Showing papers on "Vector (molecular biology) published in 2001"

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics

Abstract: Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.

Nonviral Vectors in the New Millennium: Delivery Barriers in Gene Transfer

Abstract: Development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired diseases by gene therapy. To this end, various nonviral vectors have been designed and developed, and some of them are in clinical trials. The simplest approach is naked DNA injection into local tissues or systemic circulation. Physical (gene gun, electroporation) and chemical (cationic lipid or polymer) approaches have also been utilized to improve the efficiency and target cell specificity of gene transfer by plasmid DNA. After administration, however, nonviral vectors encounter many hurdles that result in diminished gene transfer in target cells. Cationic vectors sometimes attract serum proteins and blood cells when entering into blood circulation, which results in dynamic changes in their physicochemical properties. To reach target cells, nonviral vectors should pass through the capillaries, avoid recognition by mononuclear phagocytes, emerge from the blood vessels to the interstitium, and bind to the surface of the target cells. They then need to be internalized, escape from endosomes, and then find a way to the nucleus, avoiding cytoplasmic degradation. Successful clinical applications of nonviral vectors will rely on a better understanding of barriers in gene transfer and development of vectors that can overcome these barriers.

Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery

Abstract: In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease.

Gene Therapy: Promises and Problems

Abstract: Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Based on the nature of the viral genome, these gene therapy vectors can be divided into RNA and DNA viral vectors. The majority of RNA virus-based vectors have been derived from simple retroviruses like murine leukemia virus. A major shortcoming of these vectors is that they are not able to transduce nondividing cells. This problem may be overcome by the use of novel retroviral vectors derived from lentiviruses, such as human immunodeficiency virus (HIV). The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.

Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria

Abstract: Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue-white screening via lacZalpha; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors

Abstract: Gene therapy using recombinant adeno-associated virus vectors (rAAV) is generally considered safe. During the course of a study designed to determine the long-term efficacy of rAAV-mediated gene therapy initiated in newborn mice with the lysosomal storage disease, mucopolysaccharidosis type VII (MPSVII), a significant incidence of hepatocellular carcinomas and angiosarcomas was discovered. A hepatocellular carcinoma was first detected in a 35-week-old mouse and by 72 weeks of age, three out of five rAAV-treated MPSVII mice had similar lesions. These types of tumors had not been seen previously in long-term studies of MPSVII mice using recombinant enzyme or bone marrow transplantation. In an attempt to ascertain whether mouse strain or GUSB expression confers susceptibility to tumor formation, we histopathologically examined untreated normal mice of the same strain, untreated MPSVII mice, and normal mice overexpressing human GUSB for the presence of tumors and increased hepatocyte replication. The results of these studies do not indicate that MPSVII mice or mice overexpressing human GUSB are susceptible to tumor formation; however, the number of animals examined is too small to draw definitive conclusions. Results from quantitative PCR performed on the tumor samples suggest that the tumors are probably not caused by an insertional mutagenesis event followed by the clonal expansion of a transformed cell. In a separate study, a relatively large group of mice injected with varying doses and types of rAAV vectors had no evidence of hepatic or vascular tumors. Although the mechanism of tumor formation is currently unknown, the tumorigenic potential of rAAV vectors must be rigorously determined in long-term in vivo studies.

“Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

Abstract: Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.

Silencing of a meristematic gene using geminivirus-derived vectors.

Abstract: Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants.

Silencing of gene expression: implications for design of retrovirus vectors.

