Lineage Analysis Using Retroviral Vectors

Knowledge of the genealogical relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Genealogical relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. The use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues is described. An overview of the relevant aspects of the retroviral life cycle is given, and the strategies and current methods in use in our laboratory are described.     

Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.     

Structure-Activity Relationships of Adenosine Deaminase Inhibitors

Abstract

Adenosine deaminase (ADA) is an important catabolic enzyme which converts adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. ADA exists in two different isoenzymes, namely ADA1 and ADA2, whose balance in monocytes-macrophages seems to guarantee the homeostasis of adenine nucleosides. Modifications of the purine moiety or/and substitution of the sugar moiety of adenosine with aliphatic chains led to derivatives which are good ADA inhibitors.     

Development of Retroviral Vectors for Tissue-Restricted Expression in Chicken Embryonic Gonads

The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.     

A Role for Adenosine Deaminase in Drosophila Larval Development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.     

Induction of Adenosine Deaminase in Escherichia coli

Supplementing the salts-glucose medium of Escherichia coli with adenine initiates induction of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), growth inhibition, and an increased potential for the net deamination of adenine. The extent and duration of these events are proportional to the initial adenine concentration and are dependent upon adenylate pyrophosphorylase and repression of histidine biosynthesis for maximal expression. The conversion of adenine to hypoxanthine, though limited in rate, occurs concurrently with induction and accounts for the progressively decreasing rate of deaminase induction, since hypoxanthine is a relatively ineffective inducer. The subsequent decrease in deaminase activity is due to dilution by continued cell division and by enzyme inactivation which occurs during the late-log and early-stationary phases. The partially purified deaminase is labile to a number of environmental conditions, particularly to phosphate buffers of pH 6.8 or less. A disproportionately slow rate of adenine deamination by cells utilizing lactate permits a more prolonged period of induction and, consequently, a greater quantity of enzyme to be synthesized; cell division, but not enzyme inactivation, reduces enzyme concentration. The adenosine deaminases of Aerobacter aerogenes and Salmonella typhimurium are not inducible.     

Retroviral Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus

Pseudotyping can improve retroviral vector stability and transduction efficiency. Here, we describe a novel pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV). This pseudotype was stable during ultracentrifugation and infected several cell lines from different species. Moreover, LCMV glycoproteins were not cell toxic.     

Ontogenesis of Adenosine Deaminase Activity in Rat Brain

The activity of adenosine deaminase (ADA) was determined in whole brain of rats at the embryonic age of 15 days through to adulthood and in nine brain regions in rats 1 day old through to adulthood. In 1-day-old rats, the highest activity was seen in olfactory bulbs (550 +/- 15 nmol/mg protein/30 min) and this was 4.5-fold higher than that in the pons, which was the lowest. In adult animals, olfactory bulb still contained the greatest activity, which was about eightfold higher than hippocampus, which had the lowest. Except for hypothalamus, where ADA activity increased nearly twofold in rats between the ages of 1 and 50 days, significant decreases of as much as fivefold were found in whole brain, superior colliculus, cortex, hippocampus, cerebellum, olfactory bulbs, and olfactory nucleus. In contrast, ADA activity in pons and subcortex remained relatively constant throughout the developmental period. The Km values for ADA in whole brain at 18 days gestation (48 +/- 5 microM) were not significantly different from that observed in adult rats (38 +/- 7 microM), whereas the Vmax values decreased significantly from 339 +/- 9 to 108 +/- 8 nmol/mg protein/30 min. Taken together, the developmental patterns observed in the various brain regions appear not to correspond to any one particular process such as periods of rapid cell proliferation, cell death, synaptogenesis, or myelination. Nor do they correspond to known developmental profiles of transmitters, their receptors, or their metabolic enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deamination of Cyclaradine by Adenosine Deaminase Under High Pressure#

Abstract

The deamination of cyclaradine corresponding to a carbocyclic analogue of ara-A having anti-HSV activity by adenosine deaminase was examined under various pressure. The deamination of (+)- and (±)-cyclaradine was remarkably facilitated by high pressure, and the rate was increased with increasing of pressure. However, (-)-cyclaradine was not deaminated even under high pressure.

     

小鼠腺苷脱氨酶mRNA特异Ribozyme计算机设计

小鼠腺苷脱氨酶mRNA经计算机分析,包括:ribozyme切点位置选择,二级结构预测,基因生物学功能和基因同源性分析,筛选出四个锤头结构ribozyme.结果表明上述ribozyme底物切点两翼碱基形成发夹结构,切点位于单链环区,切点所在基因片段位于该基因生物功能区内,并同已知小鼠其它基因不同源.     

Targeted Gene Delivery by Intravenous Injection of Retroviral Vectors

Specifically and effectively directing a therapeutic gene to its intended site of action is a critical issue for translation of basic genomics to clinical gene therapy. Delivering gene therapy vectors to specific cells or tissues through intravenous injection is the most desirable method for this purpose. In 2001, we reported successful targeted gene transduction in vitro utilizing both oncoretroviral and lentiviral vectors pseudotyped with a chimeric Sindbis virus envelope (ZZ SINDBIS). However, these pseudotypes mediated non-specific gene transduction to liver and spleen in vivo. To address this problem we generated the modified ZZ SINDBIS (termed m168) with significantly less non-specific infectivity. To investigate the ability of m168 pseudotyped lentiviral vector to mediate targeted gene transduction in vivo, we utilized a metastatic tumor model by using mouse melanoma cells engineered to express human P-glycoprotein. We administered the m168 pseudotyped vector conjugated with anti-P-glycoprotein antibody into the mice intravenously to target metastatic melanoma. The m168 pseudotyped vector selectively infected metastatic melanoma cells demonstrating successful targeted gene transduction in vivo. Targeting technology based upon m168 can be further modified for application not only to cancer but also potentially to genetic, neurologic, infectious and immune diseases, thereby expanding the future application of gene therapy.     

反转录病毒基因治疗载体及其安全性评价

反转录病毒载体是目前基因治疗中应用最广泛的基因运载工具之一,具有能稳定整合入宿主细胞、持久表达目的基因、感染细胞范围广、基因容量较大等优点,但其生物安全性还存在争议。在简要介绍反转录基因治疗病毒载体的基础上,对反转录病毒载体中外源目的基因的表达调控及反转录病毒载体的安全性评价进行了综述。     

混合Ribozyme联合切割小鼠腺苷脱氨酶mRNA研究

采用计算机辅助设计得到针对小鼠腺苷脱氨酶ｍＲＮＡ的４种ｒｉｂｏｚｙｍｅ,它们均能在各自的切点切割ＲＮＡ分子,４种ｒｉｂｏｚｙｍｅ经组合后成混合ｒｉｂｏｚｙｍｅ,它们分别含有２种、３种和４种ｒｉｂｏｚｙｍｓ。通过对体外转录靶ＲＮＡ分子的联合切割研究表明,随着ｒｉｂｏｚｙｍｅ种数的增加,其中以（Ｒｚ２６２＋Ｒｚ４５５＋Ｒｚ５８３）组成最为理想,由此证明混合ｒｉｂｏｚｙｍｅ中,各种ｒｉｂｏｚｙｍｅ不仅保