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At least 1,477 records · Page 82Linked to original sources
PubMed1997

Gene therapeutic strategies for neuroprotection: implications for Parkinson's disease.

Gene transfer methodologies are being explored as strategies to restore and preserve neuronal function in Parkinson's Disease. This technology represents a new therapeutic modality, holding promise for continuous and localized delivery, of neuroprotective molecules. Two primary approaches for gene transfer have emerged: in vivo and ex vivo. Recent advances in the construction and characterization of gene transfer vectors have generated more efficient vehicles to deliver and express candidate therapeutic genes. Direct gene transfer into the CNS can be achieved with replication-deficient viral vectors of several types: adenovirus, adeno-associated virus, and herpes simplex virus. These vector systems are being evaluated in models of Parkinson's disease. Strategies to deliver genes include those that either augment dopamine biosynthesis or attenuate loss of dopaminergic neurons. A discussion of the various approaches is detailed.

Adenoviridae↗
PubMed2006

Spinal gene transfer using ultrasound and microbubbles.

Spinal gene therapy is a promising option for treating various spinal-related disorders. Several previous studies using viral vectors reported successful transfer of therapeutic genes into the spinal nerve system. However, because of the considerable immunogenicity related to the use of viruses, non-viral gene transfer still needs to be developed. One possible approach is the combined use of ultrasound and echo-contrast microbubbles. The present study shows that this method can be applied for targeted intrathecal gene delivery. We intrathecally injected a mixture of plasmid-DNA encoded with luciferase and commercially available albumin microbubbles by needle puncture at the lower lumbar intervertebral space in mice. Subsequent percutaneous ultrasonication on the lumbar vertebrae significantly enhanced the luciferase expression, analyzed by imaging luciferin bioluminescence, in the dorsal meningeal cells at the insonated region. No apparent neurological damages were induced by the present spinal interventions. In addition to the general benefits of the combined use of ultrasound and microbubbles, our approach can offer some advantages specific to spinal gene transfection including minimal invasiveness of simple percutaneous dural puncture, targetability due to the limited access of ultrasound waves through anatomical apertures of the vertebrae, and possible paracrine delivery of therapeutic molecules to the spinal nerve system.

Animals↗
PubMed2000

DNA-carrier proteins for targeted gene delivery.

The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Significant progress has been made in the construction of non-viral vectors that combine different functions required for gene transfer in an artificial complex. To some extent this can be achieved by complexing plasmid DNA with synthetic compounds such as lipids and polycations. Alternative approaches rely on the activities of natural or recombinant DNA-carrier proteins to achieve uptake and intracellular delivery of plasmid DNA. Nuclear proteins such as histones and members of the high mobility group protein family have been shown to condense DNA and transfect cultured cells. Some structural proteins of DNA viruses spontaneously assemble with plasmid DNA and form transfection-competent pseudocapsids. In addition, chimeric fusion proteins have been engineered that incorporate in a single polypeptide chain heterologous protein domains which facilitate binding to plasmid DNA, specific recognition of target cells, induction of receptor-mediated endocytosis, and DNA transport through intracellular compartments.

Bacterial Toxins↗
PubMed2001

The basis for HIV immunotherapeutic vaccines.

The drug treatments introduced in recent years for HIV infection have enabled a marked reduction in morbidity and prolongation of life. These treatments, however, are often associated with acute and chronic toxicities, the development of resistant virus can limit their effectiveness, and they are too expensive and difficult to administer in most third world settings. A successful HIV immunotherapeutic vaccine has the potential to overcome these problems, and would be a valuable advance. The most promising approaches have induced the type of immune response found to correlate with reduced activity of HIV in man, especially cytotoxic T-cell responses, or have led to reduced HIV or SIV viral load and increased CD4 counts in non-human primates or man. The agents that have led to one or both of these effects have been selected for review, and include inactivated envelope depleted virus, recombinant envelope glycoprotein, DNA vaccines utilising HIV peptides or gene products, viral vectors, such as canarypox or attenuated vaccinia, with HIV core proteins. There are other approaches, such as alloimmunity, for which no candidate products yet exist, but which conceptually appear promising. Currently, however, only a few phase III studies of HIV therapeutic vaccines have been completed in man, and there has been a modest therapeutic effect. Further development of both existing and new candidates remains one of the key priorities in our fight against HIV.

AIDS Vaccines↗
PubMed2002

Gene therapy for treatment of cerebral ischemia using defective recombinant adeno-associated virus vectors.

