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PubMed2004

Cell specificity and efficiency of the Semliki forest virus vector- and adenovirus vector-mediated gene expression in mouse cerebellum.

Establishing efficient gene transfer and expression in post-mitotic neurons is important in understanding the genetic basis of neural circuits with cellular complexity. This study evaluates the properties of exogenous green fluorescent protein (GFP) expression mediated by the Semliki forest virus (SFV) and adenovirus (Ad) vectors in dissociated and slice cultures of the mouse cerebellum. Infection with SFV-GFP resulted in early-onset and high-level GFP expression in about 90% of Purkinje cells and in about 40% of granule cells in dissociated cultures at 1 day after infection. Two days after infection, GFP-positive cells showed signs of SFV-derived cytotoxicity. Ad-GFP infected almost all astrocytes and granule cells in dissociated cultures, and showed a steady increase in GFP fluorescence with a plateau at around 2 days post-infection. Ad vector-mediated GFP expression lasted for several weeks with no significant cell damage. In the slice cultures, both viral vectors mainly infected astroglial cells, but also showed a similar cell preference as that in dissociated cultures. These data indicate that the use of different viral vectors and infection conditions offers a powerful means of expressing exogenous genes in cerebellar cultures with different cell-type specificity and timing and duration of expression.

Adenoviridae↗
PubMedDate not supplied

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector.

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.

Cell Line, Tumor↗
PubMed2001

Human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase-mediated suicide gene therapy.

To evaluate human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase (HSV-TK) -mediated gene therapy, we tested the utility of different viral vectors on three human cell lines DU-145, LNCaP, and PC-3. Our viral vectors were carrying a fusion gene of HSV-TK and green fluorescent protein for accurate determination of the gene transfer rate and its contribution to the treatment outcome in each case. We observed that adenoviral and lentiviral vectors were efficient vehicles for all the cell lines, whereas Semliki Forest virus and Sindbis virus vectors yielded only a few percent of transgene-positive cells. Despite sufficient gene transfer rates (25-45%) in the ganciclovir (GCV) sensitivity experiment, only DU-145 cells were efficiently destroyed under clinically relevant GCV concentrations. This was shown to be due to low level of "bystander effect" in PC-3 and LNCaP cells. Our data demonstrate that human prostate tumors can be good targets for adenovirus- or lentivirus-mediated HSV-TK/GCV gene therapy, but each tumor should be investigated for gene transfer rate and bystander effect to warrant a sufficient treatment result.

Adenoviridae↗
PubMedDate not supplied

Restoration of wild-type p53 activity enhances the sensitivity of pleural metastasis to cisplatin through an apoptotic mechanism.

We attempted to develop a new strategy of gene transfer in human metastatic cells avoiding viral vectors. We demonstrated the feasibility of the nlsLacZ gene-liposome (DOTAP) complex transfection in lung cancer cell lines H358 and Calu-1 carrying homozygous deletion of p53 and in primary cultures of human pleural metastatic tumor cells (n = 10). The efficiency of transfection in pleural cells was high with a mean of 78 +/- 22% (range 40-100%) compared to H358 (30%) and Calu-1 cells (50%). In this study, we report that growth of pC53-SN3-transfected Calu-1 and pleural metastatic cells was greatly suppressed whereas neither liposomes or Neo-gene affected cells growth. We tempted to determine whether restoration of wtp53 increased the chemosensitivity of cells that normally lack p53 expression. The pC53-SN3 transfected cells (H358 and Calu-1) were more sensitive to CDDP than the parental cells by 14 and 1.2 fold, respectively. In addition, the sensitization ratio due to the transfection of wtp53 in pleural cells (n = 6) varied from 1.2 to 6 fold. This sensitization remained even 21 days after transfection and was accompanied by increase of p53 positive cells and of the proportion of apoptotic bodies. In conclusion our results suggested that DOTAP is an efficient vector for mediating gene transfer in pleural metastatic cells offering advantages compared to viral vectors, while tumor suppressor genes such as p53 may be good candidates in combination with conventional therapy with CDDP that could be further developed for their use in local cancer gene therapy.

Antineoplastic Agents↗
PubMed1997

Gene therapy for renal diseases.

Gene therapy is a promising therapeutic approach for a variety of renal diseases including both inherited and acquired diseases. In vivo gene transfer in the kidney using viral or non-viral vectors have been reported. These approaches have been tested in a few animal models of renal diseases, including experimental glomerulonephritis, ischemic renal failure, and carbonic anhydrase II deficiency. Selection of vectors, routes, and therapeutic genes is critical to the success of gene therapy targeted to the specific compartment of the kidney. Limitations of gene therapy for renal diseases exist and consist of: duration of transgene expression is short, transfection efficiency is not adequate, immune reactions are induced by adenoviral vector, and insertional mutagenesis may be caused by retroviral and adeno-associated viral vectors. Further studies are needed for improvement of gene delivery, minimization of side effects and development of cell-specific and long-term regulated gene expression.

