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首页> 外文学位 >Characterization and optimization of the production of adeno-associated viral vectors using a baculovirus expression vector/insect cell system.
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Characterization and optimization of the production of adeno-associated viral vectors using a baculovirus expression vector/insect cell system.

机译：使用杆状病毒表达载体/昆虫细胞系统表征和优化腺相关病毒载体的生产。

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摘要

Adeno-associated viruses exist as a class of virus that have shown promising characteristics as gene therapy vectors. They have further been shown in some instances to also illicit a strong immune response against more threatening viruses like the human immunodeficiency virus. Although recent improvements in the ability to produce these vectors at a reasonable scale in suspension by transient transfection have been reported, their production in insect cells using baculoviruses still promises to be one of the most efficient and scalable platforms to achieve the goal of producing enough material for clinical trials.;It was also found that using high MOIs of BacRep may be problematic in the long run. Instabilities with this baculovirus construct that are atypical of passaging other recombinant baculoviruses resulted in a significant decrease in expression levels over fewer passages. It has been speculated that the instability ensues from the palindromic sequence resulting front the head-to-head orientation of the Rep genes (Kohlbrenner et al., 2005a). The use of low passage BacRep minimizes this effect. An alternate four-baculovirus system developed by Kohlbrenner et al. (2005a) increases the stability by separating the two Rep genes into two baculoviruses. Unlike the triple infection system, it was found, in this work, that capsids are likely the limiting factor when using the quadruple infection system.;The total number of capsids produced always significantly exceeded the number of functional AAV vectors and vector genomes. This implied that there was a potential to increase the efficiency of genome encapsidation. To try and address this limitation using culture parameters, the temperature of the process was modulated. Increasing the temperature during the production phase of the process from 27°C to 30°C increased replication proteins early in the culture, which is thought to be a key reason for the observed increase in SS progeny DNA, ∼ two-fold increase in DNAse resistant particles and ∼ three-fold increase in functional vectors produced. The most significant replication protein increase was Rep78, which inay suggest that the attenuated expression of the Rep78, designed because of its detrimental effects in mammalian cells, may be over-attenuated in the insect cell system.;To fully exploit the insect cell system, the maximum density at which AAV could be produced was investigated. Without adding nutrients during the culture, the synthesis per cell significantly decreased beyond 4x10 6 cells/ml. Medium renewal at the time of infection allowed production at ∼ 1x107 cells/ml although the synthesis per cell was only a fraction of the maximum achieved at lower cell densities. Supplementing the media using a nutrient cocktail increased the specific production but did not achieve the maximum level seen at lower cell densities. Resuspending cells from the early exponential phase, in fresh media, to higher cell densities, maintained specific production levels up to ∼ 1x107 cells/ml. Cell "age" therefore played a role in achieving the highest production levels. The strategies developed in this work yield ∼6.5x1012 functional AAV particles/L of cell culture. (Abstract shortened by UMI.);To optimize the cellular and volumetric production a detailed characterization of the system was undertaken. To gauge the effect of each baculovirus vector used for the production of AAV vectors, the quantity of each, the ratio between them and the time at which they were added, were investigated. The highest titers were achieved when BacRep and BacCap were added at high multiplicities of infection. Manipulating expression levels with the multiplicity of infection is difficult since there is inherent competition between the baculoviruses. This is believed to be the reason why offsetting the ratio of BacRep to BacCap always led to lower production of AAV vectors. Furthermore, the highest AAV production was achieved when all three baculoviruses infected the cell at relatively the same time, otherwise a significant reduction occurred. To minimize the amount of total baculovirus added to the system, BacITR, which only supplies the AAV vector genome, could be added at lower multiplicities of infection without significantly affecting the production of AAV vectors.
机译：腺伴随病毒作为一类病毒而存在，已作为基因治疗载体表现出令人鼓舞的特性。在某些情况下，它们还被证明对诸如人类免疫缺陷病毒等更具威胁性的病毒也具有强大的免疫反应。尽管最近报道了通过瞬时转染以合理规模悬浮生产这些载体的能力的最新改进，但是使用杆状病毒在昆虫细胞中生产它们仍然有望成为实现生产足够材料的目标的最有效和可扩展的平台之一用于临床试验。还发现，从长远来看，使用高BacRep的MOI可能会有问题。这种杆状病毒构建体的不典型性是传代其他重组杆状病毒的不稳定性，导致在较少的传代中表达水平显着降低。据推测，回文序列导致不稳定性，从而导致Rep基因从头到头的定向（Kohlbrenner等，2005a）。低通量BacRep的使用可最大程度地减少这种影响。 Kohlbrenner等人开发的备用四杆状病毒系统。 （2005a）通过将两个Rep基因分成两个杆状病毒来增加稳定性。与三重感染系统不同，在这项工作中发现，衣壳可能是使用四重感染系统的限制因素。产生的衣壳总数始终明显超过功能性AAV载体和载体基因组的数目。这暗示着有可能提高基因组壳体化的效率。为了尝试使用培养参数解决此限制，对过程温度进行了调节。在该过程的生产阶段将温度从27°C升高到30°C，会使培养初期的复制蛋白增加，这被认为是观察到的SS后代DNA增加，DNAse增加约两倍的主要原因。抗性颗粒和产生的功能载体增加约三倍。复制蛋白增加最明显的是Rep78，这表明由于昆虫细胞系统中的有害表达而设计的Rep78的减毒表达可能在昆虫细胞系统中被过度减毒。要充分利用昆虫细胞系统，研究了可以生产AAV的最大密度。在培养期间不添加营养的情况下，每个细胞的合成量显着下降，超过4x10 6细胞/ ml。感染时的培养基更新允许产生约1x107细胞/ ml的产量，尽管每个细胞的合成只是在较低细胞密度下获得的最大值的一小部分。使用营养混合物补充培养基可增加单位产量，但未达到较低细胞密度时的最高水平。在新鲜培养基中，从早期指数阶段重悬细胞到更高的细胞密度，可以维持高达1x107个细胞/ ml的比生产水平。因此，细胞“年龄”在实现最高产量方面发挥了作用。这项工作中开发的策略可产生约6.5x1012个功能性AAV颗粒/ L细胞培养物。 （摘要由UMI缩短。）；为了优化细胞和体积的生产，对系统进行了详细的表征。为了评估用于生产AAV载体的每种杆状病毒载体的效果，研究了每种杆状病毒的数量，它们之间的比例以及添加时间。在高感染复数下添加BacRep和BacCap时，可达到最高滴度。由于杆状病毒之间存在内在竞争，因此难以控制具有多种感染的表达水平。认为这是为什么抵消BacRep与BacCap的比率总是导致AAV载体产量降低的原因。此外，当所有三种杆状病毒相对同时感染细胞时，AAV产量最高。为了最大程度地减少添加到系统中的杆状病毒总量，可以以较低的感染复数添加仅提供AAV载体基因组的BacITR，而不会显着影响AAV载体的生产。

著录项

  • 作者

    Aucoin, Marc Gordon.;

  • 作者单位

    Ecole Polytechnique, Montreal (Canada).;

  • 授予单位 Ecole Polytechnique, Montreal (Canada).;
  • 学科 Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 279 p.
  • 总页数 279
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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