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PubMed2004

Analysis of the production efficiency and titration of various recombinant adeno-associated viruses.

Recombinant adeno-associated virus type 2 (rAAV2) viral vector, a non-pathogenic human parvovirus, has recently emerged as a gene transfer vehicle for cancer gene therapy. To utilize rAAV2 properly and safely while carrying out preclinical and clinical studies, it is crucial to exactly titer the virus. We therefore compared biological infectious rAAV2 titers with physical titers of rAAV2 vectors encoding various transgenes with different sized viral genomes. Biological rAAV2 infectivity was assayed by measuring the number of virus particles able to transduce Hela cells using several detection methods, including X-gal staining and immunocytostaining. Physical titers of rAAV2 were determined using a commercially available rAAV2 particle-specific enzyme-linked immunosorbent assay. We found that total rAAV2 particle production was consistent within the limited size variations of the rAAV2 genome, regardless of the difference in transgenes. In contrast, the infectious titer of rAAV2 differed greatly, even for the same viruses, due to variation in the sensitivity of the relevant assays. Thus, the results suggest that both infectious virus titer and total virus particle should be precisely measured for rAAV2 vector utilized in each study.

DNA, Viral↗
PubMedDate not supplied

Therapy of peritoneal carcinomatosis from colon cancer with oncolytic adenoviruses.

BACKGROUND: The major emphasis on the safety of viral vectors for gene delivery has led to the generally accepted approach of disabling their ability to replicate and thus their potential capacity to spread throughout a tumor by consecutive rounds of infection and lysing neighboring cells. METHODS: In this study we evaluated three herpes simplex virus-1 thymidine kinase (HSV-tk) carrying replication-competent adenoviral vectors with and without the Ad5 E1B 55-kDa gene, wild-type adenovirus type 5 (Ad5wt), and a prototypical replication-deficient adenovirus expressing HSV-tk (Ad.TK) for their cytoreductive effects in a peritoneal carcinomatosis model from human HT-29 colon cancer cells in nude mice. RESULTS: The survival of nude mice treated with the replication-defective adenoviral vector Ad.TK was enhanced when followed by GCV. In contrast, administration of GCV diminished the anti-tumor efficacy of the replication-competent HSV-tk expressing vectors. However, the intrinsic oncolytic effect of all replication-competent viruses was superior to that of Ad.TK+GCV. Furthermore, the oncolysis of the E1B 55-kDa-positive viruses was significantly greater than that of the E1B 55-kDa-deleted vector. CONCLUSIONS: The more efficient diffusion of viral particles in the peritoneal cavity, when compared to the microenvironment of solid tumors and the virostatic effects of GCV, most likely antagonized the anticipated enhanced cytotoxicity of the replication-competent vectors from the use of its gene directed enzyme prodrug system. Nevertheless, in a clinical setting, the HSV-tk/GCV system allows efficient termination of viral replication in the case of a runaway infection. The results of this study warrant further evaluation of controllable viral replication as a treatment modality for cancer, especially in combination with conventional therapies (e.g. chemotherapy).

Adenoviridae↗
PubMed2006

Characterization of transient expression system for retroviral vector production.

The production of retroviral vectors using a transient expression system has been improved to obtain a high-titer virus preparation that is difficult to produce using packaging cell lines due to the cytotoxic or cytostatic effect of transgenes. Here, we used one such production method, the so-called Q-vector system, and examined its potential for virus production. The Q-vector system could produce a similar level of viral vectors compared with the packaging cell system but the production seemed to depend on the size and nature of transgenes. In the process of investigation of the quantitative difference in viral components between the transient expression system and the packaging cell system, we found that the Q-vector system could express higher amounts of viral RNA and proteins compared with the packaging cell system. However, this did not lead to a higher virus titer compared with that produced by the packaging cell system. This suggests that retroviral RNA transcribed from the plasmid in the transient system seemed to be used mainly for translation and only some of the RNA molecules were packaged in viral particles.

Animals↗
PubMed1989

Protective efficacy of a recombinant vaccinia virus in vaccinia-immune mice.

Recombinant viral vectors offer a potential means of vaccinating against diseases for which there are no current safe vaccines. One of the criteria on which a viral vaccine vector would be selected is that it either circulates in the human or livestock population without producing overt disease (e.g. adenovirus) or has a history as a safe vaccine (e.g. vaccinia virus). However, this selection criterion also means that the target population is likely to have circulating antibodies that are specific to the vaccine vector. Since a percentage of the world's population has been vaccinated during the World Health Organization's Smallpox Eradication Campaign, such antibody titres, which are likely to lower vaccine efficacy, have been raised as an objection to the use of recombinant vaccinia viruses as vaccines. We have tested the effect of vaccinia-specific immunity on the protective efficacy of a recombinant virus, VV-PR8-HA6 (1) which expresses the haemagglutinin of the influenza virus A/PR/8/34.

