Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.     

Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line

Magnetic resonance imaging (MRI) was used in 13 patients with peripheral lymphedema and 2 patients with extensive cavernous lymphangioma of the limb for the purpose of evaluating its role in diagnosis of lymphatic disorders. In chronic lymphedema, MRI showed deformity of lymphatics at different tissue levels. In the subcutis, MRI characteristically displayed diffuse edema or a honeycombed pattern consistent with reticular lymphangiectasis and "lakes" with a marked increase in signal intensity with T2-weighted imaging. In lymphedema hyperplasia and chylous reflux, MRI depicted dilated retroperitoneal lymphatic collectors and lumbar trunks. In cavernous lymphangiomatosis, MRI demonstrated a prominent lattice-like pattern which had lower signal intensity on T1-weighted imaging and higher intensity on T2-weighted imaging. The findings of MRI are valuable not only for accurate assessment of lymphatic dysplasia syndromes but also provide a blueprint for treatment options.     

Gene transfer to human cells using retrovirus vectors produced by a new polytropic packaging cell line

We report here the construction of a new packaging cell line, called MPAC, that packages defective retroviral vectors in viral particles with envelope proteins derived from a Moloney mink cell focus-inducing (MCF) polytropic virus. We characterized the tropism of MPAC-packaged retroviral vectors and show that some human cell lines can be infected with these vectors while others cannot. In addition, we show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication.     

Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.     

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAI or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of anti-atherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAI or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAI or LCAT by transduced cultures was two- to five-fold higher using AAV-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AAV-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAI and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAI and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.     

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.     

Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complexes

DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.     

Gene transfer into human dendritic antigen-presenting cells by vaccinia virus and adenovirus vectors

In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors. The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene. DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim. The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells. The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ. In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs.     

Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors

A number of packaging materials are being used not only to contain food during distribution but also to serve as the cooking container. The higher temperatures that these materials reach led the US Food and Drug Administration (FDA) to issue an intent to publish new regulations in 1989. The food and packaging industries responded by conducting extensive research and submitting the results to FDA. The methods used and results obtained are discussed. Most of the data were focused on microwave susceptors and the volatile compounds generated. One project showed that for a specific product, popcorn, there was no transfer into the food. Work is continuing to validate methods to test for non-volatile compounds. In addition to susceptors, various paper and plastic materials are used in dual ovenable (microwave and conventional ovens) applications. Most of the research on these materials has investigated the food contact temperatures on testing for migrants. An update on the current regulatory status of packaging materials intended for high temperature use in the US is discussed.     

rAAV/CEA转染树突状细胞诱导特异性CTL杀伤MCF-7细胞系CD44+ CD24-/low乳腺癌干细胞

目的:携带癌胚抗原(carcinocmbryonic antigen,CEA)基因的重组人腺相关病毒(reconstructive human adeno-association virus,rh-AAV)感染树突状细胞(dendritic cell,DC)诱导获得抗原特异性细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL),体外检测其对CD44+CD24-/low乳腺癌干细胞的杀伤效果.方法:分离供者外周血单个核细胞,以细胞因子白介素-4(interleukin-4,IL-4)、粒细胞-巨噬细胞克隆刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)诱导培养获得DC,细胞因子白介素-2(interleukin-2,IL-2),刺激培养获得T淋巴细胞.含CEA基因的rh-AAV感染培养DC,诱导成熟后将DC和T细胞混合培养获得CTL细胞.流式分选MCF-7和MA-MDB-231细胞系中CD44+CD24-/low乳腺癌干细胞,MTT法检测CTL细胞对CD44+CD24-/low乳腺癌干细胞的杀伤效果.结果:MCF-7和MDA-MB-231中CD44+/CD24-/low群比例分别为5.1%和76.3%.CEA基因转染DC诱导的CTL细胞对表达CEA抗原的MCF-7杀伤率为46.5%±15.0%,与未转染组相比,差异有统计学意义(P=0.009);对MCF-7细胞中分选的CD44+CD24-/low乳腺癌干细胞的杀伤率为44.7%±28.2%,明显高于未转染组.对非乳腺癌干细胞杀伤率为50.6%±22.2%,与未转染组相比,差异有统计学意义(P=0.05).在不表达CEA基因的MDA-MB-231乳腺癌细胞,CEA转染诱导的CTL细胞对未分选细胞、分选的CD44+/CD24-/low亚群和非干细胞亚群的杀伤率与未转染的对照组相比,差异均无统计学意义(P＞0.05).结论:携带CEA基因rh-AAV转染DC诱导产生的抗原特异性CTL细胞可杀伤表达CEA的乳腺癌细胞,对CD44+CD24-/low乳腺癌干细胞也具有一定的杀伤活性,提示免疫治疗可能是治疗乳腺癌干细胞潜在有效的手段.     

Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells

Human immunodeficiency virus type 1 (HIV-1) can readily accept envelope (Env) glycoproteins from distantly related retroviruses. However, we previously showed that the HIV-1 Env glycoprotein complex is excluded even from particles formed by the Gag proteins of another lentivirus, visna virus, unless the matrix domain of the visna virus Gag polyprotein is replaced by that of HIV-1. We also showed that the integrity of the HIV-1 matrix domain is critical for the incorporation of wild-type HIV-1 Env protein but not for the incorporation of a truncated form which lacks the 144 C-terminal amino acids of the cytoplasmic domain of the transmembrane glycoprotein. We report here that the C-terminal truncation of the transmembrane glycoprotein also allows the efficient incorporation of HIV-1 Env proteins into viral particles formed by the Gag proteins of the widely divergent Moloney murine leukemia virus (Mo-MLV). Additionally, pseudotyping of a Mo-MLV-based vector with the truncated rather than the full-length HIV-1 Env allowed efficient transduction of human CD4+ cells. These results establish that Mo-MLV-based vectors can be used to target cells susceptible to infection by HIV-1.     

Expression of a functional tagged human thyrotropin receptor in HeLa cells using recombinant vaccinia virus

The aim of the present study was to assess the prognostic relevance of relatives' interactive behaviour towards the patient, as covered by the Münster Family Interview (MFI), to the further course of the schizophrenic illness. The MFI is a family interview (of the whole family, including the patient) designed to record the emotional family atmosphere based on the concept of expressed emotion (EE). The ratings take place directly after the interview on five scales (criticism, hostility, overinvolvement, resignation and warmth), the resignation scale being added to the 'classic' EE scales. Ninety-nine families of outpatients diagnosed with schizophrenia according to the DSM-III were examined with the MFI during a home visit. The patients were seen 1 and 2 years after the first examination. The target criteria selected for the prognostic significance of the interaction measurements were: rehospitalisation within 2 years; extent of symptoms after 1 year, and psychosocial skills after 1 year. The significance of the interaction dimensions was verified in regression models. The control variable used in the regression models was the Strauss-Carpenter scale. Regression models were produced for the total group and for a subgroup of moderately ill patients. All target criteria yielded serviceable prediction models. The most important variable for prediction was the control variable, the Strauss-Carpenter scale. However the interaction variables made additional contributions to the prognosis, especially in the subgroup of moderately ill patients. The best MFI scale for all the outcome criteria was resignation; criticism predicted only the symptomatology, and emotional overinvolvement the level of social functioning after 1 year. In conclusion, practical work with families of schizophrenic patients should emphasise the protective function of relatives towards patients more strongly.     

Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3

OBJECTIVE: Elderly women with proximal femur fracture show a prolonged increase in plasma cortisol, which could have undesirable catabolic effects. Suppression of cortisol by dexamethasone is impaired, suggesting resistance to glucocorticoid effects at feedback inhibitory sites. We therefore wished to find out whether peripheral glucocorticoid sensitivity is normal. DESIGN: Peripheral blood mononuclear leucocytes were used as a model tissue. Blood samples were taken from elderly women about 2 weeks after hip fracture and from elderly control women. Each patient was then given 1 mg dexamethasone at 2300 h followed by further sampling at 0800 and 1600 h the next day. METHODS: Glucocorticoid-receptor binding parameters were measured by incubating whole cells with [3H]dexamethasone for 2 h at 37 degrees C. Inhibition of cell proliferation by dexamethasone was assessed by addition of [3H]thymidine to cells cultured for 65 h with concanavalin A. Cortisol and dexamethasone concentrations were measured in the dexamethasone suppression test. RESULTS: As expected, the hip-fracture patients had raised morning cortisol concentrations and impaired suppression by dexamethasone. The cells of the patients had similar numbers of glucocorticoid receptors to those of the control subjects but higher values for Kd (i.e. a lower binding affinity). The cells of the patients incorporated less [3H]thymidine than the control cells in the absence of dexamethasone. The percentage inhibition by a saturating concentration of dexamethasone was unchanged but the concentration giving half-maximal inhibition was decreased (sensitivity was increased) at the higher of the two concanavalin A concentrations used. CONCLUSIONS: These experiments in mononuclear leucocytes give no evidence of peripheral resistance to glucocorticoids in hip-fracture patients with impaired suppression of cortisol by dexamethasone.     

CD4 is a critical component of the receptor for human herpesvirus 7: interference with human immunodeficiency virus

In this study, we demonstrate that the glycoprotein CD4, a member of the immunoglobulin superfamily, is a critical component of the receptor for human herpesvirus 7 (HHV-7), a recently discovered T-lymphotropic human herpesvirus. A selective and progressive downregulation of the surface membrane expression of CD4 was observed in human CD4+ T cells in the course of HHV-7 infection. Various murine monoclonal antibodies to CD4 and the recombinant soluble form of human CD4 caused a dose-dependent inhibition of HHV-7 infection in primary CD4+ T lymphocytes. Moreover, radiolabeled HHV-7 specifically bound to cervical carcinoma cells (HeLa) expressing human CD4. A marked carcinoma cells (HeLa) expressing human CD4. A marked reciprocal interference was observed between HHV-7 and human immunodeficiency virus (HIV), the retrovirus that causes the acquired immunodeficiency syndrome and also uses CD4 as a receptor. Previous exposure of CD4+ T cells to HHV-7 dramatically interfered with infection by both primary and in vitro-passaged HIV-1 isolates. Reciprocally, persistent infection with HIV-1 or treatment with the soluble form of gp120, the CD4-binding envelope glycoprotein of HIV-1, rendered CD4+ T cells resistant to HHV-7 infection. These data indicate that CD4 is critically involved in the receptor mechanism for HHV-7. The antagonistic effect between HHV-7 and HIV could be exploited to devise therapeutic approaches to AIDS.     

Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium

BACKGROUND: Home training in self-lowering of blood pressure using continuous blood pressure feedback has not previously been reported. Enhancement of laboratory-learned skills was hypothesized on the basis of outcomes from other intellectual, emotional and physical endeavours. OBJECTIVE: To examine the supplementary effect of home blood pressure biofeedback training. DESIGN: Thirty unmedicated, mild hypertensives participated in a randomized, double-blinded, modified contingency placebo-controlled study. METHOD: After suitable screening and baseline blood pressure measurements subjects undertook eight laboratory biofeedback sessions and then 12 home training sessions over 4 weeks using continuous finger blood pressure monitoring. RESULTS: In the laboratory those being administered active therapy (n=16) lowered systolic pressures by 5 +/- 5.4 mmHg compared with a lowering of 4 +/- 4.2 mmHg with placebo (NS). During the fourth week at home lowering for the active group (11 +/- 8 mmHg) was greater than that with placebo (4 +/- 6.2 mmHg, P=0.017). Arm-cuff blood pressures were not statistically different for groups and with time but that of the active group was lower by 9 +/- 15.4/7 +/- 10.2 mmHg, which is a clinically relevant change, after home biofeedback. CONCLUSIONS: The efficacy of self-lowering of systolic blood pressure in mild hypertensives by continuous feedback was enhanced by 6 mmHg with 4 weeks of practice at home. Standard arm-cuff blood pressure was reduced by a clinically relevant amount. The home environment proved cost effective for this 'high-tech' approach.     

HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells

Recent studies have opened the possibility that quiescent, G0/G1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G0/G1. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G0/G1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP+ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.