In this review we present our results and experiences in performing gene therapy of cerebral stroke using recombinant adeno-associated virus (rAAV) vectors in a rat model. The methodologies involving the production of AAV vectors, gene transfer to the brain, and a trivessel ligation model of focal ischemic cerebral stroke in rats are described. Furthermore, a brief description of other viral vectors and candidates of therapeutic transgenes used for gene therapy of cerebral stroke are presented. The potential advantages and limitations of stroke gene therapy are also discussed with the intention of outlining the design of more appropriate experiments.

Adenoviridae↗
PubMed1993

Characterization of a bicistronic retroviral vector composed of the swine vesicular disease virus internal ribosome entry site.

We cloned the 5' nontranslated region (NTR) from the genome of swine vesicular disease virus (SVDV), a member of the family Picornaviridae, and used it to construct a bicistronic retroviral vector. The vector is characterized by coexpression of two genes from a single transcript. We found that inclusion of the 5' NTR of SVDV did not negate the viral vector titer. Protein analysis indicated that the 5' NTR could efficiently direct internal initiation, thus allowing the downstream gene to be translated. Translation of the internally initiated porcine growth hormone gene was about 30-fold less than that when the porcine growth hormone gene was at the upstream position in NIH 3T3 cells but was about equivalent to that in HeLa cells, implying that some cellular factors that stimulated internal initiation of the SVDV 5' NTR are present in HeLa cells. However, in G418-selected clones, the Neor-encoding gene was expressed with equivalent efficiency either at a downstream position or at an upstream position in either NIH 3T3 or HeLa cells. Compared with the conventional double-gene vector or the U3-based vector, the bicistronic vector coexpressed two genes much more efficiently, owing to elimination of promoter interference. Furthermore, this type of vector infected and expressed the target genes efficiently in two primary cell lines, rat embryo and human skin fibroblast cells, which we tested. These experimental data suggest a better design for the retroviral vector and provide evidence that internal initiation of the SVDV 5' NTR was stimulated cell specifically.

3T3 Cells↗
PubMed1997

Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice.

About 70% of hepatocellular carcinomas are known to express alpha-fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the alpha-fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

Animals↗
PubMed2004

Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography.

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 microg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.

Amino Acid Sequence↗
PubMed1995

[Gene therapy for hereditary and acquired human diseases].

Cystic Fibrosis (CF) and AIDS are primary candidate disorders to be treated by gene therapy, owing to their lethality and the absence of efficient clinical treatments. Treatment of CF by gene therapy will require the transfer of the functional CFTR cDNA into the diseased human airway epithelia since mutations within the CFTR gene are responsible for CF. We have therefore cloned the human CFTR cDNA and developed a recombinant E1-deleted adenoviral vector carrying a CFTR expression cassette. We demonstrated in vitro the ability of this vector to efficiently transduce human lung cells isolated from CF patients and to correct their phenotype. Efficient in, vivo delivery of the CFTR cDNA to the airways of cotton rats and rhesus monkeys was also obtained and no dissemination of the recombinant viral vector in other tissues than the airways was observed. We have therefore designed a phase I clinical trial involving CF patients. In contrast to the monogenic CF disease, the mechanisms of AIDS pathogenesis still remain poorly understood. Such limited knowledge of the disease constitutes a serious restriction to the development of a rational gene therapy strategy for AIDS. Since HIV, the causative agent of AIDS, predominantly infects cells of the hematopoietic system, pluri- or multipotent stem cells may constitute potential targets for the introduction of a foreign anti-HIV gene that will inhibit HIV replication and/or spread. Reimplantation of the genetically modified stem cells into asymptomatic HIV-infected patients should theoretically allow the repopulation of the host's immune system with mature CD4+ cells expressing novel molecules that interfere with viral replication, thus slowing the progression of AIDS. We identified several new transdominant inhibitors derived from the viral TAT and REV proteins and showed their ability to confer to human CD4 lymphocytes resistance against HIV1 infection. Retroviral vectors carrying these potential therapeutic genes have been developed and are currently being tested in vivo in newly developed transgenic animal models, in humanized SCID mice and in macaques.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Direct priming and cross-priming contribute differentially to the induction of CD8+ CTL following exposure to vaccinia virus via different routes.

