RESUMO
Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF--an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression--would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats--under cyclosporin immunosuppression--received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits.
Assuntos
Adenoviridae , Terapia Genética/métodos , Hiperalgesia/terapia , Traumatismos da Medula Espinal/terapia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Dedos de Zinco , Animais , Feminino , Células HEK293 , Humanos , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Neovascularização Fisiológica/genética , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).
Assuntos
Linfócitos T CD4-Positivos/transplante , Terapia Genética , Infecções por HIV/terapia , Transfusão de Linfócitos , Receptores CCR5/genética , Adulto , Terapia Antirretroviral de Alta Atividade , Transfusão de Sangue Autóloga , Linfócitos T CD4-Positivos/química , Terapia Combinada , DNA Viral/sangue , Feminino , Terapia Genética/efeitos adversos , Terapia Genética/métodos , HIV/genética , HIV/isolamento & purificação , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Reto/imunologia , Carga ViralRESUMO
Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.
Assuntos
Enzimas de Restrição do DNA/genética , Vetores Genéticos/normas , Infecções por HIV/genética , Infecções por HIV/imunologia , Receptores CCR5/genética , Dedos de Zinco/genética , Adenovírus Humanos/genética , Transferência Adotiva , Animais , Antígenos CD28/imunologia , Complexo CD3/imunologia , Enzimas de Restrição do DNA/metabolismo , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Vetores Genéticos/genética , Infecções por HIV/terapia , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Fenótipo , Receptores CCR5/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética/métodos , Transdução Genética/normas , Transplante HeterólogoRESUMO
Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5 atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72 h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery.
Assuntos
Lesões Encefálicas/terapia , Terapia Genética , Fator A de Crescimento do Endotélio Vascular/genética , Dedos de Zinco/genética , Animais , Western Blotting , Lesões Encefálicas/patologia , Lesões Encefálicas/psicologia , Região CA1 Hipocampal/patologia , Capilares/patologia , Dependovirus/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Vetores Genéticos , Marcação In Situ das Extremidades Cortadas , Potenciação de Longa Duração , Masculino , Microinjeções , Neovascularização Fisiológica/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/terapia , Fatores de Transcrição/metabolismo , Dedos de Zinco/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Insuficiência Cardíaca/genética , Humanos , Cinética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Dedos de Zinco/genéticaRESUMO
Recent studies have identified anti-apoptotic functions for vascular endothelial growth factor (VEGF) in the central nervous system (CNS). However, VEGF therapy has been hampered by a tendency to promote vascular permeability, edema, and inflammation. Recently, engineered zinc finger proteins (ZFPs) that upregulate multiple forms of VEGF in their natural biological ratios, have been developed to overcome these negative side effects. We used retinal trauma and ischemia models, and a cortical pial strip ischemia model to determine if VEGF upregulating ZFPs are neuroprotective in the adult CNS. Optic nerve transection and ophthalmic artery ligation lead to the apoptotic degeneration of retinal ganglion cells (RGCs) and are, respectively, two highly reproducible models for CNS trauma or ischemia. Adeno-associated vectors (AAV) vectors encoding VEGF-ZFPs (AAV-VEGF-ZFP) significantly increased RGC survival by â¼twofold at 14 days after optic nerve transection or ophthalmic artery ligation. Furthermore, AAV-VEGF-ZFP enhanced recovery of the pupillary light reflex. RECA-1 immunostaining demonstrated no appreciable differences between retinas treated with AAV-VEGF-ZFP and controls, suggesting that AAV-VEGF-ZFP treatment did not affect retinal vasculature. Following pial strip of the forelimb motor cortex, brains treated with an adenovirus encoding VEGF ZFPs (AdV-ZFP) showed higher neuronal survival, accelerated wound contraction, and reduced lesion volume between 1 and 6 weeks after injury. Behavioral testing using the cylinder test for vertical exploration showed that AdV-VEGF-ZFP treatment enhanced contralateral forelimb function within the first 2 weeks after injury. Our results indicate that VEGF ZFP therapy is neuroprotective following traumatic injury or stroke in the adult mammalian CNS.
