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Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors.

Dias Florencio G, Precigout G, Beley C, Buclez PO, Garcia L, Benchaouir R - Mol Ther Methods Clin Dev (2015)

Bottom Line: Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity.Moreover, this approach leads to the reduction of the total process duration.This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.

View Article: PubMed Central - PubMed

Affiliation: U1179 UVSQ-INSERM, Université de Versailles Saint-Quentin en Yvelines, UFR des Sciences de la Santé, Equipe Biothérapies des Maladies Neuromusculaires , Montigny-le-Bretonneux, France.

ABSTRACT
Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.

No MeSH data available.


Related in: MedlinePlus

Comparison of in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) recovered from CP, SN, or TC processes. (a) Schematic representation of the in vivo experimental protocol. C57Bl/10 adult mice are intramuscularly injected (Tibialis anterior (TA)) with the same suboptimal doses (1010 vg/TA) of CP, SN, or TC-derived rAAV, and blood is drawn weekly for one month before animal sacrifice and TA muscles recovery. Refer to Materials and Methods for detailed in vivo protocol. (b) The mSEAP revelation on TA muscle cryosections harvested 4 weeks post-transduction. Compared to PBS-injected muscle (bottom right), TC-derived vectors offer widespread TA mSEAP staining (bottom left) at level visually comparable to that of CP or SN vectors (top left and right respectively). Larger sections views are shown at bottom right of each TA muscle cryosections. Scale bar = 2 mm. (c) For more quantitative monitoring, in vivo mSEAP concentration is evaluated in blood-derived sera. From the second week postinjection, the mSEAP serum concentration average appears systematically higher in mice injected with TC-derived rAAV than with injections performed from CP- or SN-derived vectors. A stabilization of the mSEAP concentration is observed from the third week postinjection. (b) and (c) are obtained from mice injected with suboptimal doses of rAAV. Refer to Supplementary Table S3 and Supplementary Figure S1b for more detailed table and pictures from TA muscles injected at higher rAAV doses.
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fig4: Comparison of in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) recovered from CP, SN, or TC processes. (a) Schematic representation of the in vivo experimental protocol. C57Bl/10 adult mice are intramuscularly injected (Tibialis anterior (TA)) with the same suboptimal doses (1010 vg/TA) of CP, SN, or TC-derived rAAV, and blood is drawn weekly for one month before animal sacrifice and TA muscles recovery. Refer to Materials and Methods for detailed in vivo protocol. (b) The mSEAP revelation on TA muscle cryosections harvested 4 weeks post-transduction. Compared to PBS-injected muscle (bottom right), TC-derived vectors offer widespread TA mSEAP staining (bottom left) at level visually comparable to that of CP or SN vectors (top left and right respectively). Larger sections views are shown at bottom right of each TA muscle cryosections. Scale bar = 2 mm. (c) For more quantitative monitoring, in vivo mSEAP concentration is evaluated in blood-derived sera. From the second week postinjection, the mSEAP serum concentration average appears systematically higher in mice injected with TC-derived rAAV than with injections performed from CP- or SN-derived vectors. A stabilization of the mSEAP concentration is observed from the third week postinjection. (b) and (c) are obtained from mice injected with suboptimal doses of rAAV. Refer to Supplementary Table S3 and Supplementary Figure S1b for more detailed table and pictures from TA muscles injected at higher rAAV doses.

Mentions: Beyond the in vitro proof of concept, we decided to evaluate the in vivo functionality of our rAAV vectors. Intramuscular delivery of CP-, SN-, or TC-derived vectors was performed in C57BL/10 adult mice. Two different doses were tested in order to select non saturating mSEAP concentrations and to allow better comparison of transduction efficiency (Figure 4b and Supplementary Figure S1b). Suboptimal and optimal doses were chosen based on previous studies,22 and rAAV2/9-mSEAP vectors were injected in Tibialis anterior (TA) muscles. About 50 µl of blood was weekly withdrawn from the tail vein during 4 weeks (Figure 4a). After sacrifice, cryosections of TA were stained for mSEAP product detection. Microscopic observations show that whatever the purification process used, rAAV vectors transduced the muscle with good efficiency. Nevertheless, we observed that TC-derived vectors gave better spreading of mSEAP-positive fibers and higher staining intensity around the site of injection than CP- and SN-derived vectors (Figure 4b). To confirm the histological data, a quantitative assessment was performed by measuring the kinetics of mSEAP concentration in blood during the 4 weeks postinjection (Figure 4c and Supplementary Table S3). Surprisingly, TC-derived rAAV vectors performed better in vivo mSEAP secretion after intramuscular delivery than those issued from other classical methods. These in vivo results consolidate the in vitro observations and confirm that our new purification method gives more functional rAAV vectors, probably due to better conservation of the vector quality and integrity.


Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors.

Dias Florencio G, Precigout G, Beley C, Buclez PO, Garcia L, Benchaouir R - Mol Ther Methods Clin Dev (2015)

Comparison of in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) recovered from CP, SN, or TC processes. (a) Schematic representation of the in vivo experimental protocol. C57Bl/10 adult mice are intramuscularly injected (Tibialis anterior (TA)) with the same suboptimal doses (1010 vg/TA) of CP, SN, or TC-derived rAAV, and blood is drawn weekly for one month before animal sacrifice and TA muscles recovery. Refer to Materials and Methods for detailed in vivo protocol. (b) The mSEAP revelation on TA muscle cryosections harvested 4 weeks post-transduction. Compared to PBS-injected muscle (bottom right), TC-derived vectors offer widespread TA mSEAP staining (bottom left) at level visually comparable to that of CP or SN vectors (top left and right respectively). Larger sections views are shown at bottom right of each TA muscle cryosections. Scale bar = 2 mm. (c) For more quantitative monitoring, in vivo mSEAP concentration is evaluated in blood-derived sera. From the second week postinjection, the mSEAP serum concentration average appears systematically higher in mice injected with TC-derived rAAV than with injections performed from CP- or SN-derived vectors. A stabilization of the mSEAP concentration is observed from the third week postinjection. (b) and (c) are obtained from mice injected with suboptimal doses of rAAV. Refer to Supplementary Table S3 and Supplementary Figure S1b for more detailed table and pictures from TA muscles injected at higher rAAV doses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4502676&req=5

fig4: Comparison of in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) recovered from CP, SN, or TC processes. (a) Schematic representation of the in vivo experimental protocol. C57Bl/10 adult mice are intramuscularly injected (Tibialis anterior (TA)) with the same suboptimal doses (1010 vg/TA) of CP, SN, or TC-derived rAAV, and blood is drawn weekly for one month before animal sacrifice and TA muscles recovery. Refer to Materials and Methods for detailed in vivo protocol. (b) The mSEAP revelation on TA muscle cryosections harvested 4 weeks post-transduction. Compared to PBS-injected muscle (bottom right), TC-derived vectors offer widespread TA mSEAP staining (bottom left) at level visually comparable to that of CP or SN vectors (top left and right respectively). Larger sections views are shown at bottom right of each TA muscle cryosections. Scale bar = 2 mm. (c) For more quantitative monitoring, in vivo mSEAP concentration is evaluated in blood-derived sera. From the second week postinjection, the mSEAP serum concentration average appears systematically higher in mice injected with TC-derived rAAV than with injections performed from CP- or SN-derived vectors. A stabilization of the mSEAP concentration is observed from the third week postinjection. (b) and (c) are obtained from mice injected with suboptimal doses of rAAV. Refer to Supplementary Table S3 and Supplementary Figure S1b for more detailed table and pictures from TA muscles injected at higher rAAV doses.
Mentions: Beyond the in vitro proof of concept, we decided to evaluate the in vivo functionality of our rAAV vectors. Intramuscular delivery of CP-, SN-, or TC-derived vectors was performed in C57BL/10 adult mice. Two different doses were tested in order to select non saturating mSEAP concentrations and to allow better comparison of transduction efficiency (Figure 4b and Supplementary Figure S1b). Suboptimal and optimal doses were chosen based on previous studies,22 and rAAV2/9-mSEAP vectors were injected in Tibialis anterior (TA) muscles. About 50 µl of blood was weekly withdrawn from the tail vein during 4 weeks (Figure 4a). After sacrifice, cryosections of TA were stained for mSEAP product detection. Microscopic observations show that whatever the purification process used, rAAV vectors transduced the muscle with good efficiency. Nevertheless, we observed that TC-derived vectors gave better spreading of mSEAP-positive fibers and higher staining intensity around the site of injection than CP- and SN-derived vectors (Figure 4b). To confirm the histological data, a quantitative assessment was performed by measuring the kinetics of mSEAP concentration in blood during the 4 weeks postinjection (Figure 4c and Supplementary Table S3). Surprisingly, TC-derived rAAV vectors performed better in vivo mSEAP secretion after intramuscular delivery than those issued from other classical methods. These in vivo results consolidate the in vitro observations and confirm that our new purification method gives more functional rAAV vectors, probably due to better conservation of the vector quality and integrity.

Bottom Line: Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity.Moreover, this approach leads to the reduction of the total process duration.This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.

View Article: PubMed Central - PubMed

Affiliation: U1179 UVSQ-INSERM, Université de Versailles Saint-Quentin en Yvelines, UFR des Sciences de la Santé, Equipe Biothérapies des Maladies Neuromusculaires , Montigny-le-Bretonneux, France.

ABSTRACT
Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.

No MeSH data available.


Related in: MedlinePlus