To explore the relative importance of direct presentation vs cross-priming in the induction of CTL responses to viruses and viral vectors, we generated a recombinant vaccinia vector, vUS11, expressing the human CMV (HCMV) protein US11. US11 dislocates most allelic forms of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum into the cytosol, where they are degraded by proteasomes. Expression of US11 dramatically decreased the presentation of viral Ag and CTL recognition of infected cells in vitro without significantly reducing total cell surface MHC class I levels. However, because US11 is an endoplasmic reticulum resident membrane protein, it cannot block presentation by non-infected cells that take up Ag through the cross-priming pathway. We show that the expression of US11 strongly inhibits the induction of primary CD8(+) CTLs when the infection occurs via the i.p. or i.v. route, demonstrating that direct priming is critical for the induction of CTL responses to viral infections introduced via these routes. This effect is less dramatic following i.m. infection and is minimal after s.c. or intradermal infection. Thus, classic MHC class I Ag presentation and cross-priming contribute differentially to the induction of CD8(+) CTLs following exposure to vaccinia virus via different routes.

Animals↗
PubMed2001

Rhesus monkey model for fetal gene transfer: studies with retroviral- based vector systems.

Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human. Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days). Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer). PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth. Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.

Animals↗
PubMed2001

Specific binding of baculoviruses displaying gp64 fusion proteins to mammalian cells.

Viral vectors displaying specific ligand binding moieties have raised an increasing interest in the area of targeted gene therapy. In this report, we describe baculovirus vectors displaying either a functional single chain antibody fragment (scFv) specific for the carcinoembryonic antigen (CEA) or the synthetic IgG binding domains (ZZ) derived from protein A of Staphylococcus aureus. In addition, the vectors were engineered to incorporate a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the transcriptional regulation of the cytomegalovirus (CMV) IE promoter. Display of the targeting moieties on the viral surface was achieved through fusion to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus (AcNPV). Specific binding of the gp64 fusion viruses to mammalian target cells was demonstrated by using monoclonal anti-gp64 antibodies followed by fluorescence and/or confocal microscopy. The anti-CEA scFv displaying baculovirus was shown to bind specifically to CEA expressing cells (PC-3). Similarly, the virus displaying the ZZ domains of protein A was targeted to BHK cells via binding of an appropriate IgG antibody. In all cases, the reporter gene was expressed in the transduced mammalian cells as shown by fluorescence microscopy and flow cytometric analyses.

Animals↗
PubMed2003

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.

Adenoviridae↗
PubMed1998

Peroral gene therapy of lactose intolerance using an adeno-associated virus vector.

Gene therapy is usually reserved for severe and medically refractory disorders because of the toxicity, potential long-term risks and invasiveness of most gene transfer protocols. Here we show that an orally administered adeno-associated viral vector leads to persistent expression of a beta-galactosidase transgene in both gut epithelial and lamina propria cells, and that this approach results in long-term phenotypic recovery in an animal model of lactose intolerance. A gene 'pill' associated with highly efficient and stable gene expression might be a practical and cost-effective strategy for even relatively mild disorders, such as lactase deficiency.

Administration, Oral↗
PubMed2001

The molecular genetics of lentiviral vectors--current and future perspectives.

Gene therapy is a promising endeavor for the treatment of disease in the 21st century. The key to capitalize on this venture lies in the availability of efficient gene transfer and expression tools. Viral vectors are useful vehicles for the delivery of foreign genes into target cells, and retroviral vectors have been popular because of their ability to integrate into the host cell genome and maintain persistent gene expression. Recent studies of the human immunodeficiency virus (HIV) have demonstrated that lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and post-mitotic stages of the cell cycle. This article aims to analyze the molecular genetics, review existing systems and applications, and address problems as well as potential future developments of the lentiviral vector systems.

Forecasting↗
PubMed2000

IAP family proteins delay motoneuron cell death in vivo.

Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.

Adenoviridae↗
PubMed2004

Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis.

Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function.

Animals↗
PubMed1997

Tumor-selective transgene expression in vivo mediated by an E2F-responsive adenoviral vector.

Recent data suggest that many tumors, such as malignant gliomas, have disrupted pRB function, either because of RB-1 gene mutations or as a result of mutations affecting upstream regulators of pRB such as cyclin D1 or p16/INK4a/MTS1 (ref. 1-5). Tumor suppression by pRB has been linked to its ability to repress E2F-responsive promoters such as the E2F-1 promoter. Thus, a prediction, which has not yet been demonstrated experimentally in vivo, is that E2F-responsive promoters should be more active in tumor cells relative to normal cells because of an excess of "free" E2F and loss of pRB/E2F repressor complexes. We demonstrate that adenoviral vectors that contain transgenes driven by the E2F-1 promoter can mediate tumor-selective gene expression in vivo, allowing for eradication of established gliomas with significantly less normal tissue toxicity than seen with standard adenoviral vectors. Our data indicate that de-repression of the E2F-1 promoter occurs in cancer cells in vivo, a finding that can be exploited to design viral vectors that mediate tumor-selective gene expression.

Adenoviruses, Human↗