Limits...
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

Most of the rAAV6 particles in rAAV6-S fraction are bound to HuG3BP. rAAV6-C (left panel) or rAAV6-S (right panel) were centrifuged either at 2,000 g for 10 minutes or at 10,000 g for 30 minutes and the supernatant was carefully discarded. The quantities of rAAV6 and G3BP protein before centrifugation (B), in the supernatant (S) and precipitate (P) were estimated by the respective western blot analyses. Vectors were equally recovered in both supernatant and precipitate after rAAV6-C fraction centrifugation. After centrifugation of rAAV6-S fraction at 2,000 g 70% of rAAV6 was found in the pellet fraction and at 10,000 g, the percentage of rAAV6 in the precipitate increased up to 90%. The remaining in the supernatant rAAV6 was captured with AVB beads. Importantly, recovering of the remaining in the supernatant rAAV6 by AVB beads (S+AVB) leaded to the complete clearance of huG3BP protein from the supernatant (Supernatant). The percentage of the precipitated rAAV6 was calculated by using a ratio of VP1 intensities in rAAV6 before centrifugation and in the supernatant. Upper panels: western blot analysis using anti-VP antibodies; lower panels: corresponding western blot analysis of hu-G3BP. Positions of G3BP and viral capsid proteins are indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362353&req=5

fig5: Most of the rAAV6 particles in rAAV6-S fraction are bound to HuG3BP. rAAV6-C (left panel) or rAAV6-S (right panel) were centrifuged either at 2,000 g for 10 minutes or at 10,000 g for 30 minutes and the supernatant was carefully discarded. The quantities of rAAV6 and G3BP protein before centrifugation (B), in the supernatant (S) and precipitate (P) were estimated by the respective western blot analyses. Vectors were equally recovered in both supernatant and precipitate after rAAV6-C fraction centrifugation. After centrifugation of rAAV6-S fraction at 2,000 g 70% of rAAV6 was found in the pellet fraction and at 10,000 g, the percentage of rAAV6 in the precipitate increased up to 90%. The remaining in the supernatant rAAV6 was captured with AVB beads. Importantly, recovering of the remaining in the supernatant rAAV6 by AVB beads (S+AVB) leaded to the complete clearance of huG3BP protein from the supernatant (Supernatant). The percentage of the precipitated rAAV6 was calculated by using a ratio of VP1 intensities in rAAV6 before centrifugation and in the supernatant. Upper panels: western blot analysis using anti-VP antibodies; lower panels: corresponding western blot analysis of hu-G3BP. Positions of G3BP and viral capsid proteins are indicated by arrows.

Mentions: By using known quantities of huG3BP as standard, we identified the ratio (number of rAAV6 physical particles)/(number of huG3BP molecules) in rAAV6-S fraction as 1/5 (Figure 2). This ratio is very close to the previously determined ratio AAV6/huG3BP in the complex (one rAAV-6 particle bound up to six huG3BP monomers),2 suggesting that all or nearly all rAAV6 particles in the secreted fraction are bound to huG3BP. To further approve this statement, we used the propensity of rAAV6/huG3BP aggregates to precipitate after low-speed centrifugation.2 According to this experimental procedure, 60 µl of rAAV6-S or rAAV6-C were centrifuged either at 2,000 g for 10 minutes or at 10,000 g for 30 minutes and the supernatant (50 µl) was carefully discarded. Ten microliter aliquots from the supernatant and precipitate were analyzed by SDS-PAGE. Vectors were equally recovered in both supernatant and precipitate after rAAV6-C fraction centrifugation. After centrifugation of rAAV6-S fraction at 2,000 g, 70% of rAAV6 was found in the pellet fraction and at higher speed (10,000 g) the percentage of rAAV6 in the precipitate increased up to 90% thus indicating that at least 90% of rAAV6 in rAAV6-S fraction are bound to huG3BP (Figure 5). More efficient precipitation of rAAV6/huG3BP complexes at higher speed can be due to different sizes of the complexes, where bigger complexes precipitate first while the smaller need higher speed/more time to be precipitated.


