| Autor: |
Galli A; Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy., Iaia I; Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy., Milella MS; Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy., Cipriani F; Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy., Della Latta V; Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy., Giacca M; School of Cardiovascular Medicine & Sciences, British Heart Foundation Centre of Research Excellence, King's College London, SE5 9NU London, United Kingdom.; Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.; Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy., Zentilin L; Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy., Cervelli T; Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy. tizicerv@ifc.cnr.it. |
| Abstrakt: |
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 10 3 and 10 5 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. |
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