Abstract: Transcriptional silencing of retroviruses poses a major obstacle to their use as gene therapy vectors. Silencing is most pronounced in stem cells which are desirable targets for therapeutic gene delivery. Many vector designs combat silencing through cis-modifications of retroviral vector sequences. These designs include mutations of known retroviral silencer elements, addition of positive regulatory elements and insulator elements to protect the transgene from negative position effects. Similar strategies are being applied to lentiviral vectors that readily infect non-dividing quiescent stem cells. Collectively these cis-modifications have significantly improved vector design but optimal expression may require additional intervention to escape completely the trans-factors that scan for foreign DNA, establish silencing in stem cells and maintain silencing in their progeny. Cytosine methylation of CpG sites was proposed to cause retroviral silencing over 20 years ago. However, several studies provide evidence that retrovirus silencing acts through methylase-independent mechanisms. We propose an alternative silencing mechanism initiated by a speculative stem cell-specific "somno-complex". Further understanding of retroviral silencing mechanisms will facilitate better gene therapy vector design and raise new strategies to block transcriptional silencing in transduced stem cells.

Ti-Plasmid Vectors Useful for Functional Analysis of Rice Genes

Abstract: We developed new binary vectors, pPZP2H-lac and pPZP2Ha3, for the transformation of rice plants These binary vectors contain multiple cloning sites and the hygromycin B phosphotransferase gene as a plant-selectable marker After Agrobacterium-mediated transformation, transgenic plants containing a T-DNA could be tightly selected by hygromycin B The vectors are powerful tools for the functional analysis of genes

High-capacity 'gutless' adenoviral vectors.

Abstract: Publisher Summary This chapter discusses the technical aspects and applications related to the production and the design of high-capacity "gutless" adenoviral (HC-Ad) vectors. Safety and expression features of HC-Ad vectors are improved over earlier-generation adenoviral vectors. The increased capacity may allow coexpression of different therapeutic genes and improved control of gene expression. A critical issue that stands between the current status of HC-Ad vector development and clinically useful applications for human patients is at the level of vector production. Addition of stuffer DNA and vector capsid modification are some of the other aspects that need consideration during the production of these vectors. Retargeting strategies will increase the spectrum of therapeutic applications and will further add to the safety of HC-Ad vectors. These vectors find their application in various fields such as liver gene transfer, gene transfer into skeletal muscle, gene transfer into the eye and into the central nervous system (CNS), etc.

Time course of gene expression after plasmid DNA gene transfer to the liver.

Abstract: Background High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter-driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression. Methods Histological methods were used to determine the presence and extent of liver damage and hepatocyte proliferation. Viral and liver-specific promoters were tested to study promoter shut-off, Southern blotting was performed to determine the loss of the pDNA vector over time, and several mouse models were used to study the host immunological response. Results pDNA is lost rapidly early after injection, but remains at a relatively stable copy number after Day 4. Southern blotting experiments showed that plasmid DNA could be detected for at least 12 weeks after injection (0.2 copies per genome). The early rapid decline of expression is promoter dependent. A liver-specific albumin promoter resulted in similar levels of expression on Days 1 and 7, suggesting that promoter inactivation may be responsible for the instability of CMV promoter-driven expression. The slow decline in expression levels after 1 week appears to be the result of an immune response directed against the expressed transgene. Expression was much prolonged in immunosuppressed, immunodeficient, or antigen-tolerized mice. Conclusion The present data suggest that if promoter inactivation can be overcome, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo. Copyright © 2001 John Wiley & Sons, Ltd.

Multiple immediate-early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous system in vivo.

Abstract: Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604–5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.

Enhancement of suicidal DNA vaccine potency by linking Mycobacterium tuberculosis heat shock protein 70 to an antigen

Abstract: Naked DNA vaccines represent an attractive approach for generating antigen-specific immunity because of their stability and simplicity of delivery. There are particular concerns with DNA vaccines however, such as potential integration into the host genome, cell transformation, and limited potency. The usage of DNA-based alphaviral RNA replicons (suicidal DNA vectors) may alleviate the concerns of integration or transformation since suicidal DNA vectors eventually cause lysis of transfected cells. To improve further the potency of suicidal DNA vaccines, we evaluated the effect of linking Mycobacterium tuberculosis heat shock protein 70 (Hsp70) to human papillomavirus type 16 (HPV-16) E7 as a model antigen on antigen-specific immunity generated by a DNA-based Semliki Forest virus (SFV) RNA vector, pSCA1. Our results indicated that this suicidal DNA vaccine containing E7/Hsp70 fusion genes generated significantly higher E7-specific T cell-mediated immune responses than vaccines containing the wild-type E7 gene in vaccinated mice. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing metastatic tumors. The antitumor effect was predominantly CD8-dependent. These results indicate that linkage of Hsp70 to the antigen may greatly enhance the potency of suicidal DNA vaccines.

