A Recombinant Baculovirus Efficiently Generates Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae.
ABSTRACT: Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters. The yields of rAAV2, rAAV8, and rAAV9 derived from the BEV-infected Sf9 cells exceeded 105 vector genomes (VG) per cell. The BEV was shown to be stable and showed no apparent decrease of rAAV yield after at least four serial passages. The rAAVs derived from the new Bac system displayed high-quality and high-transduction activity. Additionally, rAAV2 could be efficiently generated from BEV-infected beet armyworm larvae at a per-larvae yield of 2.75 ± 1.66 × 1010 VG. The rAAV2 derived from larvae showed a structure similar to the rAAV2 derived from HEK293 cells, and it also displayed high-transduction activity. In summary, the novel BEV is ideally suitable for large-scale rAAV production. Further, this study exploits a potential cost-efficient platform for rAAV production in insect larvae.
Project description:Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 105 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.
Project description:Production of clinical-grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. Integrated genes remain silent until the cell is infected with a single baculovirus expression vector (BEV). The induction of expression results from a combination of the amplification of integrated resident genes (up to 1,200 copies per cell) and the enhancement of the expression mediated by the immediate-early trans-regulator 1 (IE-1) encoded by BEV. The integration cassette incorporates an IE-1 binding target sequence from wild-type Autographa californica multiple nuclear polyhedrosis virus, a homologous region 2 (hr2). A feed-forward loop is initiated by one of the induced proteins, Rep78, boosting the amplification of the integrated genes. The system was tested for the coordinated expression of 7 proteins required to package recombinant adeno-associated virus (rAAV)2 and rAAV1. The described arrangement provided high levels of Rep and Cap proteins, thus improving rAAV yield by 10-fold as compared with the previously described baculovirus/rAAV production system.
Project description:BACKGROUND:Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. METHODS:We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. RESULTS:There was ~?40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~?4.7?×?1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~?2.0?×?1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. CONCLUSIONS:Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.
Project description:The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1?×?1014 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid ?-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1?×?1015 VG in crude cell harvests and an average of 3.5?×?1014 total VG of purified rAAV9 material, resulting in greater than 1?×?105 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.
Project description:Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
Project description:Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as coreceptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this study, we examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. Mn(++)-induced integrin activation significantly enhanced (~17-fold) the efficiency of rAAV2 transduction, without altering viral binding or endocytosis. rAAV2 subcellular localization studies demonstrated that Mn(++) promotes increased clustering of rAAV2 on integrins and recruitment of intracellular vinculin (an integrin effector) to sites of rAAV2 binding at the cell surface. Focal adhesion kinase (FAK), a downstream effector of integrin signals, was essential for rAAV2/integrin complex internalization and transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and AAV6).
Project description:The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.
Project description:Different strategies are being worked out for engineering the original baculovirus expression vector (BEV) system to produce cost-effective clinical biologics at commercial scale. To date, thousands of highly variable molecules in the form of heterologous proteins, virus-like particles, surface display proteins/antigen carriers, heterologous viral vectors and gene delivery vehicles have been produced using this system. These products are being used in vaccine production, tissue engineering, stem cell transduction, viral vector production, gene therapy, cancer treatment and development of biosensors. Recombinant proteins that are expressed and post-translationally modified using this system are also suitable for functional, crystallographic studies, microarray and drug discovery-based applications. Till now, four BEV-based commercial products (Cervarix®, Provenge®, Glybera® and Flublok®) have been approved for humans, and myriad of others are in different stages of preclinical or clinical trials. Five products (Porcilis® Pesti, BAYOVAC CSF E2®, Circumvent® PCV, Ingelvac CircoFLEX® and Porcilis® PCV) got approval for veterinary use, and many more are in the pipeline. In the present chapter, we have emphasized on both approved and other baculovirus-based products produced in insect cells or larvae that are important from clinical perspective and are being developed as preventive, diagnostic or therapeutic agents. Further, the potential of recombinant adeno-associated virus (rAAV) as gene delivery vector has been described. This system, due to its relatively extended gene expression, lack of pathogenicity and the ability to transduce a wide variety of cells, gained extensive popularity just after the approval of first AAV-based gene therapy drug alipogene tiparvovec (Glybera®). Numerous products based on AAV which are presently in different clinical trials have also been highlighted.
Project description:Viral vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently shown immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a 'ceiling' for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and, more strikingly, only approximately 1 of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy.
Project description:The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.