Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 10³ and 10⁵ viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

腺相关病毒 (AAV) 是一种小型无包膜病毒，用作基因治疗中的载体，主要在人体细胞和杆状病毒系统中产生。对这些平台的深入研究导致产生了每个细胞的病毒基因组 (vg)在 10 ³和 10 ⁵之间的滴度的载体。尽管如此，仍需提高载体产量以满足临床试验和未来商业化的高产品需求。我们的研究和其他小组的研究探索了利用酿酒酵母的可能性生产 rAAV。我们之前已经证明酵母支持 AAV 基因组复制和衣壳组装。本研究的目的是评估封装的 AAV DNA 的质量。这里，我们报告的酵母菌株的从一个整合拷贝表达Rep68 / 40的结构代表的酵母组成ADH启动子的控制和衣壳蛋白：从下基因第受诱导型 GAL 启动子控制的基因。我们的结果表明，该系统产生的一部分 AAV 颗粒含有衣壳化的 AAV DNA。然而，大部分衣壳化 DNA 由转基因盒的片段化区域组成，其中 ITR 是最具代表性的序列。总而言之，这些数据表明，在酵母中，衣壳化的效率很低，并且 rAAV 类似于临床级 rAAV 制剂中存在的假载体。