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Determination of anti-adeno-associated virus vector neutralizing antibody titer with an in vitro reporter system.

Meliani A, Leborgne C, Triffault S, Jeanson-Leh L, Veron P, Mingozzi F - Hum Gene Ther Methods (2015)

Bottom Line: However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration.In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration.Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples.

View Article: PubMed Central - PubMed

Affiliation: 1 Genethon, Evry 91000, France .

ABSTRACT
Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors.

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Related in: MedlinePlus

Transduction assay for the determination of the optimal MOI to be used in an AAV8 neutralization assay. The 2V6.11 cells were seeded at 2 × 104 cells/well in a 96-well microplate with ponasterone A at 1 μg/ml to induce the expression of the adenoviral gene E4. The next day, the cells were transduced with increasing MOIs of AAV8-luciferase vector and the signal was measured 24 hr later. Results are expressed as mean RLU/sec/well/optical density (Bradford protein assay)±standard deviation (error bars).
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Figure 3: Transduction assay for the determination of the optimal MOI to be used in an AAV8 neutralization assay. The 2V6.11 cells were seeded at 2 × 104 cells/well in a 96-well microplate with ponasterone A at 1 μg/ml to induce the expression of the adenoviral gene E4. The next day, the cells were transduced with increasing MOIs of AAV8-luciferase vector and the signal was measured 24 hr later. Results are expressed as mean RLU/sec/well/optical density (Bradford protein assay)±standard deviation (error bars).

Mentions: Additionally, for every new serotype it is necessary to select an optimal MOI to be used in the NAb assay. In Fig. 3 we determined the optimal MOI to be used in an AAV8 NAb assay. 2V6.11 cells were transduced with an increasing MOIs of AAV8 and the luciferase expression was measured 24 hr later. Based on the absolute luciferase signal and variability measured across wells transduced with the same amount of virus, an MOI of ~200 was chosen for the AAV8 NAb assay. In general, the optimal MOI to be used in the NAb assay should correspond to the lowest amount of virus resulting in a reporter gene signal above background and not saturated; for example, for luciferase a signal of ~104 relative light units (RLU) will allow for efficient detection of the reporter gene signal, well remaining below the signal saturation level (e.g., > 106 RLU).


Determination of anti-adeno-associated virus vector neutralizing antibody titer with an in vitro reporter system.

Meliani A, Leborgne C, Triffault S, Jeanson-Leh L, Veron P, Mingozzi F - Hum Gene Ther Methods (2015)

Transduction assay for the determination of the optimal MOI to be used in an AAV8 neutralization assay. The 2V6.11 cells were seeded at 2 × 104 cells/well in a 96-well microplate with ponasterone A at 1 μg/ml to induce the expression of the adenoviral gene E4. The next day, the cells were transduced with increasing MOIs of AAV8-luciferase vector and the signal was measured 24 hr later. Results are expressed as mean RLU/sec/well/optical density (Bradford protein assay)±standard deviation (error bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403012&req=5

Figure 3: Transduction assay for the determination of the optimal MOI to be used in an AAV8 neutralization assay. The 2V6.11 cells were seeded at 2 × 104 cells/well in a 96-well microplate with ponasterone A at 1 μg/ml to induce the expression of the adenoviral gene E4. The next day, the cells were transduced with increasing MOIs of AAV8-luciferase vector and the signal was measured 24 hr later. Results are expressed as mean RLU/sec/well/optical density (Bradford protein assay)±standard deviation (error bars).
Mentions: Additionally, for every new serotype it is necessary to select an optimal MOI to be used in the NAb assay. In Fig. 3 we determined the optimal MOI to be used in an AAV8 NAb assay. 2V6.11 cells were transduced with an increasing MOIs of AAV8 and the luciferase expression was measured 24 hr later. Based on the absolute luciferase signal and variability measured across wells transduced with the same amount of virus, an MOI of ~200 was chosen for the AAV8 NAb assay. In general, the optimal MOI to be used in the NAb assay should correspond to the lowest amount of virus resulting in a reporter gene signal above background and not saturated; for example, for luciferase a signal of ~104 relative light units (RLU) will allow for efficient detection of the reporter gene signal, well remaining below the signal saturation level (e.g., > 106 RLU).

Bottom Line: However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration.In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration.Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples.

View Article: PubMed Central - PubMed

Affiliation: 1 Genethon, Evry 91000, France .

ABSTRACT
Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors.

Show MeSH
Related in: MedlinePlus