Animals↗
PubMed2001

Analysis of recombinant adenoviruses using an integrated microfluidic chip-based system.

The use of recombinant virus for gene therapy requires rigorous quality control methods to ensure that the viral vector preparations are functional and safe. A viral identity test is performed in which the viral payload, or transgene, is PCR amplified, followed by digestion with restriction enzymes that yields a characteristic "fingerprint." These DNA fragments are typically analyzed by agarose gel electrophoresis. The ethidium bromide-stained gels are photographed or scanned and the results are sufficient for a qualitative or semiquantitative identity confirmation of the viral product. We have investigated the use of an integrated microfluidic chip-based system as a new tool in the quality control testing of a recombinant, adenoviral, gene therapy product. The chip-based method was found to be very sensitive, requiring 100-fold less sample and only one-third the time compared to the agarose gel method. The automated data analysis sizes and quantitates the DNA fragments, thus yielding a more thorough, reproducible, sensitive, and rapid analysis.

Adenoviruses, Human↗
PubMed1999

A novel anti-herpes simplex virus type 1-specific herpes simplex virus type 1 recombinant.

A recombinant herpes simplex virus (HSV) capable of inhibiting its own replication as well as the replication of wild-type virus would have greatly increased safety as a general purpose vector for in vivo gene transfer, antitumor therapy, and viral vaccine against HSV infection. By using a tetracycline repressor (tetR)-mediated HSV-1 viral replication switch [Yao and Eriksson (1999). Hum. Gene Ther. 10, 419-427], we have generated a novel anti-HSV-1-specific HSV-1 recombinant (CJ83193) that expresses a trans-dominant negative HSV-1 UL9 origin-binding protein, UL9-C535C. The de novo synthesis of CJ83193 can be suppressed by UL9-C535C by at least 1 x 10(6)-fold in non-tetR-expressing cells, and is subject to tetracycline regulation over a range of four to five orders of magnitude in a tetR-expressing osteosarcoma line. In particular, the UL9-C535C peptides expressed from the CJ83193 genome can inhibit the replication of wild-type HSV-1 by 100- to 200-fold in single-step growth assays. The construction of CJ83193 creates a new general strategy for developing recombinant viral vectors able to function as an intracellular therapy against wild-type viral infections.

Animals↗
PubMed2005

Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification.

Large scale purification of viruses and viral vectors for gene therapy applications and viral vaccines is a major separation challenge. Here tangential flow microfiltration and ultrafiltration using flat sheet membranes has been investigated for concentration of human influenza A virus. Ultrafiltration membranes with molecular weight cutoffs of 100 and 300 kDa as well as 0.1, 0.2 and 0.45 microm microfiltration membranes have been tested. The results indicate that use of 300 kDa membranes not only concentrate the virus particles but also lead to a significant removal of host cell proteins and DNA in the permeate. Tangential flow filtration may be used to fractionate virus particles. Human influenza A virus particles are spherical with an average size of 100 nm. Use of a 0.1 microm membrane leads to passage of virus particles less than 100 nm into the permeate and an increase of larger particles in the retentate. These results suggest that control of the transmembrane pressure, membrane pore size and pore size distribution could enable isolation of intact virus particles from damaged virions. Isolation of the virus particles of interest from viral fragments and other particulate matter could result in simplification of subsequent purification steps. Larger pore size membranes such as 0.45 microm that allow the passage of all virus particles may be used to remove host cell fragments. In addition virus particles attached to these fragments will be removed. Careful selection of membrane morphology and operating conditions will be essential in order to maximize the benefit of tangential flow filtration steps in the purification of viral products from cell cultures.

Animals↗
PubMed2005

A calcium phosphate-based gene delivery system.

