RESUMO
Hypercholesterolemia remains one of the leading risk factors for the development of cardiovascular disease. Many large double-blind studies have demonstrated that lowering low-density lipoprotein (LDL) cholesterol using a statin can reduce the risk of having a cardiovascular event by approximately 30%. However, despite the success of statins, some patient populations are unable to lower their LDL cholesterol to meet the targeted lipid levels, due to compliance or potency issues. This is especially true for patients with heterozygous familial hypercholesterolemia who may require additional upregulation of the low-density lipoprotein receptor (LDLR) to reduce LDL cholesterol levels below those achievable with maximal dosing of statins. Here we identify a series of small molecules from a genomic DNA reporter screen that upregulate the LDLR in mouse and human liver cell lines at nanomolar potencies (EC50 = 39 nM). Structure-activity relationship studies carried out on the lead compound, OX03771 [(E)-N,N-dimethyl-3-(4-styrylphenoxy)propan-1-amine], led to the identification of compound OX03050 [(E)-3-(4-styrylphenoxy)propan-1-ol], which had similar potency (EC50 = 26 nM) but a much-improved pharmacokinetic profile and showed in vivo efficacy. Compounds OX03050 and OX03771 were found to inhibit squalene synthase, the first committed step in cholesterol biosynthesis. These squalene synthase inhibitors were shown to act cooperatively with statins to increase LDLR expression in vitro. Overall, we demonstrated here a novel series of small molecules with the potential to be further developed to treat patients either alone or in combination with statins.
Assuntos
Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Testes Genéticos/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Receptores de LDL/biossíntese , Bibliotecas de Moléculas Pequenas/administração & dosagem , Regulação para Cima/fisiologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos , Farnesil-Difosfato Farnesiltransferase/metabolismo , Humanos , Masculino , Camundongos , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
NSW Health is implementing genomics as a mainstream component of clinical care. The strategic, holistic approach is considering infrastructure, data governance and management, workforce, education, service planning and delivery. This work is generating insights about how to realise the promise of genomics in healthcare, highlighting the need for strong foundations, real-world application, accessibility and a focus on people using genomic information in clinical care.
Assuntos
Atenção à Saúde , Instalações de Saúde , Humanos , Recursos Humanos , GenômicaRESUMO
Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Receptores de LDL/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Humanos , Hiperlipoproteinemia Tipo II/terapia , Fígado/metabolismo , Camundongos , Modelos Genéticos , Receptores de LDL/genética , Receptores de LDL/fisiologia , Esteróis/farmacologiaRESUMO
The principal aim of gene therapy for recessive genetic diseases is to supplement the loss of function of an endogenous gene. For the treatment of many diseases regulation of transgene expression at physiological levels, expression of multiple splice variants, and correct tissue specificity are of utmost importance for effective therapy. We therefore believe the use of a complete genomic locus, in which the native promoter and regulatory regions drive and control expression, is an elegant and effective alternative to traditional complementary DNA (cDNA) vectors utilising heterologous promoters. Viral vectors have proved, over the years, to be an effective means of gene delivery in vitro and in vivo, but the size of complete genomic loci precludes their use in most viral systems. One notable exception comprises the amplicon-type vectors based on human herpesviruses, such as the herpes simplex virus type I (HSV-1) amplicon vector. The large genome of HSV-1 (152 kb) confers upon HSV-1 amplicons a very large transgene capacity sufficient to accommodate approximately 95% of human genomic loci. The combination of the large transgene capacity, a broad range of cell tropism, and the ability to infect dividing and non-dividing cells makes HSV-1 amplicons an excellent vector system to develop for the delivery of large genomic loci. Here we review recent work which has shown that HSV-1 amplicons can be used for the delivery and expression of large genomic inserts >100 kb to cells in culture to rescue phenotypes in cellular models of genetic disease. We then discuss applications for high capacity HSV-1 amplicons in vivo and their potential to facilitate the use of large genomic inserts in gene therapy treatment regimes.