A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors

Abstract: A novel baculovirus genome has been generated that can be maintained in Escherichia coli and which facilitates the rapid and efficient generation of recombinant baculovirus expression vectors. This genome, designated bAcGOZA, lacks a portion of the essential ORF1629 gene but includes a miniF replicon and selectable kanamycin-resistance marker. The bAcGOZA can replicate only in E. coli and can be rescued by recombination with a transfer vector containing an intact ORF1629. There is no background of non-recombinant virus.

Delivery systems intended for in vivo gene therapy of cancer: targeting and replication competent viral vectors

Abstract: Cancer gene therapy represents one of the most rapidly evolving areas in pre-clinical and clinical cancer research Application of gene transfer techniques in clinical trials has made increasingly obvious that several issues will need to be addressed prior to meaningful incorporation of gene therapy in the care of cancer patients Two of the most important problems to overcome are lack of selectivity of the existing vectors and low efficiency of gene transfer This review focuses on use of targeting and replication competent vectors in order to overcome these obstacles Targeted gene therapy of malignancies can be achieved through vector targeting or transcriptional targeting and can improve the therapeutic index of gene transfer by preventing damage of normal tissues, an important requirement if systemic gene delivery is contemplated Replication competent viral vectors can improve the efficiency of gene transfer Provisionally replicating viruses can also improve the therapeutic index by targeting toxicity to tumor cells A variety of provisionally replicating viruses, such as the attenuated adenovirus ONYX-015, the adenovirus CN706 that selectively replicates in prostate cancer cells, the double mutant herpes simplex virus G207, the human reovirus, and the Newcastle disease virus are currently in clinical trials Early clinical results and limitations in the application of these vectors are discussed

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.

Abstract: Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus–adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 × 108p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in ∼100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.

Wild-type adenovirus decreases tumor xenograft growth, but despite viral persistence complete tumor responses are rarely achieved--deletion of the viral E1b-19-kD gene increases the viral oncolytic effect.

Abstract: Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was ...

Plasmids for therapy and vaccination

Abstract: Plasmids Structures of Plasmid DNA Genetic Vaccination with Plasmid Vectors Phase I Plasmid Gene Therapy Trials Today pAH9 - A Vector for the Treatment of Restenosis pUT649 - A Cancer Gene Therapy Vector pCMV-S2S - A Curative Immunization Approach against Hepatitis B Virus pXY53 - A Malaria Vaccine Polyvalent/Polycistronic Plasmid Vectors From Research Data to Clinical Trials with Plasmid DNA Plasmid Manufacturing - An Overview Quality Criteria for a Clinical Batch of pDNA Plasmids in Fish Vaccination.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Abstract: Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors.

Abstract: A major obstacle in cancer gene therapy is the limited efficiency of in vivo gene transfer by replication-defective retrovirus vectors in current use. One strategy for circumventing this difficulty would be to use vectors capable of replication within tumor tissues. We have developed a replication-competent retrovirus (RCR) vector derived from murine leukemia virus (MuLV). This vector utilizes a unique design strategy in which an internal ribosome entry site-transgene cassette is positioned between the env gene and the 3′ long terminal repeat (LTR). The ability of this vector to replicate and transmit a transgene was examined in culture and in a solid tumor model in vivo. The RCR vector exhibited replication kinetics similar to those of wildtype MuLV and mediated efficient delivery of the transgene throughout an entire population of cells in culture after an initial inoculation with 1 plaque-forming unit (PFU) of vector per 2000 cells. After injection of 6 × 103 PFU of vector into established subcutaneous...