Although nonviral vectors have lower transfection efficiency than viral vectors, the excellent safety profile of nonviral vectors is appealing for gene therapy. An efficient, simple nonviral vector gene delivery system has been designed that includes plasmid DNA-calcium phosphate precipitates (pDNA-CaP) and porous collagen spheres (Cultispherestrade mark). The hypothesis for this study was the pDNA-CaP would achieve efficient plasmid DNA transfection and the porous collagen spheres would provide a suitable delivery carrier system for three-dimensional (3D) administration. To test the hypothesis, plasmid DNA including the LacZ reporter gene encoding beta-galactosidase was precipitated with CaP to form particles of compacted LacZ-CaP and delivered directly or by Cultispherestrade mark to cells in vitro. The transfection efficiency was determined by beta-galactosidase gene expression. Results indicated that pLacZ-CaP promoted 25-84% of transfection efficiency in a broad cell line spectrum and in flexible experimental conditions. Maximum transfection efficiency was achieved by having mostly nano-sized partles (50-200 nm in diameter) of pDNA-CaP precipitates. Seeding density of 0.7-4 x 10(4) cells/cm2 provided sufficient transfection efficiency, and storage of pDNA-CaP at 4 degrees C was most efficient to preserve transfection efficacy for up to 3 days. The pDNA-CaP worked well in the presence of serum and serum-free conditions and was less cytotoxic than the liposomes. Cultispherestrade mark carrying plasmid LacZ-CaP was an effective 3D system for gene delivery. The technique described here is a simple and safe procedure to deliver genes, and may have application to regenerate bone and other tissues.

Animals↗
PubMed2002

Gene therapy and reproductive medicine.

OBJECTIVE: To review the literature on the principles of gene therapy and its potential application in reproductive medicine. DESIGN: Literature review. SETTING: Gene therapy involves transfer of genetic material to target cells using a delivery system, or vector. Attention has primarily focused on viral vectors. Significant problems remain to be overcome including low efficacy of gene transfer, the transient expression of some vectors, safety issues with modified adenoviruses and retroviruses, and ethical concerns. If these issues can be resolved, gene therapy will be applicable to an increasing spectrum of single and multiple gene disorders, as the Human Genome Project data are analyzed, and the genetic component of human disease becomes better understood. Gynecologic gene therapy has advanced to human clinical trials for ovarian carcinoma, and shows potential for the treatment of uterine leiomyomata. Obstetric applications of gene therapy, including fetal gene therapy, remain more distant goals. CONCLUSION(S): Concerns about the safety of human gene therapy research are being actively addressed, and remarkable progress in improving DNA transfer has been made. The first treatment success for a genetic disease (severe combined immunodeficiency disease) has been achieved, and ongoing research efforts will eventually yield clinical applications in many spheres of reproductive medicine.

Adenoviridae↗
PubMed2006

Therapy of cancer by cytokines mediated by gene therapy approach.

Gene therapy offers a new approach for treatment of cancer. Transfer of genes encoding immunostimulatory cytokines has been used with remarkable success to eliminate cancer in animals. However, clinical trials in patients with this strategy had limited efficacy. Therefore, improvement of gene transfer vector system is necessary. A hybrid viral vector, consisting of SFV replicon with either murine IL-12 or reporter LacZ gene, was constructed. This hybrid vector showed specificity and high level of expression in HCC both in vitro and in vivo. In a rat orthotropic liver tumor model, treatment of established tumors by the hybrid vector with mIL-12 gene resulted in a strong anti-tumor activity without accompanying toxicity. Subsequently, a helper-dependent adenovirus vectors containing a mifepristone (RU486) inducible system was constructed for controlled and liver-specific expression of human interleukin 12 (hIL-12) (HD-Ad/RUhIL-12) and mouse IL-12 (mIL-12) (HD-Ad/RUmIL-12). Data showed that high and sustained serum levels of hIL-12 could be attained by continuing administration of RU486 every 12 or 24 h. Repetitive induction of hIL-12 could be obtained over, at least, a period of 48 weeks after a single injection of HD-Ad/RUhIL-12. Treatment of liver metastases with of HD-Ad/RUmIL-12 plus RU846 resulted in complete tumor regression in all animals. Then, different cytokine genes were inserted into conditional replicative adenoviruses vectors (also called oncolytic adenovirus). Replication of adenovirus in tumor cells would kill tumor cells and release viruses, which infect surrounding tumor cells. The combination of cytopathic effect by oncolytic adenovirus and biological effect of transgene would exert strong antitumor activity. These new types of vectors may provide a potent and safe tool for cancer gene therapy.

Animals↗
PubMed2006

Gene therapy that safely targets and kills tumor cells throughout the body.

The authors studied the therapeutic value of Sindbis vectors for advanced metastatic cancer by using a variety of clinically accurate mouse models and demonstrated through imaging, histological, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in vivo, leading to significant suppression of tumor growth and enhanced survival. Use of two different bioluminescent genetic markers for the IVIS Imaging System (Xenogen Corp., Alameda, CA) permitted demonstration of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor, which is elevated in tumor versus normal cells, and downregulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. Laser capture microdissection of mouse tumor implants showed equivalent laminin receptor expression levels in the different tumor metastases in the peritoneal cavity. Incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer.

Animals↗
PubMed2000

Functional characterization of the dimer linkage structure RNA of Moloney murine sarcoma virus.

Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255-7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.

