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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

Systemic injections of rAAV6-S induce immune response against huG3BP. 1 × 10E11 vg of either rAAV6-S or rAAV6-C or rAAV6-C preincubated with serum huG3BP coding for the MuSEAP under the CMV promoter were injected in the tail vein. The level of anti-huG3BP IgG was determined by enzyme-linked immunosorbent assay 2 weeks after injection.
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fig7: Systemic injections of rAAV6-S induce immune response against huG3BP. 1 × 10E11 vg of either rAAV6-S or rAAV6-C or rAAV6-C preincubated with serum huG3BP coding for the MuSEAP under the CMV promoter were injected in the tail vein. The level of anti-huG3BP IgG was determined by enzyme-linked immunosorbent assay 2 weeks after injection.

Mentions: Human and mouse G3BP proteins have 68% of homology, and it is expected that injection of human protein can induce a humoral response in mice. Indeed, the quantity of huG3BP protein associated with rAAV6-S was able to induce an immune response in mice, while no anti huG3BP antibody formation could be detected when the animals received the rAAV6-C vector. Consistently, addition of serum huG3BP to rAAV6-C confirmed the immunogenicity of huG3BP in the mouse model (Figure 7).


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)

Systemic injections of rAAV6-S induce immune response against huG3BP. 1 × 10E11 vg of either rAAV6-S or rAAV6-C or rAAV6-C preincubated with serum huG3BP coding for the MuSEAP under the CMV promoter were injected in the tail vein. The level of anti-huG3BP IgG was determined by enzyme-linked immunosorbent assay 2 weeks after injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Systemic injections of rAAV6-S induce immune response against huG3BP. 1 × 10E11 vg of either rAAV6-S or rAAV6-C or rAAV6-C preincubated with serum huG3BP coding for the MuSEAP under the CMV promoter were injected in the tail vein. The level of anti-huG3BP IgG was determined by enzyme-linked immunosorbent assay 2 weeks after injection.
Mentions: Human and mouse G3BP proteins have 68% of homology, and it is expected that injection of human protein can induce a humoral response in mice. Indeed, the quantity of huG3BP protein associated with rAAV6-S was able to induce an immune response in mice, while no anti huG3BP antibody formation could be detected when the animals received the rAAV6-C vector. Consistently, addition of serum huG3BP to rAAV6-C confirmed the immunogenicity of huG3BP in the mouse model (Figure 7).

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