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PubMed2001

Retargeting of viral vectors to the folate receptor endocytic pathway.

Viral vectors with high transfection efficiencies are not always those with optimal target cell binding specificities. As a consequence, virus pseudotyping has been developed to endow transfection competent viruses with improved cell binding specificities and affinities. We have hypothesized that chemical conjugation of a virus to a cell specific ligand might also alter its target cell specificity and produce a virus that would transfect only the desired cell type. To test this concept, an ecotropic replication-defective myeloproliferative sarcoma retrovirus and an amphotropic murine adenovirus containing the gene for beta-galactosidase were chemically derivatized with folic acid. As expected from its strong ecotropism, the unmodified retrovirus did not induce beta-galactosidase expression in nonhost KB cells, while the amphotropic adenovirus yielded high levels of gene expression in the same cell line. Surprisingly, although folate derivatization enabled avid binding of both viruses to folate receptor expressing KB cells, the folate conjugation did not promote retroviral gene expression and actually prevented the normal beta-galactosidase expression seen with the adenoviral vector. The fact that co-administration of excess free folic acid to block uptake by folate receptor-mediated endocytosis restored adenoviral gene expression to the level obtained with unmodified virus suggests that folate derivatization per se does not hamper viral activity. We, therefore, conclude that neither retroviral nor adenoviral delivery via the folate endocytosis pathway is compatible with viral gene expression in KB cells.

Adenoviridae↗

PubMed2000

Immunogenicity of whole-cell tumor preparations infected with the ALVAC viral vector.

The immunogenicity of recombinant canarypox (ALVAC) viral vectors within murine whole-cell tumor vaccines was evaluated using the T cell thymic lymphoma STF10 and the B16 melanoma. Tumor cells were modified with the recombinant ALVAC vectors and injected into syngeneic mice. Control mice receiving cells alone all developed tumors, while mice injected with tumor variants bearing parental and recombinant vectors either completely rejected their tumors, or exhibited a significant delay in tumor formation. Rechallenge of mice receiving STF10-variant vaccines yielded a protective effect against parental tumor cells only when a modified regimen incorporating two vaccinations was utilized. Notably, the parental ALVAC virus was equivalent to all other recombinant ALVAC viruses in conferring antitumor immunity when using a prime-and-boost protocol. Tumorigenicity experiments in nude mice revealed that the effector mechanism mediating rejection of tumor cells bearing ALVAC vectors is multifactorial, in that the immunogenicity of STF10/ALVAC vaccines is reduced, but not completely abolished in these mice. Finally, in vitro experiments revealed that cytotoxic T cells specific for parental STF10 cells could be generated as a result of in vivo immunization with STF10/ALVAC vaccines.

Animals↗

PubMed2003

Viral vectors for dendritic cell-based immunotherapy.

Dendritic cells (DCs) constitute a specialised system of antigen-presenting cells with a high capacity to induce and to modulate the immune response against microbial, tumour and self-antigens. New techniques to generate large amounts of DCs together with the molecular identification of human tumour-associated antigens (TAA) have opened new ways for antigen-specific cancer immunotherapies. DCs loaded either with TAA-derived MHC class I-specific synthetic peptides or with whole tumour cell preparations have been used in numerous clinical trials evaluating the efficacy of DCs in patients with cancer. However, the disadvantages of DCs pulsed with synthetic peptides from TAA include the uncertainty regarding the longevity of antigen presentation, the restriction by the patient's haplotype and the relatively low number of known MHC class I and in particular of MHC class II helper cell-related epitopes. Whole tumour cell preparations are difficult to standardise, and they depend on the availability of tumour cells. Thus the utilisation of viral vectors genetically modified to express TAA for the ex vivo transduction of DCs is an attractive alternative to achieve a MHC I- and MHC II-restricted presentation of tumoural antigens. To induce protective anti-tumoural immune response an increasing number of modified viral vectors have been used to transduce DCs. Although high transduction efficacies were reported for several viruses, analysis of the interaction of viral vectors with DCs has revealed several viral mechanisms that interfere with main functions of DCs, dampening somewhat the initial optimism in the field of DC transduction. However, promising results with different vectors have been achieved. In this review we summarise available data and discuss advantages and drawbacks of currently available vectors.

Adenoviridae↗

PubMed2003

Immune response following intraocular delivery of recombinant viral vectors.

There has been significant progress in the last few years in demonstrating the utility of recombinant viral vectors in treating a variety of ocular diseases. The field has moved beyond 'proof-of-principle' and, in fact, has entered the phase where some of these vectors/paradigms are being or soon will be evaluated in human clinical trials. For this reason and also, to increase the understanding of immunological effects of transgenes/viral vectors on the eye, it is important to summarize what is known about these effects. Here, the biology of and immune responses to intraocular injection of three different recombinant viral vectors - adenovirus, adeno-associated virus (AAV), and lentivirus - are summarized. Perhaps, in part because of the unique immunological environment of the eye, the immunological effects of these viruses appear to be fairly benign. Nevertheless, a significant cell-mediated immune response can develop after intraocular administration of adenovirus. The magnitude of this response is affected by the nature of the intraocular compartment to which this virus is administered. Neither AAV nor lentivirus, however, elicit a cell-mediated response and are thus promising vectors for treatment of chronic ocular (retinal) diseases.

Adenoviridae↗

PubMed2000

Viral vector delivery in solid-state vehicles: gene expression in a murine prostate cancer model.

BACKGROUND: Although there are increasingly more clinical trials involving gene therapy, efficient gene transfer remains a major hurdle to success. To enhance the efficiency of delivery of viral vectors in gene therapy protocols, we evaluated the effect of various matrices to act as a vehicle for recombinant virus during intratumoral injection. METHODS: The ability of several vehicles (catgut spacer, polyglycolic acid, chromic catgut, and gelatin sponge matrix) to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line RM-1 was studied in vitro and in vivo. ALVAC recombinants encoding the murine cytokines interleukin 2 (IL-2), interleukin 12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha) were used to assess enhancement of antitumor activity after intratumoral inoculation. Confirmatory experiments were conducted by use of another mouse prostate cancer cell line, RM-11, and a mouse bladder cancer cell line, MB-49. All statistical tests were two-sided. RESULTS: The gelatin sponge matrix proved to be the most effective solid-state vehicle for delivering viral vectors to cells in culture. In addition, this matrix statistically significantly enhanced expression of ALVAC-delivered reporter genes in tumor models when compared with fluid-phase delivery of virus (P =.037 for the RM-1 model and P =.03 for the MB-49 model). Statistically significant growth inhibition of established tumors was observed when a combination of the three recombinant ALVAC viruses expressing IL-2, IL-12, and TNF-alpha was delivered with the matrix in comparison with 1) fluid-phase intratumoral injection of the ALVAC recombinants, 2) no treatment, or 3) treatment with parental ALVAC (all P<.05). CONCLUSIONS: Viral vector delivery in a solid-state vehicle resulted in improved recombinant gene expression in vivo and translated to greater inhibition of tumor growth in an immunotherapy protocol for heterotopic tumor nodules. The efficient delivery of reporter genes described herein may prove useful in many solid tumor gene therapy protocols.

Animals↗

PubMed2026

A randomized trial of viral vector and adjuvanted protein HBV therapeutic vaccine in people with chronic hepatitis B on nucleos(t)ide analogs.

BACKGROUND: This study assessed the safety, efficacy, and immunogenicity of a therapeutic immunization strategy aimed at reaching a functional cure for chronic hepatitis B (CHB), relying on a heterologous prime-boost with viral vectors ChAd155-hIi-HBV and MVA-HBV, combined with sequential or concomitant administration of adjuvanted recombinant HBV proteins (HBc-HBs/AS01B). METHODS: This single-blind, randomized, controlled, first-in-human, phase 1/2 trial enrolled adults aged 18-65 years with HBeAg-negative CHB, virally suppressed on nucleos(t)ide analogs (NAs), with HBsAg >50 IU/mL. Participants received NAs and the following regimens of 4 doses (8-week intervals): sequential administration of ChAd155-hIi-HBV, MVA-HBV, and 2 HBc-HBs/AS01B doses; co-administration of ChAd155-hIi-HBV+HBc-HBs/AS01B, followed by 3 co-administered MVA-HBV+HBc-HBs/AS01B doses; 4 HBc-HBs/AS01B doses; 2 placebo doses followed by ChAd155-hIi-HBV and MVA-HBV administered alone or with HBc-HBs/AS01B; or 4 placebo doses. Safety, efficacy (≥1-log decrease in quantitative (q)HBsAg or HBsAg loss 24 weeks post-dose 4 [day (D)337]), antibody, and T-cell responses were evaluated. RESULTS: In all, 134 participants were vaccinated. Grade 3 solicited adverse events (AEs) (median duration: 2-3 days) were more frequent after co-administration (systemic: 59.3%; administration-site: 33.3%) than sequential administration (systemic: 10.3%; administration-site: 12.8%) of high-dose viral vectors and proteins. No vaccine-related or fatal serious AEs were reported. After 4 doses, no participant had HBsAg loss or ≥1-log decrease in qHBsAg (D337 vs. D1). Co-administration induced the strongest anti-HBs response (73.7% achieved anti-HBs ≥10 mIU/mL 2 weeks post-dose 4 vs. 40.0% after sequential administration). Both sequential and co-administration induced HBc-specific CD4+ and CD8+ T-cell responses, with a prime-boost effect of the viral vectors. CONCLUSIONS: Heterologous prime-boost with ChAd155-hIi-HBV and MVA-HBV, combined with sequential or co-administration of HBc-HBs/AS01B, had an acceptable safety profile, were moderately immunogenic, but no participants showed the expected efficacy outcome.

Humans↗

PubMed1999

Viral vectors for gene transfer into antigen presenting cells.

Crucial insights for vaccine development have come from examining how the immune system responds to antimicrobial vaccines, as well as to viral vectors employed for gene therapy. The effectiveness of a vaccine depends upon both the method of antigen delivery and the presentation of antigen to lymphocytes. Much focus has turned to delivering antigens to dendritic cells, to promote clinically beneficial T- and B-cell responses. Recombinant viral vectors represent a powerful vehicle to deliver genes encoding microbial- or tumor-derived antigens to generate clinically beneficial immunity. Dendritic cell-based and viral vector-based vaccines are currently being evaluated in clinical trials as a means of inducing antitumor immunity.

Animals↗

PubMed2005

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank.

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.

Adenoviridae↗

PubMed2003

[Construction of a non-viral vector H1s-EGFc and a preliminary study on its function].

OBJECTIVE: To construct a non-viral vector for targeting cancer gene therapy. METHODS: The coding sequence of H1s-EGFc was inserted into the expression vectors of Pichia pastoris, and the fusion protein was expressed in secretary way. H1s-EGFc was purified by anion exchange chromatography and size exclusion chromatography. H1s-EGFc fusion protein and "killing gene" expression recombinant pKG plasmid DNA were dissolved in serum-free RPMI-1640 culture to produce H1s-EGFc/pKG complex. HeLa cells, an epidermal growth factor receptor (EGFR) highly expressing cell line, and Jurkat cells, an EGFR non-expressing cell line, were cultured and transfected with H1s-EGFc/pKG complex of different concentrations. Trypan blue staining was used to calculate the number of live cells and the killing rate of H1s-EGFc/pKG. RESULTS: H1s-EGFc fusion protein was constructed and expressed with a purity of over 90%. When the concentrations of H1s-EGFc/pKG complex were 3 microg/ml, 6 microg/ml, and 9 microg/ml respectively the killing rates were 30.6%, 36.2%, and 58.1% respectively. CONCLUSION: The fusion protein H1s-EGFc binds functional gene efficiently and targets it into specific cells. It can be used as non-viral vector in target cancer gene therapy.

ErbB Receptors↗

PubMed1999

Chimeric viral vectors--the best of both worlds?

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The vector systems currently available have not allowed the simultaneous provision of both of these goals. Several groups are now developing chimeric viral vector systems that incorporate the favorable attributes of two different viral vectors. These chimeric vectors might allow the goals for specific gene therapy applications to be realized.

Animals↗

PubMedDate not supplied

Effect of protease inhibitors on yield of HSV-1-based viral vectors.

The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors.

Culture Media↗

PubMed2005

Assessment of optimal transduction of primary human skin keratinocytes by viral vectors.

BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes.

Adenoviruses, Canine↗

PubMed2003

Gene transfer and in vivo promoter analysis of the rat progesterone receptor using a herpes simplex virus viral vector.

The progesterone receptor (PR) gene is expressed in cells of the anterior pituitary and hypothalamus, and PR levels are regulated by estrogen (E) in a tissue-specific fashion. To demonstrate that E induces transcription via the PR promoter, and to identify sequences within the PR promoter responsible for tissue-specific and hormonal regulation, we have utilized a defective herpes simplex virus vector for direct gene transfer into the rat pituitary and brain. We designed a viral amplicon expressing the beta-galactosidase gene under the regulation of a 2.1-kb PR promoter fragment to create a defective viral vector for gene transfer into the brain. Following injection of this vector into the pituitary and brain, its pattern of expression and ability to respond to estradiol 3-benzoate (EB) were examined. In the pituitary, lacZ activity was observed in cells of the anterior lobe (AL). However, no activity was seen in the neurointermediate lobe (NIL), demonstrating tissue specific transcriptional regulation. A approximately sixfold increase in cells demonstrating beta-galactosidase activity was observed in the AL following treatment with EB. Likewise, injection of defective viral vector into the hypothalamus followed by treatment with EB resulted in a approximately eightfold increase in cells demonstrating beta-galactosidase activity including the very cell groups responsible for EB-dependent reproductive behavior. In contrast, no vector dependent activity was observed in the caudate nucleus, a tissue with no endogenous expression of PR, despite polymerase chain reaction evidence demonstrating the presence of the vector in this tissue. These results demonstrate that the 2.1-kb PR promoter fragment contains the sequence information required for correct tissue and hormonal regulation of PR.

Animals↗

PubMed2003

Sodium chloride modified silica nanoparticles as a non-viral vector with a high efficiency of DNA transfer into cells.

Development of reliable vectors is a major challenge in gene therapy. Previous gene transfer methods using non-viral vectors, such as liposomes or nanoparticles, have resulted in relatively low levels (35 to approximately 50%) of gene expression. We have developed a silicon nanoparticle (SNAP) system, a novel non-viral vector, for DNA transfer into cells. SNAP was synthesized chemically and modified with sodium chloride or sodium iodide. Electronmicroscopy of SNAP and fluorescence microscopy of fluorescence-labeled SNAP revealed that they were generated uniformly, had diameters of 10-100 nm, and showed a better efficiency (about 70%) of DNA transfection into cells as well as protection of DNA against degradation. The microscopy also demonstrated the adhesion of SNAP with HT1080 cell surface and entry of SNAP into the cells without cytotoxicity. Intravenous and/or intra-abdominal administration of the SNAP to mice revealed the accumulation of SNAP in the cells of the brain, liver, spleen, lung, kidney, intestine, prostate and the testis without any pathological cell changes or mortality, suggesting that they passed through the blood-brain, blood-prostate, and blood-testis barriers. These findings indicate that the SNAP generated has good biological characteristics as a potential promising vector for gene transfer, gene therapy and drug delivery.

Animals↗

PubMed2003

Adeno-associated viral vector gene expression in the adult rat spinal cord following remote vector delivery.

The current investigation tests whether adeno-associated viral vectors (rAAV) undergo remote delivery to the spinal cord via peripheral nerve injection as previously demonstrated with adenoviral vectors. The sciatic nerves of adult rats (n = 10) were injected with either an rAAV (rAAVCMV-lacZ) or adenoviral (AdCMV-lacZ) vector (1.4 x 10(7) particles/ml). After 21 days, the rAAV group demonstrated significantly higher spinal cord viral expression than the adenoviral group (P < 0.024). A second group of rats was injected with rAAV expressing the green fluorescence protein (GFP) reporter gene. GFP was detected 21 days after unilateral sciatic nerve injection in the neurons of the dorsal root ganglion and spinal cord. The codistribution of the viral genome and transgene in CNS neurons was confirmed with in situ hybridization. In summary, rAAV genes are expressed in CNS neurons following peripheral nerve injection at levels exceeding those seen following remote adenovirus injection.

Animals↗

PubMed1999

Viral vectors for veterinary vaccines.

Whatever strategy is adopted for the development of viral vectors for delivery of veterinary vaccines there are several key points to consider: (1) Will the vectored vaccine give a delivery advantage compared to what's already available? (2) Will the vectored vaccine give a manufacturing advantage compared to what's already available? (3) Will the vectored vaccine provide improved safety compared to what's already available? (5) Will the vectored vaccine increase the duration of immunity compared to what's already available? (6) Will the vectored vaccine be more convenient to store compared to what's already available? (7) Is the vectored vaccine compatible with other vaccines? If there is no other alternative available then the answer to these questions is easy. However, if there are alternative vaccines available then the answers to these questions become very important because the answers will determine whether a vectored vaccine is merely a good laboratory idea or a successful vaccine.

Animals↗

PubMed2002

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors.

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Adenoviridae↗

PubMed2000

Replication of the herpes simplex virus type 1 RL1 mutant 1716 in primary neuronal cell cultures--possible relevance to use as a viral vector.

The herpes simplex virus type 1 (HSV-1) RL1 deletion mutant 1716 has properties that make it a promising candidate as a viral vector for gene therapy in the human nervous system. These properties include its ability to spread along neural pathways and establish a latent infection in post-mitotic neurons, while retaining a non-virulent phenotype in vivo and an inability to cause a lytic infection in stationary or fully differentiated cells. In this study, we used viral replication assays and indirect immunofluorescence to investigate the ability of 1716 to bind to, enter, express genes and produce progeny virus in dissociated neuronal cell cultures prepared from rat hippocampal, medial septal and dorsal root ganglion (DRG) tissues and in primary rat astrocyte cultures. Both heterogeneous cultures and those that had been enriched for neurons were employed. Following both low and high multiplicities of virus infection, the behaviour of 1716 was compared with its wild-type parent HSV-1 strain 17 in these cultures. It was found that the growth of 1716 was significantly impaired compared to wild type HSV-1, with these differences being magnified at lower multiplicities of viral infection as well as in neuron-enriched cultures: this impairment is likely to be due to decreased replication, as immunofluorescence assays showed that 1716 bound to, entered and expressed genes in all neuronal cell types and astrocytes with similar efficiency to the wild type virus. This ability of 1716 to enter and express genes in different neuronal populations demonstrates its potential suitability as a viral vector.

Animals↗