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Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27.1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application. [ABSTRACT FROM AUTHOR]

Recent advancements have focused on enhancing factor VIII half-life and refining its delivery methods, despite the well-established knowledge that factor VIII deficiency is the main clotting protein lacking in hemophilia. Consequently, both viral and non-viral delivery systems play a crucial role in enhancing the quality of life for hemophilia patients. The utilization of viral vectors and the manipulation of non-viral vectors through targeted delivery are significant advancements in the field of cellular and molecular therapies for hemophilia. These developments contribute to the progression of treatment strategies and hold great promise for improving the overall well-being of individuals with hemophilia. This review study comprehensively explores the application of viral and non-viral vectors in cellular (specifically T cell) and molecular therapy approaches, such as RNA, monoclonal antibody (mAb), and CRISPR therapeutics, with the aim of addressing the challenges in hemophilia treatment. By examining these innovative strategies, the study aims to shed light on potential solutions to enhance the efficacy and outcomes of hemophilia therapy. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 to 10 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization. Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system. [ABSTRACT FROM AUTHOR]

Contrast agents are commonly used in combination with magnetic resonance imaging (MRI) to monitor the distribution of molecules in the brain. Recent experiments conducted in our laboratory have shown that co-infusion of recombinant Adeno-associated virus serotype 5 (rAAV5) and the MRI contrast agent gadoteridol (Gd) enhances vector transduction in the rat striatum. The goal of this study was to determine whether gadoteridol may also be used as a tool to enhance transduction efficiency of rAAV1 and rAAV5 within the rat hippocampus. We show that Gd/rAAV1-GFP but not Gd/rAAV5-GFP co-infusion results in significantly higher distribution of the transgene both in the injected hemisphere as well as in the contralateral side and adjacent areas of cortex along the injection track. We also show that Gd/rAAV1-GFP co-infusion has no deleterious effect on hippocampal function as assessed by two tests of spatial memory formation. This work indicates that Gd can be exploited as a method to increase transduction efficiency of AAV1 in the hippocampus for animal studies. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the kinetics, mechanism and extent of MNP loading into endothelial cells and the effect of this loading on cell function. Methods: MNP uptake was examined under field on/off conditions, utilizing varying magnetite concentration MNPs. MNP-loaded cell viability and functional integrity was assessed using metabolic respiration, cell proliferation and migration assays. Results: MNP uptake in endothelial cells significantly increased under the influence of a magnetic field versus non-magnetic conditions. Larger magnetite density of the MNPs led to a higher MNP internalization by cells under application of a magnetic field without compromising cellular respiration activity. Two-dimensional migration assays at no field showed that higher magnetite loading resulted in greater cell migration rates. In a three-dimensional migration assay under magnetic field, the migration rate of MNP-loaded cells was more than twice that of unloaded cells and was comparable to migration stimulated by a serum gradient. Conclusions: Our results suggest that endothelial cell uptake of MNPs is a force dependent process. The in vitro assays determined that cell health is not adversely affected by high MNP loadings, allowing these highly magnetically responsive cells to be potentially beneficial therapy (gene, drug or cell) delivery systems. [ABSTRACT FROM AUTHOR]

Our recent studies have revealed that among the 10 different commonly used adeno-associated virus (AAV) serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface-exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single mutants in liver cancer cells in vitro. In immunodeficient mouse xenograft models, direct intratumoral injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intratumoral and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing proapoptotic genes should prove useful for the potential gene therapy of human liver cancers. [ABSTRACT FROM AUTHOR]

The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine. [ABSTRACT FROM AUTHOR]

A number of preclinical studies have shown the adeno-associated virus (AAV) to be an efficient vehicle for gene therapy. Clinical studies successfully demonstrated its potential for in vivo gene transfer. The complexity of host–vector interactions when progressing from small to large animal models, and eventually to humans, has impeded translation of AAV technology to the clinic. One approach to address this complexity has been to explore the biological characteristics of variations in AAV capsid structure. Initial strategies characterized the naturally occurring capsid variants from mammalian species. The structural and functional knowledge gathered on these natural AAV variants as vectors has led to the first series of second-generation vectors that aim at specifically improving certain properties by rational design of the capsid. A third exciting approach uses directed evolution to isolate vectors that are able to overcome selective pressures applied in the laboratory and thereby steer the capsid to evolve toward improved functionality.Gene Therapy (2009) 16, 311–319; doi:10.1038/gt.2008.170; published online 4 December 2008 [ABSTRACT FROM AUTHOR]

To gain acceptance as a medical treatment, adeno-associated virus (AAV) vectors require a scalable and economical production method. Recent developments indicate that recombinant AAV (rAAV) production in insect cells is compatible with current good manufacturing practice production on an industrial scale. This platform can fully support development of rAAV therapeutics from tissue culture to small animal models, to large animal models, to toxicology studies, to Phase I clinical trials and beyond. Efforts to characterize, optimize and develop insect cell-based rAAV production have culminated in successful bioreactor-scale production of rAAV, with total yields potentially capable of approaching the ‘exa-(1018) scale.’ These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases such as Duchenne muscular dystrophy, for which large amounts of recombinant vector are essential for successful outcome.Gene Therapy (2008) 15, 823–830; doi:10.1038/gt.2008.61; published online 10 April 2008 [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units. [ABSTRACT FROM AUTHOR]