A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 ampticon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.     

Production of clinical-grade recombinant adeno-associated virus vectors

Recombinant adeno-associated viral (rAAV) vectors are being evaluated in animal models and humans. Pre-clinical data demonstrating vector safety, efficiency and efficacy have been used to initiate human clinical trials. The clinical manufacture of rAAV vectors has supported phase I and phase II trials, showing that adeno-associated virus serotype 2 vectors are safe when administered to humans.     

Design and packaging of adeno-associated virus gene targeting vectors

Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy.     

Self-amplification system for recombinant adeno-associated virus production

A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins.     

Novel strategy for generation and titration of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.     

Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges.

The titer of retroviral vectors can be increased by cocultivation of retrovirus packaging cells that produce a vector with packaging cells having a different host range. Multiple rounds of infection occur in such cultures, producing an amplification of vector copy number and titer. Production of a vector with a very high titer of over 10(10) CFU per ml of conditioned medium has been reported, although replication-competent helper virus was also present. Since helper-free virus is a requirement for many applications of retroviral vectors, we repeated this procedure with a modified vector and achieved a 2- to 10-fold amplification of vector titer in the absence of helper virus, up to 2 x 10(7) CFU/ml. We have also repeated these experiments with the same vector and methods described previously or have assayed virus from the high-titer vector-producing cell line reported previously and observed maximum titers of 10(8) CFU/ml, invariably accompanied by helper virus. Thus, while amplification of vector titer in the absence of helper virus is possible, some unexplained difference in the assays for virus titer must account for our inability to obtain the exceptionally high vector titers that were reported previously.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors

We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A(1) prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.     

Adenovirus and adeno-associated virus vectors

Recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) are among the most extensively used vectors in gene therapy studies to date. These two vectors share some similar features such as a broad host range and ability to infect both proliferating and quiescent cells. However, they also possess their own unique set of properties that render them particularly attractive for gene therapy applications. rAd vectors can accommodate larger inserts, mediate transient but high levels of protein expression, and can be easily produced at high titers. Development of gutted rAd vectors has further increased the cloning capacity of these vectors. The gaining popularity of rAAV use in gene therapy can be attributed to its lack of pathogenicity and added safety due to its replication defectiveness, and its ability to mediate long-term expression in a variety of tissues. Site-specific integration, as occurs with wild-type AAV, will be a unique and valuable feature if incorporated into rAAV vectors, further improving their safety. This paper describes these properties of rAd and rAAV vectors, and discusses further development and vector improvements that continue to extend the utility of these vectors, such as cell retargeting by capsid modification, differential transduction by use of serotypes, and extension of the cloning capacity of rAAV vectors by dual vector heterodimerization.     

High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids

Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.     

Immune responses following salivary gland administration of recombinant adeno-associated virus serotype 2 vectors

BACKGROUND: Gene transfer to salivary glands (SGs) can be accomplished in a minimally invasive manner, resulting in stable, long-term secretion of the transgene product. Therefore, SGs provide a novel target site for several potentially useful clinical gene therapeutics applications. Previous studies have indicated that intravenous, intramuscular and intranasal administration of recombinant adeno-associated virus serotype 2 (rAAV2) vectors induce host immune responses. There are no reported studies on immune responsiveness of rAAV2 vector administration to SGs. MATERIAL AND METHODS: Vectors were administered by retrograde infusion to the SGs of Balb/c mice in various combinations. Thereafter, transgene expression was determined, and evaluations of host innate and adaptive immune responsiveness performed over a 56-day period. RESULTS: Histological examination of SGs from vector-treated mice showed no significant changes in appearance from controls, including the frequency of activated macrophage detection. There were also no differences in salivary flow rates among experimental groups. In vitro stimulation of splenocytes from mice administered rAAV2 showed elevated interferon-gamma levels in culture media. Significant titers of neutralizing antibodies to rAAV2 were detected in serum of mice following rAAV2 vector administration. While SGs could be transduced with low doses of vector it was not possible to repeat the administration and detect transduction with the same serotype at low doses. However, repeat administration was possible with an alternative serotype (rAAV4). CONCLUSIONS: Following a single administration of rAAV2 vectors to SGs there is no significant innate immune response. However, rAAV2 vector administration to SGs results in both cellular and humoral immune responses. The latter may interfere with the efficacy of repeated rAAV2 vector administration.     

Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors.

The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.     

Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands

The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1alpha receptor (FGFR1alpha), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1alpha-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.     

Production and titering of recombinant adeno-associated viral vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.     

Targeted Genome Editing by Recombinant Adeno-Associated Virus （rAAV） Vectors for Generating Genetically Modified Pigs

Recombinant adeno-associated virus(rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer(SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.