microRNA调控的靶向性病毒载体在基因治疗中的应用

安全、有效、具有靶向性的病毒载体是基因治疗药物在临床上得以应用的关键。microRNA是一类单链、内源性的转录后调控小分子,它的发现为开发具有靶向性调控能力的病毒载体提供了新的研究方法。以下在介绍microRNA调节病毒载体靶向性原理的基础上,着重介绍microRNA在清除复制能力病毒的污染、消除转基因特异性免疫、增强肿瘤靶向性基因治疗、开发活体疫苗等领域的应用。     

Developmental fate of a human insulin gene in a transgenic mouse

Summary A plasmid containing a genomic human insulin clone was microinjected into a pronucleus of the fertilised mouse egg. Eggs were subsequently transferred into oviducts of pseudopregnant Swiss/Alb females. Embryos developed to term and the DNA was extracted from different organs. Southern blotting analyses revealed 1 transgenic female out of 96 animals born after microinjection of C57BL/6 mouse eggs. A tandem integration was found at one locus within the mouse genome and molecular rearrangement was found within this locus. The structure of the entire locus was identical in DNA from all tissues. Both the human insulin gene sequences and the pBR322 sequences were found to be extensively methylated, although some sites were hypomethylated in the pancreas and liver. The transgenic female produced ten offspring, none of which retained the insulin gene sequences. Seven offspring retained some pBR322 sequences which were stably transmitted to the F2 and F3 generations. Homozygous F3 pBR/pBR animals were obtained, which showed neither visible defects nor sterility. The loss of the tandem locus in the F1 generation did not seem to be due to mosaicism, but involved excision due to recombination. Sequences close to the ends of the tandem locus were involved in this event. A mechanism implying excision during germ cell formation is discussed.     

A mouse model of the fragile gene FHIT: From carcinogenesis to gene therapy and cancer prevention

Mouse models of tumor suppressors are increasingly useful to investigate biomedical aspects of cancer genetics. Some tumor suppressor genes are located at common fragile sites that are specific chromosomal regions highly susceptible to DNA lesions. The tumor suppressor gene FHIT, at the fragile site FRA3B, is the first fragile gene with a developed and characterized mouse knockout model. The human gene FHIT is frequently deleted in cancers and cancer cell lines of many epithelial tissues, and Fhit protein is absent or reduced in most cancers. The mouse Fhit ortholog is also located at a common fragile site, Fra14A2 on murine chromosome 14, and sustains homozygous deletions in murine cancer cell lines. The Fhit knockout mouse is, therefore, an adequate model to study human FHIT function. To establish an animal model and to explore the role of FHIT in tumorigenesis, we have developed a mouse strain carrying one or two inactivated Fhit alleles. Insights into Fhit mouse genetics that have emerged in the last 7 years, and are reviewed in the present article, allowed for development of new tools in carcinogenesis and gene delivery studies.     

A model for evaluation of the peroral insulin therapy: short-term treatment of alloxan diabetic rats with oral water-in-oil-in-water insulin emulsions.

Alloxan diabetic rats with fasting blood glucose levels above 300 mg/100 ml were treated with oral administration of water-in-oil-in-water (W/O/W) insulin emulsions at a dose of 50 U/100 g body weight, three times daily for 10 to 14 days. The course of diabetes was followed by determinations of glucose levels in blood and urine. During treatment with oral W/O/W insulin emulsions, daily excretion of urinary glucose decreased by about 30 to 40% (2 to 3 g/day) in all of the five rats studied, and returned to the pre-treatment levels after the treatment being discontinued. During treatment, a significant reduction in fasting blood glucose levels was observed in 4 out of 5 rats, giving the decrease by 18 to 44%. Quantitative estimates suggested that the effectiveness of 50 U/100 g of oral W/O/W insulin emulsions was comparable to that after intramuscular regular insulin at a dose of 0.5 U/100 g. Although oral W/O/W insulin emulsions are still of low efficiency, these results would indicate that diabetes can be controlled by effective oral insulin preparations.     

Effective gene therapy in a mouse model of prion diseases

Classical drug therapies against prion diseases have encountered serious difficulties. It has become urgent to develop radically different therapeutic strategies. Previously, we showed that VSV-G pseudotyped FIV derived vectors carrying dominant negative mutants of the PrP gene are efficient to inhibit prion replication in chronically prion-infected cells. Besides, they can transduce neurons and cells of the lymphoreticular system, highlighting their potential use in gene therapy approaches. Here, we used lentiviral gene transfer to deliver PrPQ167R virions possessing anti-prion properties to analyse their efficiency in vivo. Since treatment for prion diseases is initiated belatedly in human patients, we focused on the development of a curative therapeutic protocol targeting the late stage of the disease, either at 35 or 105 days post-infection (d.p.i.) with prions. We observed a prolongation in the lifespan of the treated mice that prompted us to develop a system of cannula implantation into the brain of prion-infected mice. Chronic injections of PrPQ167R virions were done at 80 and 95 d.p.i. After only two injections, survival of the treated mice was extended by 30 days (20%), accompanied by substantial improvement in behaviour. This delay was correlated with: (i) a strong reduction of spongiosis in the ipsilateral side of the brain by comparison with the contralateral side; and (ii) a remarkable decrease in astrocytic gliosis in the whole brain. These results suggest that chronic injections of dominant negative lentiviral vectors into the brain, may be a promising approach for a curative treatment of prion diseases.     

