Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae

Alvaro Galli; Ilenia Iaia; Maria Serena Milella; Filippo Cipriani; Veronica Della Latta; Mauro Giacca; Lorena Zentilin; Tiziana Cervelli

doi:10.1007/s12033-020-00294-4

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae

Alvaro Galli, Ilenia Iaia, Maria Serena Milella, Filippo Cipriani, Veronica Della Latta, Mauro Giacca, Lorena Zentilin, Tiziana Cervelli^*

^*Corresponding author for this work

Cardiovascular Sciences

Research output: Contribution to journal › Article › peer-review

6 Citations (Scopus)

Abstract

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.

Original language	English
Pages (from-to)	156-165
Number of pages	10
Journal	Molecular Biotechnology
Volume	63
Issue number	2
DOIs	https://doi.org/10.1007/s12033-020-00294-4
Publication status	Published - Feb 2021

Keywords

Adeno-associated virus
Encapsidation
Gene therapy
rAAV
Saccharomyces cerevisiae
Viral genome

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10.1007/s12033-020-00294-4

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@article{f9593f82c86f4c90bea050def2446e38,

title = "Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae",

abstract = "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.",

keywords = "Adeno-associated virus, Encapsidation, Gene therapy, rAAV, Saccharomyces cerevisiae, Viral genome",

author = "Alvaro Galli and Ilenia Iaia and Milella, {Maria Serena} and Filippo Cipriani and {Della Latta}, Veronica and Mauro Giacca and Lorena Zentilin and Tiziana Cervelli",

note = "Funding Information: This work was supported by the Grant 127/16 funded by the “Fondazione Pisa” assigned to AG. We thank Dr Milena Rizzo for helpful suggestions on qPCR. We thanks also Marina Dapas and Michela Zotti of the AVU (AAV vector unit) core facility of ICGEB, Trieste, for AAV production in human cells. Publisher Copyright: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = feb,

doi = "10.1007/s12033-020-00294-4",

language = "English",

volume = "63",

pages = "156--165",

journal = "Molecular Biotechnology",

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AU - Galli, Alvaro

AU - Iaia, Ilenia

AU - Milella, Maria Serena

AU - Cipriani, Filippo

AU - Della Latta, Veronica

AU - Giacca, Mauro

AU - Zentilin, Lorena

AU - Cervelli, Tiziana

N1 - Funding Information: This work was supported by the Grant 127/16 funded by the “Fondazione Pisa” assigned to AG. We thank Dr Milena Rizzo for helpful suggestions on qPCR. We thanks also Marina Dapas and Michela Zotti of the AVU (AAV vector unit) core facility of ICGEB, Trieste, for AAV production in human cells. Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/2

Y1 - 2021/2

N2 - 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.

AB - 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.

KW - Adeno-associated virus

KW - Encapsidation

KW - Gene therapy

KW - rAAV

KW - Saccharomyces cerevisiae

KW - Viral genome

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M3 - Article

AN - SCOPUS:85098560484

SN - 1073-6085

VL - 63

SP - 156

EP - 165

JO - Molecular Biotechnology

JF - Molecular Biotechnology

IS - 2

ER -

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae

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Keywords

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