Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates
Vectors based on recombinant adeno-associated viruses (rAAV) attract a growing interest for human gene therapy. Recently, it was shown that many rAAV serotypes produced by transient transfection of human embryonic kidney 293 cell line (HEK293) are efficiently released into culture medium and functio...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2014-01-01
|
| Series: | Molecular Therapy: Methods & Clinical Development |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050116300985 |
| _version_ | 1819062808081334272 |
|---|---|
| author | Jerome Denard Christine Jenny Véronique Blouin Philippe Moullier Fedor Svinartchouk |
| author_facet | Jerome Denard Christine Jenny Véronique Blouin Philippe Moullier Fedor Svinartchouk |
| author_sort | Jerome Denard |
| collection | DOAJ |
| description | Vectors based on recombinant adeno-associated viruses (rAAV) attract a growing interest for human gene therapy. Recently, it was shown that many rAAV serotypes produced by transient transfection of human embryonic kidney 293 cell line (HEK293) are efficiently released into culture medium and functionally equivalent to those purified from cell lysates. Here, we report that HEK293 cells produce and secrete Galectin 3-binding protein (huG3BP), a protein that efficiently binds rAAV6 in vivo. Importantly, intracellular G3BP and secreted G3BP have different properties: while the secreted protein had the same electrophoretic mobility as serum huG3BP and interacted with rAAV6, intracellular protein migrated faster and did not bind rAAV6. Consequently, rAAV6 purified from culture medium (secreted, rAAV6-S) was physically associated with huG3BP while rAAV6 harvested from cell lysates (cellular, rAAV6-C) was huG3BP-free. After systemic injections, rAAV6-S bound to huG3BP was 3 times less efficient compared to rAAV6-C and induced an immune response against huG3BP protein. Our findings show that protein content of rAAVs purified from culture medium or from cell lysates can be different and these differences may impact vector efficacy and/or immune response. |
| first_indexed | 2024-12-21T15:04:39Z |
| format | Article |
| id | doaj.art-571489f5df1c4a6fb1aa395317632f9e |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-21T15:04:39Z |
| publishDate | 2014-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-571489f5df1c4a6fb1aa395317632f9e2022-12-21T18:59:28ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012014-01-011C10.1038/mtm.2014.31Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysatesJerome Denard0Christine Jenny1Véronique Blouin2Philippe Moullier3Fedor Svinartchouk4Biomarkers Department, Genethon, 1 bis rue de l'Internationale, Evry, FranceBiomarkers Department, Genethon, 1 bis rue de l'Internationale, Evry, FranceUniversité de Nantes–Inserm UMR649, Nantes, FranceUniversité de Nantes–Inserm UMR649, Nantes, FranceBiomarkers Department, Genethon, 1 bis rue de l'Internationale, Evry, FranceVectors based on recombinant adeno-associated viruses (rAAV) attract a growing interest for human gene therapy. Recently, it was shown that many rAAV serotypes produced by transient transfection of human embryonic kidney 293 cell line (HEK293) are efficiently released into culture medium and functionally equivalent to those purified from cell lysates. Here, we report that HEK293 cells produce and secrete Galectin 3-binding protein (huG3BP), a protein that efficiently binds rAAV6 in vivo. Importantly, intracellular G3BP and secreted G3BP have different properties: while the secreted protein had the same electrophoretic mobility as serum huG3BP and interacted with rAAV6, intracellular protein migrated faster and did not bind rAAV6. Consequently, rAAV6 purified from culture medium (secreted, rAAV6-S) was physically associated with huG3BP while rAAV6 harvested from cell lysates (cellular, rAAV6-C) was huG3BP-free. After systemic injections, rAAV6-S bound to huG3BP was 3 times less efficient compared to rAAV6-C and induced an immune response against huG3BP protein. Our findings show that protein content of rAAVs purified from culture medium or from cell lysates can be different and these differences may impact vector efficacy and/or immune response.http://www.sciencedirect.com/science/article/pii/S2329050116300985 |
| spellingShingle | Jerome Denard Christine Jenny Véronique Blouin Philippe Moullier Fedor Svinartchouk Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates Molecular Therapy: Methods & Clinical Development |
| title | Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates |
| title_full | Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates |
| title_fullStr | Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates |
| title_full_unstemmed | Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates |
| title_short | Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates |
| title_sort | different protein composition and functional properties of adeno associated virus 6 vector manufactured from the culture medium and cell lysates |
| url | http://www.sciencedirect.com/science/article/pii/S2329050116300985 |
| work_keys_str_mv | AT jeromedenard differentproteincompositionandfunctionalpropertiesofadenoassociatedvirus6vectormanufacturedfromtheculturemediumandcelllysates AT christinejenny differentproteincompositionandfunctionalpropertiesofadenoassociatedvirus6vectormanufacturedfromtheculturemediumandcelllysates AT veroniqueblouin differentproteincompositionandfunctionalpropertiesofadenoassociatedvirus6vectormanufacturedfromtheculturemediumandcelllysates AT philippemoullier differentproteincompositionandfunctionalpropertiesofadenoassociatedvirus6vectormanufacturedfromtheculturemediumandcelllysates AT fedorsvinartchouk differentproteincompositionandfunctionalpropertiesofadenoassociatedvirus6vectormanufacturedfromtheculturemediumandcelllysates |