Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Chen, Ling; Yin, Zifei; Li, Jun; Zhang, Dongke; Aslanidi, George; Srivastava, Arun

doi:10.1038/mtm.2016.29

Cited by 24 publications

(25 citation statements)

References 45 publications

(49 reference statements)

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“…The rep gene and ITRs are generally derived from AAV2 since they are compatible with the capsid of other serotypes. However, it is possible to obtain better yields by employing ITRs, rep and cap derived from the same serotype .…”

Section: Production Of Aav In Mammalian Cellsmentioning

confidence: 99%

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Robert

Chahal

Audy

et al. 2017

Biotechnology Journal

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Manufacturing practices for recombinant adeno-associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10 -10 vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small-scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol-step gradient whereas large-scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead-end filtration) and chromatography based-methods are used for large-scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.

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Section: Production Of Aav In Mammalian Cellsmentioning

confidence: 99%

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Robert

Chahal

Audy

et al. 2017

Biotechnology Journal

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“…AAV3 vector has been studied less because of its poor transduction efficiency in mouse liver. However, recent studies have indicated that AAV3 efficiently transduced human hepatocytes [30,31]. Therefore, we attempted to make haploid viruses that were produced by VP1/2 from AAV8 or AAV9 and VP3 from AAV3 for enhancing AAV3 transduction.…”

Section: Discussionmentioning

confidence: 99%

Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors

Chai

Zhang

Dobbins

et al. 2019

Viruses

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Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better “communication” between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.

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“…The human hepatoma cell lines of BEL7404 were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum (FBS) [ 13 , 14 ] and incubated under the condition of 5% CO2 humidified atmosphere at 37°C with saturated humidity [ 15 , 16 ].…”

Section: Methodsmentioning

confidence: 99%

Extraction of Amana edulis Induces Liver Cancer Apoptosis

Fan

Hou

Guo

et al. 2018

Evidence-Based Complementary and Alternative Medicine

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HCC is one of the fastest-rising causes of cancer-related death. Novel therapeutic approaches for treatment are warranted. The goal of this study is to find effective components from Chinese herbal medicines, which is an important alternative source of anticancer medicine. To this end, six different herbs were selected from various traditional literatures. Soxhlet extractor was used to distill the strong polar and weak polar components of each herb. The inhibitive effect of each component was determined using liver cancer cells BEL7404. From total of 12 extractions, it was found that the combined crude lysate of Amana edulis from water and ethanol system had the best efficacy. At the concentration of 0.1 mg/mL, this component has the highest inhibition rate up to 70%. To investigate the underlying molecular reasons, we observed that the component can significantly induce the liver cancer cells apoptosis and retard the cell reproduction at G2/M stage. Verification experiments showed that this component also has apparent inhibitive effects on other liver cancer cells, such as HepG2 and Huh7. On the other hand, it has less effectiveness on another cell line HepaRG, which retains many characteristics of primary human hepatocytes. The results suggested that there might be highly efficient antihepatoma ingredient in the water and ethanol extraction of Amana edulis. The pure substances remain to be isolated and further research on their targets is required.

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Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Cited by 24 publications

References 45 publications

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors

Extraction of Amana edulis Induces Liver Cancer Apoptosis

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