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)

Most of the rAAV6 particles in rAAV6-S fraction are bound to HuG3BP. rAAV6-C (left panel) or rAAV6-S (right panel) were centrifuged either at 2,000 g for 10 minutes or at 10,000 g for 30 minutes and the supernatant was carefully discarded. The quantities of rAAV6 and G3BP protein before centrifugation (B), in the supernatant (S) and precipitate (P) were estimated by the respective western blot analyses. Vectors were equally recovered in both supernatant and precipitate after rAAV6-C fraction centrifugation. After centrifugation of rAAV6-S fraction at 2,000 g 70% of rAAV6 was found in the pellet fraction and at 10,000 g, the percentage of rAAV6 in the precipitate increased up to 90%. The remaining in the supernatant rAAV6 was captured with AVB beads. Importantly, recovering of the remaining in the supernatant rAAV6 by AVB beads (S+AVB) leaded to the complete clearance of huG3BP protein from the supernatant (Supernatant). The percentage of the precipitated rAAV6 was calculated by using a ratio of VP1 intensities in rAAV6 before centrifugation and in the supernatant. Upper panels: western blot analysis using anti-VP antibodies; lower panels: corresponding western blot analysis of hu-G3BP. Positions of G3BP and viral capsid proteins are indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Most of the rAAV6 particles in rAAV6-S fraction are bound to HuG3BP. rAAV6-C (left panel) or rAAV6-S (right panel) were centrifuged either at 2,000 g for 10 minutes or at 10,000 g for 30 minutes and the supernatant was carefully discarded. The quantities of rAAV6 and G3BP protein before centrifugation (B), in the supernatant (S) and precipitate (P) were estimated by the respective western blot analyses. Vectors were equally recovered in both supernatant and precipitate after rAAV6-C fraction centrifugation. After centrifugation of rAAV6-S fraction at 2,000 g 70% of rAAV6 was found in the pellet fraction and at 10,000 g, the percentage of rAAV6 in the precipitate increased up to 90%. The remaining in the supernatant rAAV6 was captured with AVB beads. Importantly, recovering of the remaining in the supernatant rAAV6 by AVB beads (S+AVB) leaded to the complete clearance of huG3BP protein from the supernatant (Supernatant). The percentage of the precipitated rAAV6 was calculated by using a ratio of VP1 intensities in rAAV6 before centrifugation and in the supernatant. Upper panels: western blot analysis using anti-VP antibodies; lower panels: corresponding western blot analysis of hu-G3BP. Positions of G3BP and viral capsid proteins are indicated by arrows.
Mentions: By using known quantities of huG3BP as standard, we identified the ratio (number of rAAV6 physical particles)/(number of huG3BP molecules) in rAAV6-S fraction as 1/5 (Figure 2). This ratio is very close to the previously determined ratio AAV6/huG3BP in the complex (one rAAV-6 particle bound up to six huG3BP monomers),2 suggesting that all or nearly all rAAV6 particles in the secreted fraction are bound to huG3BP. To further approve this statement, we used the propensity of rAAV6/huG3BP aggregates to precipitate after low-speed centrifugation.2 According to this experimental procedure, 60 µl of rAAV6-S or rAAV6-C were centrifuged either at 2,000 g for 10 minutes or at 10,000 g for 30 minutes and the supernatant (50 µl) was carefully discarded. Ten microliter aliquots from the supernatant and precipitate were analyzed by SDS-PAGE. Vectors were equally recovered in both supernatant and precipitate after rAAV6-C fraction centrifugation. After centrifugation of rAAV6-S fraction at 2,000 g, 70% of rAAV6 was found in the pellet fraction and at higher speed (10,000 g) the percentage of rAAV6 in the precipitate increased up to 90% thus indicating that at least 90% of rAAV6 in rAAV6-S fraction are bound to huG3BP (Figure 5). More efficient precipitation of rAAV6/huG3BP complexes at higher speed can be due to different sizes of the complexes, where bigger complexes precipitate first while the smaller need higher speed/more time to be precipitated.

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