Animals↗
PubMed2001

Gene therapy for superficial bladder cancer.

In this review, the basics of gene therapy and the strategies to increase the therapeutic effect of gene therapy for superficial bladder cancer are discussed. Strategies considered in detail are modification of the structure of vectors, modification of the promoters of viral vectors and the timing and route of vector administration. Although all of these modifications have shown some degree of improvement for gene transfer, the use of polyamides intravesically in conjunction with an adenoviral system shows the most promise and the greatest potential to supplement or even replace the current treatment modalities for superficial bladder cancer.

Adenoviridae↗
PubMed1986

Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons.

To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion.

Chimera↗
PubMed2004

Reducing the immunostimulatory activity of CpG-containing plasmid DNA vectors for non-viral gene therapy.

The mammalian innate immune system has the ability to recognise and direct a response against incoming foreign DNA. The primary signal that triggers this response is unmethylated CpG motifs present in the DNA sequence of various disease-causing pathogens. These motifs are rare in vertebrate DNA, but abundant in bacterial and some viral DNAs. Because gene therapy generally involves the delivery of DNA from either plasmids of bacterial origin or recombinant viruses, an acute inflammatory response of variable severity inevitably results. The response is most serious for non-viral gene delivery vectors composed of cationic lipid-DNA complexes, producing adverse effects at lower doses and lethality at higher doses of complex. This review examines the role of immunostimulatory CpG motifs in the acute inflammatory response to non-viral gene therapy vectors. Strategies to neutralise or eliminate CpG motifs within plasmid DNA vectors, and the existing limitations of CpG reduction on improving the safety profile of non-viral vectors, will be discussed.

Animals↗
PubMed2004

Gene therapy of liver diseases.

Many liver diseases lack satisfactory treatment and alternative therapeutic options are urgently needed. Gene therapy is a new mode of treatment for both inherited and acquired diseases, based on the transfer of genetic material to the tissues. Genes are incorporated into appropriate vectors in order to facilitate their entrance and function inside the target cells. Gene therapy vectors can be constructed on the basis of viral or non-viral molecular structures. Viral vectors are frequently used, due to their higher transduction efficiency. Both the type of vector and the expression cassette determine the duration, specificity and inducibility of gene expression. A considerable number of preclinical studies indicate that a great variety of liver diseases, including inherited metabolic defects, chronic viral hepatitis, liver cirrhosis and primary and metastatic liver cancer, are amenable to gene therapy. Gene transfer to the liver can also be used to convert this organ into a factory of secreted proteins needed to treat conditions that do not affect the liver itself. Clinical trials of gene therapy for the treatment of inherited diseases and liver cancer have been initiated but human gene therapy is still in its infancy. Recent progress in vector technology and imaging techniques, allowing in vivo assessment of gene expression, will facilitate the development of clinical applications of gene therapy.

Animals↗
PubMed2001

Dendritic cells transduced with HSV-1 amplicons expressing prostate-specific antigen generate antitumor immunity in mice.

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.

Animals↗
PubMed2001

Efficiency of non-viral gene delivery systems to rat lungs.

OBJECTIVE: Transient expression of therapeutic genes within lung allografts may modulate the pathological processes following allotransplantation. Whilst efficient gene transfer to lungs has been reported with viral vectors, their usefulness is limited on the grounds of safety. Since non-viral systems overcome many of these safety issues, our studies were designed to evaluate the efficiency of several non-viral gene delivery vectors for in vivo transfer of plasmid DNA to rat lungs via the airways. METHODS: Fischer rats (230--260 g) underwent a thoracotomy, right main bronchus occlusion and instillation of 300 microg naked or complexed DNA (pCIluci, luciferase gene/CMV promoter) to the left lung followed by ventilation for 10 min. Rats were divided into five treatment groups (n=5): (1) Glucose, (2) Naked DNA, (3) Linear polyethylenimine (PEI), (4) Branched PEI, (5) Lipid GL-67/DOPE and (6) DOTAP/cholesterol. Animals were sacrificed 24 h after gene delivery for measurement of reporter gene activity and gas exchange of the left lung. RESULTS: Linear PEI was the most efficient gene delivery vector and was significantly better than DOTAP/cholesterol (P=0.00002) and naked DNA (P=0.004). All gene delivery vectors impaired function of the transfected left lung compared with DNA alone. Of all the gene delivery vectors tested, lipid GL-67/DOPE exerted the least effect on lung function whilst DOTAP/cholesterol mediated the most adverse effect. CONCLUSION: Linear PEI was the most efficient vector for gene delivery to rat lungs in our experimental setting although it mediated a moderate impairment in lung function. Further studies are needed to evaluate whether this effect is transient.

Animals↗