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Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates.

Denard J, Jenny C, Blouin V, Moullier P, Svinartchouk F - Mol Ther Methods Clin Dev (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Biomarkers Department, Genethon, 1 bis rue de l'Internationale , Evry, France.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Interaction of secreted and intracellular G3PB with rAAV6. Interactions of secreted (G3BP-S) and intracellular (G3BP-C) G3BP with rAAV6 were assessed by coimmunoprecipitation assay with the vector immobilized on AVB-Sepharose beads. M, molecular weight markers; Beads-AAV6 or beads alone, immobilized rAAV6 or beads alone incubated respectively with human serum (HS), G3BP-S or G3BP-C. The same quantity of G3BP (70 ng) was used in each coimmunoprecipitation assay. Reference standard huG3BP: 100 ng of glycosylated human protein. Rec-huG3BP: 100 ng of nonglycosylated human protein. Upper panel: Coomassie staining; lower panel: corresponding western blot analysis of hu-G3BP using anti-huG3BP antibodies. Positions of G3BP and viral capsid proteins are indicated by arrows.
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fig3: Interaction of secreted and intracellular G3PB with rAAV6. Interactions of secreted (G3BP-S) and intracellular (G3BP-C) G3BP with rAAV6 were assessed by coimmunoprecipitation assay with the vector immobilized on AVB-Sepharose beads. M, molecular weight markers; Beads-AAV6 or beads alone, immobilized rAAV6 or beads alone incubated respectively with human serum (HS), G3BP-S or G3BP-C. The same quantity of G3BP (70 ng) was used in each coimmunoprecipitation assay. Reference standard huG3BP: 100 ng of glycosylated human protein. Rec-huG3BP: 100 ng of nonglycosylated human protein. Upper panel: Coomassie staining; lower panel: corresponding western blot analysis of hu-G3BP using anti-huG3BP antibodies. Positions of G3BP and viral capsid proteins are indicated by arrows.

Mentions: Using human recombinant G3BP as a reference standard, the quantities of secreted (huG3BP-S) and intracellular (huG3BP-C) proteins produced by HEK293 cells in one T75 flask were estimated as 10E13 and 2 × 10E12 molecules respectively. Taking into account that vector yield from a T75 flask does not exceed 1 × 10E12 particles (our unpublished data) and,5 it was surprising that no G3BP protein was detected in rAAV6-C harvested from cell lysates. To explain the absence of G3BP in rAAV6-C, we suggested that huG3BP-C and huG3BP-S bind rAAV6 differently. The results of a coimmunoprecipitation assay (Figure 3) fully validated this hypothesis: while huG3BP-S, similar to huG3BP from serum, was able to bind rAAV6, no binding was detected for huG3BP-C (Figure 3).


Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates.

Denard J, Jenny C, Blouin V, Moullier P, Svinartchouk F - Mol Ther Methods Clin Dev (2014)

Interaction of secreted and intracellular G3PB with rAAV6. Interactions of secreted (G3BP-S) and intracellular (G3BP-C) G3BP with rAAV6 were assessed by coimmunoprecipitation assay with the vector immobilized on AVB-Sepharose beads. M, molecular weight markers; Beads-AAV6 or beads alone, immobilized rAAV6 or beads alone incubated respectively with human serum (HS), G3BP-S or G3BP-C. The same quantity of G3BP (70 ng) was used in each coimmunoprecipitation assay. Reference standard huG3BP: 100 ng of glycosylated human protein. Rec-huG3BP: 100 ng of nonglycosylated human protein. Upper panel: Coomassie staining; lower panel: corresponding western blot analysis of hu-G3BP using anti-huG3BP antibodies. Positions of G3BP and viral capsid proteins are indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362353&req=5

fig3: Interaction of secreted and intracellular G3PB with rAAV6. Interactions of secreted (G3BP-S) and intracellular (G3BP-C) G3BP with rAAV6 were assessed by coimmunoprecipitation assay with the vector immobilized on AVB-Sepharose beads. M, molecular weight markers; Beads-AAV6 or beads alone, immobilized rAAV6 or beads alone incubated respectively with human serum (HS), G3BP-S or G3BP-C. The same quantity of G3BP (70 ng) was used in each coimmunoprecipitation assay. Reference standard huG3BP: 100 ng of glycosylated human protein. Rec-huG3BP: 100 ng of nonglycosylated human protein. Upper panel: Coomassie staining; lower panel: corresponding western blot analysis of hu-G3BP using anti-huG3BP antibodies. Positions of G3BP and viral capsid proteins are indicated by arrows.
Mentions: Using human recombinant G3BP as a reference standard, the quantities of secreted (huG3BP-S) and intracellular (huG3BP-C) proteins produced by HEK293 cells in one T75 flask were estimated as 10E13 and 2 × 10E12 molecules respectively. Taking into account that vector yield from a T75 flask does not exceed 1 × 10E12 particles (our unpublished data) and,5 it was surprising that no G3BP protein was detected in rAAV6-C harvested from cell lysates. To explain the absence of G3BP in rAAV6-C, we suggested that huG3BP-C and huG3BP-S bind rAAV6 differently. The results of a coimmunoprecipitation assay (Figure 3) fully validated this hypothesis: while huG3BP-S, similar to huG3BP from serum, was able to bind rAAV6, no binding was detected for huG3BP-C (Figure 3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Biomarkers Department, Genethon, 1 bis rue de l'Internationale , Evry, France.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus