Vector Strategies Research Articles

Engineering the Cellular Protein Secretory Pathway for Enhancement of Recombinant Tissue Plasminogen Activator Expression in Chinese Hamster Ovary Cells: Effects of CERT and XBP1s Genes

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

The Context of Gene Expression Defines the Immunodominance Hierarchy of Cytomegalovirus Antigens

Natural immunity to CMV dominates the CD4 and CD8 memory compartments of the CMV-seropositive host. This property has been recently exploited for experimental CMV-based vaccine vector strategies, and it has shown promise in animal models of AIDS and Ebola disease. Although it is generally agreed that CMV-based vaccine vectors may induce highly protective and persistent memory T cells, the influence of the gene expression context on Ag-specific T cell memory responses and immune protection induced by CMV vectors is not known. Using murine CMV (MCMV) recombinants expressing a single CD8 T cell epitope from HSV-1 fused to different MCMV genes, we show that magnitude and kinetics of T cell responses induced by CMV are dependent on the gene expression of CMV Ags. Interestingly, the kinetics of the immune response to the HSV-1 epitope was paralleled by a reciprocal depression of immune responses to endogenous MCMV Ags. Infection with a recombinant MCMV inducing a vigorous initial immune response to the recombinant peptide resulted in a depressed early response to endogenous MCMV Ag. Another recombinant virus, which induced a slowly developing "inflationary" T cell response to the HSV-1 peptide, induced weaker long-term responses to endogenous CMV Ags. Importantly, both mutants were able to protect mice from a challenge with HSV-1, mediating strong sterilizing immunity. Our data suggest that the context of gene expression markedly influences the T cell immunodominance hierarchy of CMV Ags, but the immune protection against HSV-1 does not require inflationary CD8 responses against the recombinant CMV-expressed epitope.

A Vector Space Decomposition Based Space Vector PWM Algorithm for a Three-Level Seven-Phase Voltage Source Inverter

This paper presents a novel modulation strategy for a seven-phase three-level inverter, aimed at variable-speed drive applications. Developed space vector pulsewidth modulation (SVPWM) strategy is based on vector space decomposition (VSD) approach and an analysis of multidimensional space vector projections in mutually orthogonal planes. The algorithm represents an extension of a recently developed five-phase three-level SVPWM algorithm and is considerably more complex, due to the existence of three mutually orthogonal planes in a seven-phase system. The difficulties of generalization of space vector strategies, which stem from the nature of the multiphase systems, are highlighted. Increasing the number of phases from five to seven increases the number of switching states from 35 = 243 to 37 = 2187 and the number of orthogonal planes from two to three. This considerably affects the complexity of the offline calculations. However, the final implementation of the algorithm is simple. Developed SVPWM algorithm is compared to an equivalent carrier-based strategy and it is shown that they yield identical performance. The complexity of the algorithms for real-time implementation is compared. Simulation and experimental results, obtained using neutral-point clamped inverter, verify the theoretical developments.

Recent Patents Involving Virus Nucleotide Sequences; Host Defense, RNA Silencing and Expression Vector Strategies

Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.

Gene therapy for pulmonary hypertension: prospects and challenges

Introduction: Recent evidence shows that pulmonary arterial hypertension (PAH) remains a fatal disease despite the introduction of new pharmacological treatments. New options are therefore needed and gene therapy approaches are a rational consideration based on emerging understanding of the genetic basis of PAH.Areas covered: This review briefly discusses the recent developments in clinical management of PAH and the investigation of gene delivery techniques for pulmonary vascular disease from 1997 to 2010, relating this to improved understanding of disease pathogenesis during this period. There is a focus on bone morphogenetic protein receptor type 2, as mutations in this gene are clearly linked to disease pathogenesis and outcomes. The reader will gain insight into the gene vector strategies being used, the target cells and the specific genes being delivered as candidate therapeutic approaches for PAH.Expert opinion: Various genes and strategies for delivery have achieved improvements in PAH in animal models, which is encouraging for the development of this technology for human application. The main limiting factor for clinical progress relates to gene delivery vector technology.

HIV-vaccines: lessons learned and the way forward

Abstract A safe and efficacious preventive HIV vaccine, as part of a comprehensive prevention program, remains among the highest public health priorities. It would be the best tool that could reduce the spread of HIV significantly in the long run. Current AIDS vaccine candidates are unable to induce neutralizing antibodies against primary HIV isolates or only to a very limited and narrow extent, representing a major obstacle in the development of an efficacious HIV vaccine. Clinical efforts have mainly focused on T-cell vaccines such as DNA and various recombinant vectors alone or in prime-boost regimens. The Merck Ad5 vaccine not only failed to show efficacy but also was associated with increased risk of HIV acquisition in vaccinees in a Phase IIb trial. While gp120 alone was not efficacious, the ALVAC prime and gp120 boost regimen showed 31% efficacy in a Phase III trial in Thailand. These contrasting results illustrate the limitations of available laboratory assays to assess the vaccine-induced immune responses and the lack of understanding of immune correlates of protection. Efforts should therefore focus on developing vaccine candidates inducing broadly neutralizing antibodies. Similarly, new vector strategies such as replicating vectors should be explored to induce strong and broad T-cell responses in the systemic and mucosal compartments. Innovation in immune assay development and testing algorithms is critically needed. The standardization of more relevant and predictive non-human primate models for immunogenicity and efficacy studies will contribute to better and faster vaccine assessment. HIV vaccine development requires innovative ideas and a sustained long-term commitment of the scientific community, civil society, politicians, and donors and participants for clinical research.

Yellow fever vectors’ surveillance in three satellite communities of Enugu Municipality, Nigeria

Outbreaks of yellow fever have continued to occur in various parts of Nigeria. Between 1985 and 2000, sporadic outbreaks have plagued some parts of Oyo, Ekiti, Delta, Imo, Anambra, Cross River, Lagos and Benue States of Nigeria. In addition to favourable environmental factors encouraging the development and spread of the viraemia, there is preponderance of Aedes mosquito vector populations, animal reservoir hosts and high number of non-immune human populations, which do not easily render themselves to vector and environmental management strategies. Regular vector surveillance to detect warning signs posed by vector activities and regular immunization of non-immune human populations have remained a reliable method of abating yellow fever outbreaks. In consequence, yellow fever vectors surveillance has been undertaken in three satellite villages (Obe, Nkalagu and Uwani-Uboji) of Enugu Municipality. Four immunized volunteers were used to collect adult vectors, while locally adapted CDC (Centre for Disease Control) ovitraps were used to collect their eggs. Also, house inspection to detect the breeding index of vector populations in and around the houses was undertaken. Results of house inspections in the three communities showed that out of sixty (60) houses visited, 385 containers were found with water, 221 (57.6%) water containers had Aedes larvae with Uwani-Uboji and Obe communities having 83.5% and 53.8% of their water containers with Aedes larvae. For the ovitraps, Nkalagu had an average of 22 eggs per trap. There were high numbers of Aedes africanus adults in the human bait collections at Obe and Uwani-Uboji communities. Aedes albopictus was amongst other mosquito species collected.

Flexible nanovectors

In this paper we show that the control of adhesion in highly flexible (a property that couldbe crucial for smart drug delivery but which is still ignored in the literature) nanovectorscan help in smartly targeting and delivering the drug. The existence of and the conditionsfor activating and controlling a super-adhesive state are addressed. Even if such a statehas never been observed in nanovectors, our calculations, as well as observationsin spiders and geckos, suggest its existence and feasible control. Control of thecompetition between the drag and the adhesive force is exploited to improve thetargeting ability and a hierarchical model is applied to describe a real vasculature.The high flexibility of the nanovector is used to smartly deliver the drug onlyduring adhesion by nanopumping or, as a limiting case, by the new concept of‘adhesion induced nanovector implosion’; a liquid drop analogy is utilized for thecalculations. Fast (pumping) and slow (diffusion) drug deliveries can thus be separatelycontrolled by controlling the size and shape of the nanovector. Multiple stagenanovectors are also briefly discussed, mimicking aerospace vector strategies.

Seasonal Distribution, Biology, and Human Attraction Patterns of Mosquitoes (Diptera: Culicidae) in a Rural Village and Adjacent Forested Site Near Iquitos, Peru

This study was conducted as part of a field-ecology study of arboviral and malarial activity in the Amazon Basin, Loreto Department, Peru, to determine the relative abundance, species diversity, and seasonal and vertical distributions of potential mosquito vectors. Mosquitoes were captured either by volunteers using mouth aspirators while mosquitoes attempted to land on the collectors or in dry ice-baited ABC light traps. Anopheles darlingi, the principal malaria vector in the region, was the most commonly captured anopheline mosquito in Puerto Almendra village (99%) while landing on humans, with a mean of 37.1 mosquitoes captured per 24-h period, representing nearly one half of all mosquitoes collected. An. darlingi human landing activity began shortly after sunset, peaked at 2000-2100 hours, and declined gradually until sunrise. This species readily entered houses, because 51% of the An. darlingi captured by paired collectors, stationed inside and outside houses, were captured indoors. Human landing collections provided a more accurate estimate of human attraction of An. darlingi, capturing 30 times as many as co-located dry ice-baited ABC light traps. In contrast, eight times as many Culex (Melanoconion) species, including known arbovirus vectors, were captured in light traps as by co-located human collectors. Despite being located within 300 m of the village collection site, only a few Anopheles species were captured at the forest collection site, including only 0.1 An darlingi/ 24 h, thus indicating that An. darlingi activity was directly associated with the rural village. These data provide a better understanding of the taxonomy, population density, and seasonal distribution of potential mosquito vectors of disease within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies that target vector populations.

The potential role of fowlpox virus in rational vaccine design

The design of optimal vaccines requires detailed knowledge of how protective immune responses are generated in vivo under normal circumstances. This approach to vaccine development, where the immune correlates of protection are defined and vaccines are designed to elicit the same response, is called rational vaccine design. Poxviruses are attractive candidates for inclusion in such design strategies owing to their large genome, which allows for the inclusion of multiple heterologous genes, including those encoding antigens, co-stimulatory molecules and cytokines. Fowlpox virus, the prototypical member of the Avipoxvirus genus, is particularly suitable, as it is also incapable of replicating in mammalian cells. The potential of recombinant fowlpox virus as a safe vaccine vector is being evaluated currently in a number of clinical trials for diseases, including HIV, malaria and various types of cancer. Despite their promise, intricate details regarding how fowlpox virus interacts with the host immune system have not been resolved. In this review, the issues surrounding the use of fowlpox virus as a vaccine vector and possible strategies for enhancing its efficacy are discussed.

Coexpression of the Uracil Phosphoribosyltransferase Gene with a Chimeric Human Nerve Growth Factor Receptor/Cytosine Deaminase Fusion Gene, Using a Single Retroviral Vector, Augments Cytotoxicity of Transduced Human T Cells Exposed to 5-Fluorocytosine

Donor T lymphocytes genetically engineered to express a "suicide gene" to facilitate negative selection represent a promising strategy for the management of graft-versus-host disease occurring after allogeneic hematopoietic cell transplantation (HCT). For this purpose, the herpes simplex virus thymidine kinase (HSV-tk) gene, although well studied, has limitations. Cytosine deaminase (CD), an alternative gene for negative selection, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by expression of uracil phosphoribosyltransferase (UPRT), which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate. By using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor (NGFR) for positive selection fused to the Saccharomyces cerevisiae CD gene, we investigated strategies to achieve optimal T cell eradication by CD and UPRT expression, utilizing a single retroviral vector. Three vector strategies were compared on the basis of NGFR expression by flow cytometry, western analysis, and enzymatic activity. A construct (NG/CDiU) expressing UPRT and NG/CD, using a bicistronic message, provided the greatest UPRT activity and killing, reducing the lethal dose of 5-FC sufficient to eradicate 90% of cells from 38.7 microg/ml (300 microM) (NG/CD expression alone) to 0.13 microg/ml (1 microM). This approach provides an effective alternative to the HSV-tk system for eradication of donor T lymphocytes after allogeneic HCT.

Comparison of Whole Gene and Whole Virus Scrambled Antigen Approaches for DNA Prime and Fowlpox Virus Boost HIV Type 1 Vaccine Regimens in Macaques

T cell immunity plays a critical role in controlling HIV-1 viremia, and encoding a limited set of HIV-1 genes within DNA and poxvirus vectors can, when used sequentially, induce high levels of T cell immunity in primates. However, a limited breadth of T cell immunity exposes the host to potential infection with either genetically diverse HIV-1 strains or T cell escape variants of HIV-1. In an attempt to induce maximally broad immunity, we examined DNA and recombinant fowlpox virus (rFPV) vaccines encoding all HIV-1 genes derived from a global HIV-1 consensus sequence, but expressed as multiple overlapping scrambled 30-amino acid segments (scrambled antigen vaccines, or SAVINEs). Three groups of seven pigtail macaques were immunized with sets of DNA and rFPV expressing Gag/Pol antigens only, the whole genome SAVINE antigens, or no HIV-1 antigens and T cell immunity was monitored by ELISpot and intracellular cytokine staining. High levels of cross-subtype HIV-specific T cell immunity to Gag were consistently induced in the seven macaques primed with DNA and rFPV vaccines expressing Gag/Pol as intact proteins. It was, however, difficult to repeatedly boost immunity with further rFPV immunizations, presumably reflecting high levels of anti- FPV immunity. Unfortunately, this vaccine study did not consistently achieve a broadened level of T cell immunity to multiple HIV genes utilizing the novel whole-virus SAVINE approach, with only one of seven immunized animals generating broad T cell immunity to multiple HIV-1 proteins. Further refinements are planned with alternative vector strategies to evaluate the potential of the SAVINE technology.

Convexity Analysis of Option Embedded Bonds

Interest rate risk hedge strategies usually assume that term structure of interest rates in moving under a specific way. If a real term structure is moving differently from the assumed term structure movement, the interest rate risk hedge strategies assuming the special term structure movement may incur unexpected large loss for a bond portfolio manager. Hence an interest rate risk hedge strategy which could be effective under various types of term structure movements has been strongly needed by bond portfolio managers. Duration vector strategies have been developed to satisfy this practical need. To allow various types of term structure movements, duration vector strategies assume multi-factor models for the term structure movement. When a duration vector strategy is considered as a generalization of a duration strategy which is a single factor model for the term structure movement, there will be a generalized concept which measures convexity of a bond under the duration vector model. This study identifies the convexity property of an option embedded bond portfolio under ‘key rate duration model‘ which is a kind of duration vector model suggested by Ho (1992).

Seasonal distribution, biology, and human attraction patterns of culicine mosquitoes (Diptera: Culicidae) in a forest near Puerto Almendras, Iquitos, Peru.

This study was conducted as part of a field ecology study of arboviral activity in the Amazon Basin, Peru, to determine the taxonomy, frequency, seasonal, and vertical distributions of potential mosquito vectors. In addition, the relative efficiency of human-landing collections and dry ice-baited Centers for Disease Control (CDC)-type light traps was determined for collecting mosquitoes. A total of 70 species of mosquitoes from 14 genera were collected from June 1996 through December 1997 at a forested site near Puerto Almendras, approximately 20 km west-southwest of Iquitos, Peru. Three species [Psorophora (Janthinosoma) albigenu (Peryassu), Ochlerotatus (Ochlerotatus) fulvus (Wiedemann), and Ochlerotatus (Ochlerotatus) serratus (Theobald)] accounted for 70% of all mosquitoes captured in human-landing collections. Overall, biting activity occurred throughout the 24-h cycle but was higher during the daytime, primarily because of large populations of two day-biting species, Ps. albigenu and Oc. serratus. Oc. fulvus was active throughout the 24-h cycle but was more frequently collected during the evening. Oc. fulvus, Ps. albigenu, Culex (Melanoconion) pedroi Sirivanakarn & Belkin, and a mixture of Culex (Melaonoconion) vomerifer Komp, and Culex (Melanoconion) gnomatos Sallum, Huchings & Ferreira, accounted for 73% of the mosquitoes captured during darkness) by human collectors. In general, Ochlerotatus spp. and Psorophora spp. were more commonly captured in human-landing collections, whereas most Culex spp. were more frequently collected in the dry ice-baited CDC-type light traps. In general, mosquito populations were lowest from June through August when river levels were at their lowest. Two large population peaks occurred in November-December and in February-March as a result of "flood water" mosquito populations (e.g., Ps. albigenu). These data provide a better understanding of the taxonomy, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.

914. Mutant MGMT Lentivirus Co-Transduction with a Marker Lentivirus Efficiently Enriches for Dual-Vector Expressing Cells In Vivo

Dual gene vectors are limited by insert size constraints, reduced titer, and transcriptional interference. Moreover, high multiplicities of infection (MOI) are needed to compensate for limited expression. We evaluated an alternative strategy; co-transduction with separate lentiviral (LV) vectors. We assessed whether two genes in separate LV vectors efficiently transduce cells to produce dual-positive populations. One LV expressed a mutant form of the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT-P140K), and the other LV expressed GFP (both constructs were derived from a 2nd generation SIN vector obtained from D. Kohn). Dual-positive cells were enriched by AGT-P140K mediated resistance to O6-benzylguanine (BG) and BCNU treatment. The two LV vectors were combined at varying ratios and used to transduce K562 cells with the cumulative multiplicity of infection held constant (MOI = 0.5). The level of expression achieved for each gene was proportional to the amount of the specific virus added and the fraction of dual-positive cells was equal to the product of the individual expression percentages (e.g., 9% AGT and 46% GFP yielded 6% dual positive). Equal proportions of the two vectors resulted in the highest level of co-transduction. After BG and BCNU treatment, AGT only and dual positive cells are enriched with equal efficiencies, while GFP only and untransduced cells are lost. Enrichment of co-transduced cells was higher in cultures that were initially transduced with a low AGT:GFP MOI ratio (8.8 fold for 0.05:0.45 vs. 2.5 fold for 0.25:0.25). We next evaluated whether the selective co-transduction strategy could be used to enrich dual positive murine bone marrow progenitors in vivo. 5-FU treated bone marrow cells were co-transduced with LV AGT-P140K and LV GFP (MOI of 10 each) and resulted in 31% AGT and 29% GFP expression ex vivo. These cultures were then transplanted into lethally irradiated recipients. A range of peripheral blood (PB) expression levels (12–33% AGT and 9-31% GFP) was detected and decreased over time in the absence of treatment. Following selection, expression levels increased up to 4 fold for AGT (17% to 75%) and up to 5 fold for GFP (17% to 81%). We then tested whether co-transduction would allow stem cell selection to be coupled to lineage specific GFP expression. A second generation vector (pRRL-GATA-GFP) containing the GATA-1 upstream enhancer (HS2) in place of U3 was previously shown to restrict expression to mature erythroblasts (F. Lotti et al, J Virol. 2002). Donor marrow was co-transduced with LV AGT-P140K and pRRL-GATA-GFP and transplanted into lethally irradiated recipients. After drug treatment, erythroid specific (TER119+) GFP levels were enriched in PB up to 16 fold (3–50%). These data demonstrate that co-transduction with an AGT-P140K and a marker LV can enrich dual expressing cells at limiting MOIs. Co-transduction simplifies the evaluation of in vivo selection by avoiding the constraints of dual gene vector strategies. This method is particularly suited to vector constructs containing complex regulatory sequences or therapeutic applications requiring tissue specific gene expression.