Assuntos
DNA , Vetores Genéticos , Herpesvirus Humano 1/genéticaRESUMO
The prognosis of hepatocellular carcinoma patients is usually poor, the size of tumors being a limiting factor for surgical treatments. Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors. Mice developing diethylnitrosamine-induced hepatocellular carcinoma were subjected to hydrodynamic gene delivery to overexpress Gas1 in liver. This treatment significantly (p < 0.05) reduced the number of large tumors, while the difference in the total number of lesions was not significant. Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced. These results are related with the finding that overexpression of Gas1 in Hepa 1-6 cells arrests cell cycle before S phase, with a significant (p < 0.01) and concomitant reduction in the expression of cyclin E2 gene. In addition, a triangular analysis of microarray data shows that Gas1 overexpression restores the transcription levels of 150 genes whose expression was affected in the diethylnitrosamine-induced tumors, thirteen of which are involved in the hedgehog signaling pathway. Since the in vivo Gas1 gene delivery to livers of mice carrying hepatocellular carcinoma reduces the size and proliferating activity of tumors, partially restoring the transcriptional profile of the liver, the present study opens promising insights towards a therapeutic approach for hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Fígado/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Hidrodinâmica , Fígado/patologia , Neoplasias Hepáticas/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Extrachromosomal gene expression vectors that contain native genomic gene expression elements have numerous advantages over traditional integrating mini-gene vectors. In this protocol chapter we describe our work using episomal vectors where expression of a cDNA is controlled by a 10 kB piece of genomic DNA encompassing the promoter of the low density lipoprotein receptor. We explain methods to sub-clone large genomic inserts into gene expression vectors. We also illustrate various methods employed to ascertain whether expression from these vectors is robust and physiologically relevant by investigating their sensitivity to changes in cellular milieu. Delivery of gene expression vectors in vivo is also described using hydrodynamic tail vein injection, a high pressure, high volume tail vein injection used for liver-directed gene transfer.
Assuntos
Vetores Genéticos/genética , Plasmídeos/genética , Transfecção/métodos , Animais , Células CHO , Cromossomos Artificiais Bacterianos/genética , Cricetinae , Cricetulus , DNA/genética , Expressão Gênica , Genes Reporter/genética , Engenharia Genética , Terapia Genética , Vetores Genéticos/metabolismo , Genoma/genética , Humanos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Luciferases/genética , Camundongos , Imagem Molecular , Especificidade de Órgãos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transgenes/genéticaRESUMO
BACKGROUND: The delivery of a complete genomic DNA locus in vivo may prove advantageous for complementation gene therapy, especially when physiological regulation of gene expression is desirable. Hydrodynamic tail vein injection has been shown to be a highly efficient means of non-viral delivery of plasmid DNA to the liver. Here, we apply hydrodynamic tail vein injection to deliver and express large genomic DNA inserts > 100 kb in vivo. METHODS: Firstly, a size series (12-172 kb) of bacterial artificial chromosome (BAC) plasmids, carrying human genomic DNA inserts, episomal retention elements, and the enhanced green fluorescent protein (EGFP) reporter gene, was delivered to mice by hydrodynamic tail vein injection. Secondly, an episomal BAC vector carrying the whole genomic DNA locus of the human low-density lipoprotein receptor (LDLR) gene, and an expression cassette for the LacZ reporter gene, was delivered by the same method. RESULTS: We show that the efficiency of delivery is independent of vector size, when an equal number of plasmid molecules are used. We also show, by LacZ reporter gene analysis, that BAC delivery within the liver is widespread. Finally, BAC-end PCR, RT-PCR and immunohistochemistry demonstrate plasmid retention and long-term expression (4 months) of human LDLR in transfected hepatocytes. CONCLUSION: This is the first demonstration of somatic delivery and long-term expression of a genomic DNA transgene > 100 kb in vivo and shows that hydrodynamic tail vein injection can be used to deliver and express large genomic DNA transgenes in the liver.