Carbonic Anhydrases↗
PubMed2006

Targeting the heart with gene therapy-optimized gene delivery methods.

With evolving knowledge in molecular and cellular cardiology, cardiac gene therapy has already been investigated in clinical studies. Different vector systems for cardiac gene therapy have been developed in recent years. While non-viral vectors, such as plasmid DNA, allow remarkable organ specificity, they are often limited by low transfection efficiency and transient gene expression. In contrast, adenoviral or adeno-associated virus-based vectors transfer the transgene more efficiently, but organ specificity may be reduced and immunogenic properties can limit their applicability. Using advanced transcriptional and transductional targeting strategies, viral vectors have been improved in the last few years. Recently, more efficient serotypes of adeno-associated viruses have been identified that show increased transduction rates, thus reducing the necessity for high virus titers. Combination with specific application techniques, such as intramyocardial injection, catheter-based perfusion, ultrasound targeted microbubble destruction, or retroinfusion may further enhance vector efficiency. This review article will give a broad overview of different gene delivery strategies that have been applied in experimental and clinical studies targeting the heart.

Animals↗
PubMed2001

Gene therapy for central nervous system repair.

The amelioration of the sequelae of CNS injury by gene therapy requires three post-injury events to be addressed: scarring, neuron death and axon regeneration. Administering anti-apoptotic, and axon growth promoting genes to neurons is inefficient, using nonviral vectors, because access to the transcriptional machinery is restricted by an intact nuclear membrane in G0 cells. Viral vectors have better transfection rates but a higher incidence of deleterious effects than non-viral vectors. Discrete targeted transfection of astrocytes, for example, about the wound with antifibrotic genes is essential to control scarring and for the inhibition of axon growth locally by gene products without transfecting neighboring and distant cells. Either rational or forced evolutionary design of vectors will ultimately achieve efficient safe gene transduction. Recombinant protein treatments for CNS repair have proved disappointing, probably because axotomized neurons are difficult to access in multiple disparate sites in the CNS after penetrant injury. Gene therapy has the potential to overcome these difficulties since sustained antiscarring/neurotrophic regimes are achievable after a single delivery to the site of injury by uptake by local injury responsive cells in the wound, and by axon terminals. Subsequent retrograde axonal transport delivers the therapeutic genes to all axotomized neurons throughout the CNS.

Active Transport, Cell Nucleus↗
PubMed2003

A genetically retargeted adenoviral vector enhances viral transduction in esophageal carcinoma cell lines and primary cultured esophageal resection specimens.

OBJECTIVE: To evaluate if an integrin-retargeted adenoviral vector could establish a more efficient and tumor-specific gene transfer in esophageal carcinoma cells. SUMMARY BACKGROUND DATA: Although preclinical data indicated that adenoviral gene therapy could be a promising novel treatment modality for various malignancies, clinical results are often disappointing. An important problem is the decreased tumoral expression of the Coxsackie and adenovirus receptor (CAR), which mediates adenoviral entry. Retargeting the adenoviral vector to other cellular receptors, by inserting an arginine-glycine-aspartate (RGD) tripeptide in the fiber knob, might overcome this problem. METHODS: Four esophageal carcinoma cell lines and 10 fresh surgical resection specimens were cultured. All were infected with the native adenovirus (Ad) and the retargeted adenovirus (AdRGD), encoding for the reporter genes luciferase or Green Fluorescent Protein to analyze gene transfer efficiency. RESULTS: In all cell lines, an increase in viral expression per cell and an increase in the percentage of transduced cells were seen with the retargeted adenovirus. Also, in the primary cultures of carcinoma cells, a more efficient gene transfer was seen when the retargeted vector was used. This phenomenon was less pronounced in normal cells, indicating that the RGD virus transduces tumor cells more efficiently than normal cells. CONCLUSIONS: This study demonstrates that an RGD retargeted adenovirus infects human esophageal carcinoma cells with enhanced efficiency, while in normal esophageal cells this effect is less pronounced. Therefore, this retargeted vector is expected to have a better performance in vivo, when compared with nonretargeted vectors used for cancer gene therapy so far.

Adenocarcinoma↗
PubMed2006

Hyperthermia and gene therapy: potential use of microPET imaging.