Assuntos
Lesões Encefálicas/terapia , Terapia Genética/métodos , Acidente Vascular Cerebral/terapia , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/genética , Dedos de Zinco/genética , Animais , Comportamento Animal/fisiologia , Lesões Encefálicas/genética , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/terapia , Engenharia de Proteínas , Ratos , Recuperação de Função Fisiológica/genética , Acidente Vascular Cerebral/genética , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: The objectives of the study were to evaluate retrograde axonal transport of vascular endothelial growth factor A (VEGF-A) protein to sensory neurons after intramuscular administration of an engineered zinc finger protein activator of endogenous VEGF-A (VZ+434) in an experimental model of diabetes, and to characterize the VEGF-A target neurons. RESEARCH DESIGN AND METHODS: We compared the expression of VEGF-A in lumbar (L)4/5 dorsal root ganglia (DRG) of control rats and VZ+434-treated and untreated streptozotocin (STZ)-induced diabetic rats. In addition, axonal transport of VEGF-A, activation of signal transduction pathways in the DRG, and mechanical sensitivity were assessed. RESULTS: VEGF-A immunoreactivity (IR) was detected in small- to medium-diameter neurons in DRG of control rats. Fewer VEGF-A-IR neurons were observed in DRG from STZ-induced diabetic rats; this decrease was confirmed and quantified by Western blotting. VZ+434 administration resulted in a significant increase in VEGF-A protein expression in ipsilateral DRG, 24 h after injection. VEGF-A was axonally transported to the DRG via the sciatic nerve. VZ+434 administration resulted in significant activation of AKT in the ipsilateral DRG by 48 h that was sustained for 1 week after injection. VZ+434 protected against mechanical allodynia 8 weeks after STZ injection. CONCLUSIONS: Intramuscular administration of VZ+434 increases VEGF-A protein levels in L4/5 DRG, correcting the deficit observed after induction of diabetes, and protects against mechanical allodynia. Elevated VEGF-A levels result from retrograde axonal transport and are associated with altered signal transduction, via the phosphatidylinositol 3'-kinase pathway. These data support a neuroprotective role for VEGF-A in the therapeutic actions of VZ+434 and suggest a mechanism by which VEGF-A exerts this activity.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Gânglios Espinais/fisiopatologia , Células Receptoras Sensoriais/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Dedos de Zinco/fisiologia , Animais , Glicemia/metabolismo , Peso Corporal , Regulação para Baixo , Lateralidade Funcional , Engenharia Genética , Aumento da Imagem , Imuno-Histoquímica , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Neurônios/fisiologia , Fenótipo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/deficiência , Fator A de Crescimento do Endotélio Vascular/genética , Dedos de Zinco/genéticaRESUMO
Spinal cord injury (SCI) leads to local vascular disruption and progressive ischemia, which contribute to secondary degeneration. Enhancing angiogenesis through the induction of vascular endothelial growth factor (VEGF)-A expression therefore constitutes an attractive therapeutic approach. Moreover, emerging evidence suggests that VEGF-A may also exhibit neurotrophic, neuroprotective, and neuroproliferative effects. Building on this previous work, we seek to examine the potential therapeutic benefits of an engineered zinc finger protein (ZFP) transcription factor designed to activate expression of all isoforms of endogenous VEGF-A (ZFP-VEGF). Administration of ZFP-VEGF resulted in increased VEGF-A mRNA and protein levels, an attenuation of axonal degradation, a significant increase in vascularity and decreased levels of apoptosis. Furthermore, ZFP-VEGF treated animals showed significant improvements in tissue preservation and neurobehavioural outcomes. These data suggest that activation of VEGF-A via the administration of an engineered ZFP transcription factor holds promise as a therapy for SCI and potentially other forms of neurotrauma.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/terapia , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Apoptose/genética , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Modelos Animais de Doenças , Feminino , Vetores Genéticos/farmacologia , Vetores Genéticos/uso terapêutico , Neovascularização Fisiológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/metabolismo , Fatores de Transcrição/farmacologia , Ativação Transcricional/genética , Resultado do Tratamento , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Degeneração Walleriana/terapia , Dedos de Zinco/genéticaRESUMO
PURPOSE: Self-complementary AAV (scAAV) vectors have been developed to circumvent rate-limiting second-strand synthesis in single-stranded AAV vector genomes and to facilitate robust transgene expression at a minimal dose. In this study, the authors investigated the effects of intraocular injections of type 2 scAAV.GFP in mice. METHODS: Dose-response experiments were performed to compare conventional single-strand AAV type 2 (ssAAV2) vectors with scAAV2 vectors encoding an identical expression cassette. RESULTS: Subretinal injection of 5 x 10(8) viral particles (vp) of scAAV.CMV-GFP resulted in green fluorescent protein (GFP) expression in almost all retinal pigment epithelial (RPE) cells within the area of the small detachment caused by the injection by 3 days and strong, diffuse expression by 7 days. Expression was strong in all retinal cell layers by days 14 and 28. In contrast, 3 days after subretinal injection of 5 x 10(8) vp of ssAAV.CMV-GFP, GFP expression was detectable in few RPE cells. Moreover, the ssAAV vector required 14 days for the attainment of expression levels comparable to those observed using scAAV at day 3. Expression in photoreceptors was not detectable until day 28. Dose-response experiments confirmed that onset of GFP expression was more rapid and robust after subretinal injection of scAAV.CMV-GFP than of ssAAV.CMV-GFP, resulting in pronounced expression in photoreceptors and other retinal neurons. Similar results were obtained for intravitreous injections. CONCLUSIONS: These data suggest that scAAV vectors may be advantageous for ocular gene therapy, particularly in retinal diseases that require rapid and robust transgene expression in photoreceptor cells.