Assuntos
Pareamento de Bases/genética , DNA/metabolismo , Técnicas de Transferência de Genes , Genoma Humano/genética , Receptores de LDL/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Feminino , Vetores Genéticos , Humanos , Injeções , Fígado/citologia , Fígado/metabolismo , Camundongos , Plasmídeos/metabolismo , Fatores de Tempo , Transgenes , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: To use in vivo gene transfer into the testis by electroporation to express a fluorescent recombinant form of a testis-specific gene in the mature epididymal sperm of mice and thus study the pattern of gene localization. DESIGN: Controlled animal study. SETTING: Research laboratory at the University of Oxford. ANIMAL(S): Four- to 6-week-old male mice. INTERVENTION(S): Phospholipase C zeta (PLCzeta), the putative mammalian egg activation factor, was fused to enhanced yellow fluorescent protein (EYFP), and in vivo gene transfer by electroporation was used to introduce this transgene (PLCzeta-EYFP) into mouse testis. Transgene expression in testis and sperm were analyzed at 20 and 40 days after electroporation. MAIN OUTCOME MEASURE(S): Transgene expression in testis and epididymal sperm was analyzed by fluorescence microscopy and an excitation light source suitable for EYFP. RESULT(S): Phospholipase C zeta-EYFP was successfully expressed in epididymal sperm when analyzed 40 days after gene transfer and was localized to the head and midpiece regions. CONCLUSION(S): Our results provide the first demonstration that in vivo gene transfer can be used to study the localization of proteins in mature sperm and that this represents a powerful new technique for studying male infertility and gene function in sperm.
Assuntos
Eletroporação/métodos , Proteínas Secretadas pelo Epidídimo/biossíntese , Proteínas Secretadas pelo Epidídimo/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Fosfoinositídeo Fosfolipase C , Proteínas Recombinantes de Fusão/metabolismo , Distribuição TecidualRESUMO
The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.
Assuntos
Técnicas de Transferência de Genes , Espermatozoides/metabolismo , Testículo/metabolismo , Transgenes/fisiologia , Animais , Cricetinae , Eletroporação , Epididimo/fisiologia , Técnicas de Transferência de Genes/efeitos adversos , Masculino , Microscopia de Fluorescência , Motilidade dos Espermatozoides , Espermatogênese/fisiologia , Espermatozoides/fisiologia , Testículo/fisiologiaRESUMO
Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.
Assuntos
Cálcio/metabolismo , Oócitos/metabolismo , Óvulo/metabolismo , Espermatozoides/enzimologia , Tilápia/embriologia , Fosfolipases Tipo C/metabolismo , Animais , Bioensaio , Sinalização do Cálcio , Extratos Celulares/administração & dosagem , Extratos Celulares/farmacologia , Citosol/enzimologia , Injeções , Cinética , Masculino , Camundongos , Óvulo/efeitos dos fármacos , Proteínas/administração & dosagem , Proteínas/análise , Proteínas/farmacologia , Ouriços-do-Mar/embriologia , Espermatozoides/química , Fosfolipases Tipo C/análiseRESUMO
Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, including fertilization and development of the embryo. One of the key mechanisms involved in triggering intracellular calcium release is the generation of the second messenger inositol-1,4,5-phosphate (IP(3)) by the phospholipase C (PLC) class of enzymes. Although five distinct forms of PLC have been identified in mammals (beta, gamma, delta, epsilon, and zeta), only one, PLCgamma, has thus far been detected in echinoderms. In the present study, we describe the isolation of a cDNA encoding a novel PLC isoform of the delta (delta) subclass, PLC-deltasu, from the egg of the Pacific purple sea urchin Strongylocentrotus purpuratus. We also demonstrate the presence of this PLC within the sperm and in the early embryo. The PLC-deltasu cDNA (2.44kb) encodes a 742 amino acid polypeptide with an open reading frame of 84.6kDa and a pI of 6.04. All of the characteristic domains found in mammalian PLCdelta isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) are present in PLC-deltasu. A homology search revealed that PLC-deltasu shares most sequence identity with bovine PLCdelta2 (39%). We present evidence that PLC-deltasu is expressed in unfertilized eggs, fertilized eggs, and in the early embryo. In addition to Northern and polymerase chain reaction (PCR) analyses, in situ hybridization experiments further demonstrated that the embryonic regions within which the PLC-deltasu transcript can be detected during early embryonic development are associated with the highest levels of proliferative activity, suggesting a possible involvement with metabolism or cell cycle regulation.