Promoters and control elements: designing expression cassettes for gene therapy.

It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailor-made' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

Vector Systems for the Delivery of Small Interfering RNAs: Managing the RISC

Silence is golden: Recent breakthroughs in the field of RNA interference have made it feasible to engineer DNA vectors capable of selectively silencing the expression of genes in a wide variety of cell types. For example, lentiviral vectors containing an RNA polymerase III promoter such as U6 have been developed for the expression of small RNA hairpins. These RNA hairpin transcripts serve as substrates for the cytoplasmic ribonuclease Dicer, generating small interfering RNAs (siRNAs) capable of inducing RNA interference in association with the RNA-induced silencing complex (RISC). A variety of vector strategies have been employed to achieve siRNA expression and long-term gene suppression. Recent studies have provided new insights into how these vector strategies might be refined to achieve potent and specific gene suppression, while minimizing the risks associated with these approaches.

Digital scalar pulse-width modulation: a simple approach to introduce nonsinusoidal modulating waveforms

The digital scalar pulse-width modulation (DSPWM) gathers the characteristics of simplicity of implementation found in the regular sampling with the flexibility of manipulation of the switching patterns in the space vector modulation (SVPWM). This paper establishes a correlation between the SVPWM and DSPWM techniques. It also shows how to make the DSPWM strategy equivalent to the SVPWM technique without losing its simplicity of implementation. By using such an equivalence concept a microprocessor-based scheme, which uses standard timer circuits and a simple software algorithm, is proposed to implement the DSPWM technique. The introduction of the distribution ratio in this technique, allows the development of a systematic approach for implementing either conventional or any modified vector strategies without changing the modulator scheme. This corresponds to generating any attractive nonsinusoidal modulating signals (NSMS) in the carrier-based modulation techniques. Furthermore, the simple digital blocks can be easily implemented as a specialized integrated circuit. Simulated and experimental results demonstrate the validity of the proposed methods.

Localized adenovirus gene delivery using antiviral IgG complexation.

Gene therapy with viral vectors has progressed to clinical trials. However, the localization of viral vector delivery to diseased target sites remains a challenge. We tested the hypothesis that an adenoviral vector could be successfully delivered by complexation with a specific antibody that is bound to a biodegradable matrix designed for achieving localized gene transduction. We report the first successful delivery system based upon antibody immobilization of virions in a type I collagen-avidin gel using a polyclonal biotinylated IgG specific for the adenovirus hexon. In vitro stability studies demonstrated retention of viral vector activity with antibody-complexed adenovirus collagen gel preparations, in comparison to loss of vector activity from collagen gels prepared with nonspecific biotinylated IgG. Cell culture investigations using this antibody-controlled release system for adenoviral vector transduction of rat aortic smooth muscle cells (A10) demonstrated a significantly more localized reporter expression (beta-galactosidase) compared with non-antibody-complexed controls. Herpes simplex thymidine kinase (HSVtk) adenoviral vectors were immobilized on avidin-collagen gels via this antibody-complexation approach, and ganciclovir was added to rat smooth muscle cells (A10) in culture with the gels. With complexed HSVtk adenovirus, only cells either in contact with the virus-containing gel or within 50 microm were killed. By comparison, at the same adenovirus and ganciclovir dose, non-antibody-complexed HSVtk adenoviral delivery with ganciclovir resulted in the death of virtually all cells. Myocardial gene transfer studies in pigs demonstrated significantly more efficient right ventricular adenoviral GFP expression with anti-hexon antibody-complexed matrix injections, compared with direct vector injections. Thus, our results show that matrix formulations based on antibody-complexation delivery of adenovirus resulted in site-specific localization of transgene expression that enhances the efficiency of therapeutic vector strategies and provides a potent means for localization, to avoid distal side-effects. This approach has therapeutic potential as an implantable preparation that through the means of antibody-complexation, can localize and optimize viral vector gene therapy.

Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.

Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.