Assuntos
Dependovirus/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Epitélio Pigmentado Ocular/metabolismo , Animais , Feminino , Terapia Genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Plasmídeos , TransgenesRESUMO
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.
Assuntos
Neuropatias Diabéticas/terapia , Terapia Genética/métodos , Fatores de Transcrição/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Dedos de Zinco/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/fisiopatologia , Expressão Gênica , Vetores Genéticos/genética , Humanos , Ratos , Retroviridae/genética , Estreptozocina/toxicidade , Fatores de Transcrição/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) exists in multiple isoforms. We investigated an engineered zinc finger-containing transcription factor plasmid designed to activate the endogenous VEGF gene (ZFP-VEGF). METHODS AND RESULTS: New Zealand White rabbits (n=56) underwent unilateral femoral artery ligation and excision. At day 10 postoperatively, the ischemic muscle received ZFP treatment (500 microg ZFP-VEGF plasmid) or no ZFP treatment (beta-galactosidase, empty, or no plasmid). Group 1 (n=13) was harvested 3 days after injection to examine VEGF mRNA by real-time polymerase chain reaction and protein by ELISA. Groups 2 (n=13) and 3 (n=10) were harvested 11 days after injection. Group 2 was studied by histology and group 3, by histology and changes in blood flow. Groups 4 and 5 (n=10 each) were harvested 22 and 32 days after injection, respectively, and studied for changes in blood flow. In group 1, VEGF mRNA copy numbers were significantly higher for VEGF121, VEGF165, VEGF189, and protein in the ZFP-VEGF-treatment versus no-ZFP-treatment arms. In groups 2 and 3, capillary density and proliferating cells were significantly greater and apoptosis significantly lower in the treatment versus no-treatment arms. Changes in the blood flow ratio of the ischemic to the nonischemic limb were significantly greater in the treatment versus no-ZFP-treatment groups (6.57+/-1.52% versus 3.38+/-0.87%, P<0.005; 13.15+/-1.77% versus 6.13+/-1.55%, P<0.001; and 20.16+/-2.84% versus 13.88+/-3.14%, P<0.01, for groups 3, 4, and 5, respectively). CONCLUSIONS: This engineered ZFP-VEGF-activating transcription factor may provide a novel approach to treat peripheral arterial disease.
Assuntos
Regulação da Expressão Gênica/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Neovascularização Fisiológica/genética , Engenharia de Proteínas , Fator A de Crescimento do Endotélio Vascular/biossíntese , Dedos de Zinco/fisiologia , Animais , Antígenos Virais de Tumores/genética , Apoptose , Sítios de Ligação , Capilares/patologia , Citomegalovirus/genética , DNA/metabolismo , DNA Recombinante/genética , Feminino , Artéria Femoral/lesões , Genes Sintéticos , Vetores Genéticos/administração & dosagem , Injeções Intramusculares , Isquemia/fisiopatologia , NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , Coelhos , Vírus 40 dos Símios/genética , Fator de Transcrição RelA , Fator A de Crescimento do Endotélio Vascular/genética , Dedos de Zinco/genéticaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Quinase do Ponto de Checagem 2 , DNA/genética , DNA/metabolismo , Dano ao DNA , Regulação Enzimológica da Expressão Gênica , Genoma Humano , Humanos , Regiões Promotoras Genéticas , Engenharia de Proteínas , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
The relationship between the structure of zinc-finger protein (ZFP) transcription factors and DNA sequence binding specificity has been extensively studied. Advances in this field have made it possible to design ZFPs de novo that will bind to specific targeted DNA sequences. It has been proposed that such designed ZFPs may eventually be useful in gene therapy. A principal advantage of this approach is that activation of an endogenous gene ensures expression of the natural array of splice variants. Preliminary studies in tissue culture have validated the feasibility of this approach. The studies reported here were intended to test whether engineered transcription factors are effective in a whole-organism model. ZFPs were designed to regulate the endogenous gene encoding vascular endothelial growth factor-A (Vegfa). Expression of these new ZFPs in vivo led to induced expression of the protein VEGF-A, stimulation of angiogenesis and acceleration of experimental wound healing. In addition, the neovasculature resulting from ZFP-induced expression of Vegfa was not hyperpermeable as was that produced by expression of murine Vegfa(164) cDNA. These data establish, for the first time, that specifically designed transcription factors can regulate an endogenous gene in vivo and evoke a potentially therapeutic biophysiologic effect.