The effect of repeated administration of insulin on pain threshold and gamma-aminobutyric acid level in mouse brain

In experiments on male Albino-Swiss mice weighing 18-22 g insulin given in doses of 2 i.u./kg caused no change in the time of reaction to pain, while the same dose administered daily for 7 days potentiated the analgesic action of morphine (3 mg/kg s.c.). Glucose caused no change in this effect of insulin. After 14 days of insulin treatment the time of reaction to pain in the animals subjected to the action of morphine returned to its initial value. Twenty-four hours after the last administration of morphine the level of gamma-aminobutyric acid (GABA) was found to be decreased in the animals receiving insulin with glucose. These results suggest that the central action of insulin is dependent not only on hypoglycaemia produced by it, but may be due also to its direct action on the central structures and an indirect action mediated by its effect on other neurotransmitter systems.     

Systemic administration of anti-NGF increases A-type potassium currents and decreases pancreatic nociceptor excitability in a rat model of chronic pancreatitis

We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents (I(A)), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal I(A) density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1-4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of I(A) by as yet unknown mechanisms.     

人胰岛素基因在乳酸菌中的表达及其对非肥胖糖尿病(NOD)小鼠的作用

为实现人胰岛素基因在乳酸菌中的表达及探索其用作口服疫苗治疗Ⅰ型糖尿病(T1DM)的可行性,首先将人胰岛素基因密码子替换为乳酸菌偏爱密码子,同时在A,B链序列间加入连接短肽序列,经引物退火拼接合成人胰岛素基因。克隆至乳酸菌表达载体中后,利用电击转化法实现了带信号肽SPUsp45的人胰岛素基因在乳酸乳球菌(Lactococcus lactis)MG1363和干酪乳杆菌(Lactobacillus casei)ATCC27092中的表达。Western blot检测显示重组胰岛素位于细胞壁上,当菌体生长到OD600为0.4时达到最大表达量。用含有表达重组人胰岛素的Lactobacillus caseiATCC27092/pSW501菌体饲喂非肥胖糖尿病(NOD)小鼠,发现可刺激小鼠产生特异性抗体,同时使与免疫耐受相关的细胞因子IL-4水平明显升高(38.583±2.083pg/mL,P<0.05),提示其对NOD小鼠产生免疫耐受有一定的作用,为研制乳酸菌口服疫苗防治T1DM的可行性进行了有益的探索。     

逆转录病毒载体在血友病A基因治疗中的研究进展

基因治疗是彻底治愈血友病A的最理想方法,逆转录病毒是最为常用的载体之一,本文对逆转录病毒在血友病A基因治疗中的研究进展作一综述。     

A simple stochastic model for complex coextinctions in mutualistic networks: robustness decreases with connectance

Understanding and predicting species extinctions and coextinctions is a major goal of ecological research in the face of a biodiversity crisis. Typically, models based on network topology are used to simulate coextinctions in mutualistic networks. However, such topological models neglect two key biological features of species interactions: variation in the intrinsic dependence of species on the mutualism, and variation in the relative importance of each interacting partner. By incorporating both types of variation, we developed a stochastic coextinction model capable of simulating extinction cascades far more complex than those observed in previous topological models. Using a set of empirical mutualistic networks, we show that the traditional topological model may either underestimate or overestimate the number and likelihood of coextinctions, depending on the intrinsic dependence of species on the mutualism. More importantly, contrary to topological models, our stochastic model predicts extinction cascades to be more likely in highly connected mutualistic communities.     

Systemic administration of tripeptidyl peptidase I in a mouse model of late infantile neuronal ceroid lipofuscinosis: effect of glycan modification

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ～10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.     

A model for diabetic nephropathy: advantages of the inducible cAMP early repressor transgenic mouse over the streptozotocin-induced diabetic mouse

We have previously found progressive diabetic nephropathy in inducible cAMP early repressor (ICER Igamma) transgenic (Tg) mice. The ICER Igamma Tg mouse is an interesting model of sustained hyperglycemia due to its low production of insulin and insulin-producing beta cells. Here in a longitudinal study we further analyzed diabetic nephropathy and structural and functional alterations in other organs, comparing our model with streptozotocin (STZ)-diabetic model mice. The high-dose STZ-diabetic model showed marked variation in blood glucose levels and severe toxicity of STZ in the liver and kidney. The low-dose STZ-diabetic model showed less toxicity, but the survival rate was very low. STZ-diabetic mice had much more variation of glomerular hypertrophy and sclerosis. Furthermore, non-specific toxicity of STZ or insulin injections to maintain optimal blood glucose levels might have another effect upon the diabetic renal changes. In contrast, ICER Igamma Tg mice exhibited a stable and progressive phenotype of diabetic kidney disease solely due to chronic hyperglycemia without other modulating factors. Thus, ICER Igamma Tg mouse has advantages for examining diabetic renal disease, and offers unique and very different perspectives compared to STZ model.     

Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study

This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan -60% trimethylated chitosan oligomer (TMCO-60%), C(43-45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60%>C(43-45 KDa, 87%)>C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.