In recent years, both hyperthermia and gene-therapy have been evaluated as approaches to improve cancer radiotherapy. In addition, potential exists to combine these approaches to increase the overall therapeutic efficacy. For example, it has been reported that adenovirus-mediated heat-inducible gene expression may reduce the normal tissue toxicity associated with constitutively controlled expression of therapeutic genes. In our laboratory, we have shown that adenovirus-mediated, heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo, suggesting a novel approach to use heat-activated gene-radiotherapy to radiosensitize human tumors.However, to optimize the application of heat-activated gene-radiotherapy in the clinic, we need to develop techniques to improve the delivery of the therapeutic adenovirus and to verify/monitor the delivery non-invasively. In an ongoing study we test the effect of mild hyperthermia in improving adenovirus-medicated vector delivery in a mouse tumor model. In addition, we evaluate the use of non-invasive microPET imaging to monitor the spread of the adenoviral vector. Our preliminary results show that (1) microPET imaging can be used to monitor non-invasively the viral vector delivery and dissemination, and (2) mild heat shock leads to significantly improved viral vector distribution, in other words, a wider spatial spread, in vivo.Here, we will present a short review on the current status of hyperthermia and heat-activated gene-radiotherapy, and the potential use of PET imaging in gene therapy.

Adenoviridae↗
PubMed1998

Manipulation of gene expression in the mammalian nervous system: application in the study of neurite outgrowth and neuroregeneration-related proteins.

A fundamental issue in neurobiology entails the study of the formation of neuronal connections and their potential to regenerate following injury. In recent years, an expanding number of gene families has been identified involved in different aspects of neurite outgrowth and regeneration. These include neurotrophic factors, cell-adhesion molecules, growth-associated proteins, cytoskeletal proteins and chemorepulsive proteins. Genetic manipulation technology (transgenic mice, knockout mice, viral vectors and antisense oligonucleotides) has been instrumental in defining the function of these neurite outgrowth-related proteins. The aim of this paper is to provide an overview of the above-mentioned four approaches to manipulate gene expression in vivo and to discuss the progress that has been made using this technology in helping to understand the molecular mechanisms that regulate neurite outgrowth. We will show that work with transgenic mice and knockout mice has contributed significantly to the dissection of the function of several proteins with a key role in neurite outgrowth and neuronal survival. Recently developed viral vectors for gene transfer in postmitotic neurons have opened up new avenues to analyze the function of a protein following local expression in naive adult rodents. The initial results with viral vector-based gene transfer provide a conceptual framework for further studies on genetic therapy of neuroregeneration and neurodegenerative diseases.

Animals↗
PubMed2005

Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage.

BACKGROUND: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells. RESULTS: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors. CONCLUSION: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.

Animals↗
PubMed2006

Exposure of lentiviral vectors to subneutral pH shifts the tropism from Purkinje cell to Bergmann glia.

Cerebellar Purkinje cells play an important role in cerebellar function; lesions of Purkinje cells result in the disruption of motor coordination and motor learning. Although selective gene delivery to Purkinje cells would be a powerful technique for the study of pathophysiology in the cerebellum, a method for such a delivery has not yet been established. Here we employed human immunodeficiency virus-derived lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein to transduce Purkinje cells and examined factors that critically affect the viral tropism for Purkinje cells. Viral vectors encoding GFP were generated using different protocols, and were then injected into the mouse cerebellum. At 7 days and 2 months post-transduction, the relative proportions of transduced Purkinje cells were determined. Lentiviral vectors harvested from a medium of pH 7.2 preferentially transduced Purkinje cells (about half of the transduced cells). In contrast, when the viral vector was harvested from medium of <or= pH 7.0, only 12-26% of transduced cells were identified as Purkinje cells and 68-77% as Bergmann glia. A similar decrease in the efficiency of transduction for Purkinje cells, depending on the pH of the medium at the viral harvest, was observed in dissociated cell cultures. These results indicate that lentivector tropism for Purkinje cells is extremely sensitive to pH: a subtle decrease in the pH of the medium at the harvest shifts viral tropism strikingly towards Bergmann glia.

Animals↗
PubMed2004

Factors influencing the production of recombinant SV40 vectors.

Most gene therapy approaches employ viral vectors for gene delivery. Ideally, these vectors should be produced at high titer and purity with well-established protocols. Standardized methods to measure the quality of the vectors produced are imperative, as are techniques that allow reproducible quantitation of viral titer. We devised a series of protocols that achieve high-titer production and reproducible purification and provide for quality control and titering of recombinant simian virus 40 vectors (rSV40s). rSV40s are good candidate vehicles for gene transfer: they are easily modified to be nonreplicative and they are nonimmunogenic. Further, they infect a wide variety of cells and allow long-term transgene expression. We report here these protocols to produce rSV40 vectors in high yields, describe their purification, and characterize viral stocks using quality control techniques that monitor the presence of wild-type SV40 revertants and defective interfering particles. Several methods for reproducible titration of rSV40 viruses have been compared. We believe that these techniques can be widely applied to obtain high concentrations of high-quality rSV40 viruses reproducibly.

Animals↗
PubMed2006

Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors.

