Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.

Palmer amaranth is the most problematic weed in agronomic crop production fields in the United States. A Palmer amaranth biotype was not controlled with sequential applications of glyphosate in glyphosateresistant (GR) soybean production field in south-central Nebraska. The seeds of the putative GR Palmer amaranth biotype were collected in the fall of 2015. The objectives of this study were to (1) confirm GR Palmer amaranth and determine the level of resistance in a whole-plant dose-response bioassay, (2) determine the copy number of 5-enolpyruvylshikimate-3-phosphate (EPSPS) gene, the molecular target of glyphosate, and (3) evaluate the response of GR Palmer amaranth biotype to POST corn and soybean herbicides with different modes-of-action. Based on the effective dose required to control 90% of plants (ED 90 ), the putative GR Palmer amaranth biotype was 37-to 40-fold resistant to glyphosate depending on the glyphosate-susceptible (GS) used as a baseline population. EPSPS gene amplification was present in the GR Palmer amaranth biotype with up to 32 to 105 EPSPS copies compared to the known GS biotypes. Response of GR Palmer amaranth to POST corn and soybean herbicides suggest reduced sensitivity to atrazine, hydroxyphenylpyruvate dioxygenase (HPPD)-(mesotrione, tembotrione, and topramezone), acetolactate synthase (ALS)-(halosulfuron-methyl), and protoporphyrinogen oxidase (PPO)-(carfentrazone and lactofen) inhibitors. GR Palmer amaranth was effectively controlled (>90%) with glufosinate applied at 593 g ai ha −1 with ≥95% reduction in biomass. More research is needed to determine whether this biotype exhibits multiple resistant to other group of herbicides and evaluate herbicide programs for effective management in corn and soybean. Nomenclature: 2,4-D; acetochlor; acifluorfen; atrazine; bentazon; bromoxynil; carfentrazone; chlorimuron; dicamba; fluthiacet; fomesafen; glufosinate; glyphosate; halosulfuron; imazamox; imazethapyr; lactofen; mesotrione; S-metolachlor; tembotrione; thiencarbazone; thifensulfuron; topramezone; Palmer amaranth, Amaranthus palmeri S. Wats.; corn, Zea mays L.; soybean, Glycine max (L.) Merr. Key words: EPSPS gene copy number, glyphosate-susceptible, herbicide efficacy, resistance confirmation, resistance management.Amaranthus palmeri es la malezas más problemática en campos de producción de cultivos agronómicos en los Estados Unidos. Un biotipo de A. palmeri no fue controlado con aplicaciones secuenciales de glyphosate en un campo de producción de soja resistente a glyphosate (GR) en el sur central de Nebraska. Las semillas del biotipo putativo GR de A. palmeri fueron colectadas en el otoño de 2015. Los objetivos de este estudio fueron (1) confirmar que A. palmeri es GR y determinar el nivel de resistencia en un bioensayo de respuesta a dosis con plantas completas, (2) determinar el número de copias del gen 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), el objetivo molecular de glyphosate, y (3) evaluar la respuesta del biotipo GR de A. palmeri a herbicidas POST para maíz y so...

Despite numerous advancements in production protocols, manufacturing AAV to meet exceptionally high demand (10 16 –10 17 viral genomes [VGs]) in late clinical stages and for eventual systemic delivery poses significant challenges. Here, we report an efficient, simple, scalable, robust AAV5 production process utilizing the most recent modification of the OneBac platform. An increase in volumetric yield of genomic particles by ∼6-fold and functional particles by ∼20-fold was achieved by operating a high-cell-density process in shake flasks and bioreactors that involves an Sf9-based rep/cap stable cell line grown at a density of about 10 million cells/mL infected with a single baculovirus. The overall volumetric yields of genomic (VG) and bioactive particles (enhanced transducing units [ETUs]) in representative fedbatch bioreactor runs ranged from 2.5 to 3.5 × 10 14 VG/L and from 1 to 2 × 10 11 ETU/L. Analytical ultracentrifugation analyses of affinity-purified AAV vector samples from side-by-side batch and fedbatch production runs showed vector preparations with a full and empty particle distribution of 20%–30% genomic and 70%–80% empty particles. Moreover, the stoichiometric analysis of capsid proteins from fedbatch production in shake flask and bioreactor run samples demonstrated the incorporation of higher VP1 subunits, resulting in better functionality.

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.