Artificial Transcription Factors Research Articles

Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice.

The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer's disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders.

Drug-tunable multidimensional synthetic gene control using inducible degron-tagged dCas9 effectors

The nuclease-deactivated variant of CRISPR-Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable orthologous dCas9 or Cpf1 effector proteins, thus opening options for multidimensional control of functional activities through combinations of orthogonal, drug-tunable artificial transcription factors.

ER Membrane Protein Interactions Using the Split-Ubiquitin System (SUS).

Protein-protein interactions (PPIs) play fundamental roles in all cellular processes. Especially membrane proteins facilitate a range of important biological functions in stimuli perception, signaling, and transport. Here we describe a detailed protocol for the yeast mating-based Split-Ubiquitin System (mbSUS) to study PPIs of ER membrane proteins in vivo. In contrast to the prominent Yeast Two-Hybrid, mbSUS enables analysis of full-length membrane proteins in their native cellular context. The system is based on the ubiquitin proteasome pathway leading to the release of an artificial transcription factor followed by activation of reporter genes to visualize PPIs. The mating-based approach is suitable for both small- and large-scale interaction studies. Additionally, we describe protocols to apply the recently established SUS Bridge assay (SUB) which is optimized for the detection of ternary protein interactions.

Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma.

SOX2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

Plant Gene Regulation Using Multiplex CRISPR-dCas9 Artificial Transcription Factors.

Besides genome editing, the CRISPR-Cas9-based platform provides a new way of engineering artificial transcription factors (ATFs). Multiplex of guide RNA (gRNA) expression cassettes holds a great promise for many useful applications of CRISPR-Cas9. In this chapter, we provide a detailed protocol for building advanced multiplexed CRISPR-dCas9-Activator/repressor T-DNA vectors for carrying out transcriptional activation or repression experiments in plants. We specifically describe the assembly of multiplex T-DNA vectors that can express multiple gRNAs to activate a silenced gene, or to repress two independent miRNA genes simultaneously in Arabidopsis. We then describe a "higher-order" vector assembly method for increased multiplexing capacity. This higher-order assembly method in principle allows swift stacking of gRNAs cassettes that are only limited by the loading capacity of a cloning or expression vector.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

An Arabidopsis mutant with high operating efficiency of Photosystem II and low chlorophyll fluorescence

The overall light energy to biomass conversion efficiency of plant photosynthesis is generally regarded as low. Forward genetic screens in Arabidopsis have yielded very few mutants with substantially enhanced photochemistry. Here, we report the isolation of a novel Arabidopsis mutant with a high operating efficiency of Photosystem II (φPSII) and low chlorophyll fluorescence from a library of lines harboring T-DNA constructs encoding artificial transcription factors. This mutant was named Low Chlorophyll Fluorescence 1 (LCF1). Only a single T-DNA insertion was detected in LCF1, which interrupted the expression of the full length mRNA of the gene At4g36280 (MORC2). We demonstrate that the high φPSII and low levels of chlorophyll fluorescence were due to a decrease in PSII:PSI ratio. Although LCF1 plants had decreased rosette surface area and biomass under normal growth conditions, they contained more starch per gram fresh weight. The growth defect of LCF1 was alleviated by low light and short day conditions, and growth could even be enhanced after a period of dark-induced senescence, showing that the plant can utilize its excess photosynthetic conversion capacity as a resource when needed.

RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts.

Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.

Using artificial transcription factors to induce differentiation into cardiomyocytes

Differentiation of pluripotent cells has become an effective way to study diseases in vitro as well as to engineer tissues for regenerative medicine. However, the discovery of natural transcription factors that specifies cells to desired fates is challenging, expensive, and labor‐intensive. Instead of taking a trial and error approach toward identifying key regulatory genes, artificial transcription factors (ATFs), capable of regulating genes independently of the endogenously expressed proteins, can provide a way to study the transcriptional networks of specific cell states in an unbiased manner. By using a genome‐scale library of ATFs, each comprised of a zinc finger DNA‐binding domain, thousands of genes can be tested in parallel. We applied this forward genetic approach in the differentiation of human pluripotent stem cells into cardiomyocytes. Cardiomyocyte differentiation relies on the temporal regulation of Wnt signaling via small molecule inhibitors. First, a Wnt signaling agonist (CHIR99021) is used to specify pluripotent cells into a mesodermal lineage, followed by treatment with an antagonist (IWP2) to induce cardiomyocyte differentiation. While this method of generating cardiomyocytes is efficient, the off‐target effects of the inhibitors used in this protocol remain unknown, and therefore, the gene networks activated by the inhibitors are not completely understood. By replacing the function of these inhibitors with ATFs, the specific genes involved in differentiation can be identified. The combinations of ATFs identified in the screen were capable of replacing IWP2, an inhibitor that prevents proper Wnt secretion. After confirming that these ATFs are capable of replacing the function of IWP2, cognate site identification was used to determine the optimal binding motifs preferred by the identified ATFs. These binding motifs were then mapped to the genome to identify genes with an ATF binding site within 1 kb of the transcriptional start site. The genes identified in this screen provide further insights into the transcriptional networks controlling the differentiation of human stem cells into cardiomyocytes, and the tools used in this project can be implemented in other biological contexts where cell fate‐defining regulatory networks have been more elusive.

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors.

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.

Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide

The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.

Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems.

Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). These tools are especially useful for studying gene function and interaction within various regulatory networks. First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX). When multiple chimeric dCas9-effector fusions are guided to gene regulatory regions via CRISPR gRNAs, they can modulate expression of transcript levels in planta. The protocol presented here provides a detailed procedure for activating AtPAP1 and repressing AtCSTF64 in Arabidopsis thaliana. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.

Reprogramming cell fate with a genome-scale library of artificial transcription factors

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

Angelman syndrome: Current and emerging therapies in 2016.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a loss of the maternally-inherited UBE3A; the paternal UBE3A is silenced in neurons by a mechanism involving an antisense transcript (UBE3A-AS) at the unmethylated paternal locus. We reviewed all published information on the clinical trials that have been completed as well as the publicly available information on ongoing trials of therapies in AS. To date, all clinical trials that strove to improve neurodevelopment in AS have been unsuccessful. Attempts at hypermethylating the maternal locus through dietary compounds were ineffective. The results of an 8-week open-label trial using minocycline as a matrix metalloproteinase-9 inhibitor were inconclusive, while a subsequent randomized placebo-controlled trial suggested that treatment with minocycline for 8 weeks did not result in any neurodevelopmental gains. A 1-year randomized placebo-controlled trial using levodopa to alter the phosphorylation of calcium/calmodulin-dependent kinase II did not lead to any improvement in neurodevelopment. Topoisomerase inhibitors and antisense oligonucleotides are being developed to directly inhibit UBE3A-AS. Artificial transcription factors are being developed to "super activate" UBE3A or inhibit UBE3A-AS. Other strategies targeting specific pathways are briefly discussed. We also reviewed the medications that are currently used to treat seizures and sleep disturbances, which are two of the more common complications of AS. © 2016 Wiley Periodicals, Inc.

Genome interrogation for novel salinity tolerant Arabidopsis mutants.

Soil salinity is becoming an increasingly large problem in agriculture. In this study, we have investigated whether a capacity to withstand salinity can be induced in the salinity sensitive plant species Arabidopsis thaliana, and whether it can be maintained in subsequent generations. To this end, we have used zinc finger artificial transcription factor (ZF-ATFs) mediated genome interrogation. Already within a relatively small collection Arabidopsis lines expressing ZF-ATFs, we found 41 lines that were tolerant to 100 mM NaCl. Furthermore, ZF-ATF encoding gene constructs rescued from the most strongly salinity tolerant lines were indeed found to act as dominant and heritable agents for salinity tolerance. Altogether, our data provide evidence that a silent capacity to withstand normally lethal levels of salinity exists in Arabidopsis and can be evoked relatively easily by in trans acting transcription factors like ZF-ATFs.

Genome-Editing Technologies: Principles and Applications.

Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system.

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Abstract 999: P16 DNA methylation inactivates transcription of IncRNA ANRIL

Abstract The exonic ANRIL (P15AS) is a 3.8-kb lncRNA transcribed from the antisense strand of the P14 promoter and flanking regions. Recently, we have constructed a P16 promoter DNA methyltransferase (P16-Dnmt) using the pTRIPZ vector that can specifically methylate human P16 CpG islands and found that P16 methylation directly leads to gene transcription silence [Cui et al. Genome Biology 2015, 16:252]. However, whether ANRIL transcription is affected by P16 DNA methylation has not previously been reported. In the present study, correlation between endogenous P16, P15, P14 mRNA and ANRIL levels in a panel of cell lines were determined using quantitative RT-PCR. Unexpectedly, the results showed that ANRIL transcriptional level was positively and significantly correlated with the P16 mRNA level (r = 0.774, P = 0.003), but not correlated with the P15 or P14 mRNA levels (r = 0.09 or 0.30, P = 0.78 or 0.35). These phenomena were confirmed using the public available transcriptome data in the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Furthermore, transcription of both ANRIL and P16 mRNA were observed only in cancer cell lines in which the P16 alleles are unmethylated (Caski, SGC7901, BGC823, GES1, Siha and HeLa), but not in these cell lines in which the P16 alleles are fully methylated (Colo205, PC3, MHCC97H, RKO, SW480 and AGS). These suggest the possibility that methylation of the P16 promoter may inactivate transcription of ANRIL as well as P16. To study the possible causality between P16 DNA methylation and transcriptional silence of ANRIL, we employed P16-Dnmt to induce P16-specific DNA methylation in gastric cancer BGC823 cells, and found that transcription levels of both P16 and ANRIL were significantly decreased by the induced P16 DNA methylation (P<0.001 and 0.024, respectively). Besides, transcription level of P14 was also reduced (P = 0.043) while the P14 promoter CpG islands remained to be unmethylated. In contrast, P16 DNA demethylation induced by an artificial P16-specific transcription factor [Zhang et al. Human Gene Therapy 2012, 23:1071-81] re-activated transcription of both ANRIL and P16 in H1299 cells (P<0.001). The mechanism of inactivation of ANRIL expression by P16 DNA methylation is under investigation. In conclusion, transcription of ANRIL is regulated by P16 DNA methylation. Citation Format: Ying Gan, Baozhen Zhang, Chenghua Cui, Dajun Deng. P16 DNA methylation inactivates transcription of IncRNA ANRIL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 999.

Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain

Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood–brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.

Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. DisclosuresMakala:Georgia Regents University: Employment.