Gene therapy holds great promise for the treatment of a wide range of inherited and acquired disorders. The development of viral vector systems to mediate safe and long-lasting expression of therapeutic transgenes in specific target cell populations is continually advancing. Gene therapy for the nervous system is particularly challenging due to the post-mitotic nature of neuronal cells and the restricted accessibility of the brain itself. Viral vectors based on lentiviruses provide particularly attractive vehicles for delivery of therapeutic genes to treat neurological and ocular diseases, since they efficiently transduce non-dividing cells and mediate sustained transgene expression. Furthermore, novel routes of vector delivery to the nervous system have recently been elucidated and these have increased further the scope of lentiviruses for gene therapy application. Several studies have demonstrated convincing therapeutic efficacy of lentiviral-based gene therapies in animal models of severe neurological disorders and the push for progressing such vectors to the clinic is ongoing. This review describes the key features of lentiviral vectors that make them such useful tools for gene therapy to the nervous system and outlines the major breakthroughs in the potential use of such vectors for treating neurodegenerative and ocular diseases.

Animals↗
PubMed1997

Potential use of herpes simplex virus (HSV) vectors for gene therapy of neurological disorders.

Advances in molecular biology and virus genetics have allowed the possibility of gene therapy using viral vectors for a variety of neurological diseases in which the genetic or biochemical basis is understood. A number of such vectors have now been constructed, including those derived from herpes simplex virus (HSV), adenovirus, retrovirus and adeno-associated virus, and used in preliminary in vitro experiments and in animal models. It is possible to package a foreign gene into such a vector which can then be targeted to specific regions of the nervous system. HSV is particularly appropriate for delivering genes to neurons in view of its ability to establish latent infection in these cells. Viral vectors have the potential to be used to treat such neurological conditions as malignant gliomas, Parkinson's disease, known single gene disorders and cerebral ischaemia. However, the technical problems which will need to be overcome are formidable and will not be easily solved. The problems include the efficient delivery of the vector to target cells, the maintenance and control of foreign gene expression, and the control of unwanted host immune responses.

Adenoviridae↗
PubMed1999

Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.

Adenoviridae↗
PubMed2005

A QSAR-modeling perspective on cationic transfection lipids. 1. Predicting efficiency and understanding mechanisms.

BACKGROUND: As gene therapy using viral vectors involves clinical risks, limited DNA-carrying capacity, and manufacturing problems, non-viral vectors, including cationic lipids, have been investigated. Unfortunately, these agents have significantly lower transfectional ability and, due to the complexity of the transfectional pathway, no general schemes exist for correlating cationic lipid chemistry with transfectional efficacy. METHODS: Quantitative structure-activity relationship (QSAR) analyses were carried out on sets of routinely used, experimental, and unsuccessful cationic lipid vectors taken from the literature. This approach described the amphipathic character, basicity, headgroup size, lipophilicity and shape of cationic lipids using numerical parameters. Compounds were plotted onto various parameter diagrams, and correlations were sought between numerical parameters and transfectional efficiency. RESULTS: Transfectionally effective cationic lipids fell into restricted zones in various parameter spaces, indicating that amphipathic character, lipid shape and lipophilicity were generally significant factors, whilst basicity and headgroup size were only important for certain compounds. The data supported the general significance of membrane mixing followed by induction of membrane curvature, and the more limited role of osmotic shock, as mechanisms of membrane disruption. QSAR descriptions of effective lipids permitted detailed chemical guidelines for optimizing cationic lipid structure to be given. Limitations of the approach and models are discussed. CONCLUSIONS: QSAR modeling indicated that induction of membrane curvature and osmotic shock are important mechanisms for membrane disruption by cationic lipids. The models also allowed specification of chemically detailed guidelines for selection or design of optimal cationic lipids.

Cations↗
PubMed2005

Airway gene therapy.

Given both the accessibility and the genetic basis of several pulmonary diseases, the lungs and airways initially seemed ideal candidates for gene therapy. Several routes of access are available, many of which have been refined and optimized for nongene drug delivery. Two respiratory diseases, cystic fibrosis (CF) and alpha1-antitrypsin (alpha1-AT) deficiency, are relatively common; the single gene responsible has been identified and current treatment strategies are not curative. This type of inherited disease was the obvious initial target for gene therapy, but it has become clear that nongenetic and acquired diseases, including cancer, may also be amenable to this approach. The majority of preclinical and clinical studies in the airway have involved viral vectors, although for diseases such as CF, likely to require repeated application, non-viral delivery systems have clear advantages. However, with both approaches a range of barriers to gene expression have been identified that are limiting success in the airway and alveolar region. This chapter reviews these issues, strategies aimed at overcoming them, and progress into clinical trials with non-viral vectors in a variety of pulmonary diseases.

Animals↗