Human Papillomavirus by Artificial Zinc Finger Proteins

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.

Artificial Transcription Factors Increase Production of Recombinant Antibodies in Chinese Hamster Ovary Cells

A randomized library that encodes for artificial zinc finger protein transcription factors (ZFP-TF) was constructed and screened for components that increased production of a monoclonal antibody (mAb-72) in Chinese hamster ovary (CHO) cells. One of these ZFP-TF, LK52, increased mAb-72 production approximately 10-fold at approximately 60% transduction efficiency; a mutated version of LK52, however, did not boost mAb-72 production. LK52 also increased production of other mAbs in CHO cells. These results demonstrate that ZFP-TF libraries can be used to identify components that improve antibody production in CHO cells.

Effects of linking 15-zinc finger domains on DNA binding specificity and multiple DNA binding modes

To assess the possibility of multi-connection of zinc finger domains for understanding of DNA binding mechanisms and gene regulation, the longest artificial zinc finger protein, Sp1ZF15, has been constructed. This zinc finger consists of 5 units of Sp1 zinc finger peptide connected by canonical short linker sequences (TGEKP). Recognition of the 50 base pairs of DNA and potential binding to shorter targets by Sp1ZF15 were determined. Sequence alterations of the GCG triplet to ATA at a target site clearly showed that Sp1ZF15 changes its DNA binding mode depending on the target sequences. Of special interest is the fact that Sp1ZF15 controls the number of finger domains active in DNA binding corresponding to the length and sequence of the target DNA. These results suggest that artificial transcription factors based upon these multi-zinc finger proteins have great potential for the regulation of a vast number of cellular processes.

Inhibition of Virus DNA Replication by Artificial Zinc Finger Proteins

Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.

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.

1049. Engineered ZFPs Can Prevent Limb Loss in Mice

Top of pageAbstract The ability to engineer artificial zinc finger protein (ZFP)-based transcription factors has led to a new and growing area of gene therapy, targeted regulation of endogenous genes. One potential advantage of this approach is that it facilitates expression of all natural splice variants of a targeted gene. To investigate the therapeutic implications of this approach we used an engineered ZFP (VOP32E) that activates transcription of the VEGF-A gene. An adenovirus encoding VOP32E was constructed and used to evaluate the ability of VOP32E-based VEGF-A expression to rescue hindlimb ischemia (HI) in aged C57BL/6J (WT) mice. HI was created by excision of left common femoral artery and proximal portion of saphenous artery. In aged WT mice this intervention results in a high incidence of limb loss. In addition, unlike young WT mice, blood flow does not return to normal in these mice by means of the endogenous response to ischemia. Delivery of Ad-VOP32E to the adductor muscles of these mice resulted in a significant improvement in a clinical score and reduced the incidence of limb loss as compared to mice treated with a control virus (Ad-RFP). This was accompanied by a significant increase in blood flow as measured serially (1,2 and 4 weeks) after treatment with a modified laser doppler system that measures deep tissue blood flow. Immunohistochemical staining for PECAM demonstrated an increase in capillary density in the ZFP treated limbs, and quantitative RT-PCR demonstrated an increase in total VEGF mRNA, as well as increased expression of all major splice variants. This data documents that engineered transcription factors can be effective therapeutically in mammalian systems, and specifically that endogenous VEGF-A activation by an engineered ZFP is effective in an ischemia model that does not have spontaneous recovery.

The artificial zinc finger protein ‘Blues’ binds the enhancer of the fibroblast growth factor 4 and represses transcription

The design of novel genes encoding artificial transcription factors represents a powerful tool in biotechnology and medicine. We have engineered a new zinc finger-based transcription factor, named Blues, able to bind and possibly to modify the expression of fibroblast growth factor 4 (FGF-4, K-fgf), originally identified as an oncogene. Blues encodes a three zinc finger peptide and was constructed to target the 9 bp DNA sequence: 5′-GTT-TGG-ATG-3′, internal to the murine FGF-4 enhancer, in proximity of Sox-2 and Oct-3 DNA binding sites. Our final aim is to generate a model system based on artificial zinc finger genes to study the biological role of FGF-4 during development and tumorigenesis.