Adeno-associated virus (AAV) is a single-stranded linear DNA-deficient virus. By engineering AAV, scientists have created recombinant AAV vectors (rAAV) suitable for cell transfection, which are characterized by low immunogenicity, strong targeting, wide host range and long-term stable expression of foreign genes. Thus,they have been widely used in in vivo gene manipulation experiments for gene function research.
In recent years, rAAV has become the most important delivery vector of gene therapy. The production and preparation of rAAV is a key and difficult point in gene therapy, because the packaging system and purification method of rAAV will influence the infection efficiency and the expression efficiency of the target gene, and ultimately affect the results. Therefore, the quality inspection of rAAV is an essential step. The following article will introduce the necessary quality control (QC) inspection items for rAAV after packaging and production.
Production System of rAAV
The packaging and production of rAAV generally uses a three-plasmid co-transfection system, which can be roughly divided into four steps: preparation of the target sequence and cloning into the vector, virus packaging, collection and purification, and quality inspection.
Figure 1. Production flow of rAAV.
Titer detection refers to the detection of the gene copy number of rAAV, and the total number of genome copies contained in the virus particles per milliliter of virus liquid is calculated. There are a variety of detection methods, including DNA dot-blotting, ELISA, qPCR and ddPCR, etc., usually using absolute quantification. Firstly, the linear relationship between standard DNA content and CT value is obtained by establishing the standard curve. On this basis, by detecting the CT value of the rAAV, the virus titer can be obtained.
AAV genome identification is relatively simple, as it only requires extraction of the rAAV genome to perform AGAR gel electrophoresis. Since we already know the target sequence and the size of the entire rAAV genome of the ITRs at both ends, we can tell whether the rAAV contains our target fragment from the electrophoresis results, and whether there is an incorrect nucleic acid sequence or impurities.
Taking Cyagen’s data as an example, we packaged multiple rAAV2 and rAAV8 containing different target sequences. After extracting the rAAV virus genome, agarose gel electrophoresis detection showed that the genome sizes of various combinations were in line with expectations, and there were no other hetero-bands, which indicated that the size and purity of rAAV genome were qualified.
Figure 2. Detection of viral genome size and purity. (Source: Cyagen)
Line1: rAAV2-CMV-EGFP (20220217)1E+10(ITR titer) 2623bp
Line2: rAAV8-CMV-luc(C+S) (20220225)1E+10(ITR titer) 3464bp
Line3: rAAV8-CMV-EGFP(C+S) (20220225)1E+10(ITR titer) 2623p
Line4: rAAV2-IRES-hrGFP (20210907)1E+10(ITR titer) 3544bp
Line5: rAAV2-CMV-EGFP (20220217)2E+10(ITR titer) 2623bp
Line6: rAAV8-CMV-luc(C+S) (20220225)2E+10(ITR titer) 3464bp
Line7: rAAV8-CMV-EGFP(C+S) (20220225)2E+10(ITR titer) 2623bp
Line8: rAAV2-IRES-hrGFP (20210907)2E+10(ITR titer) 3544bp
Since rAAV capsid protein determines the infectivity of rAAV, it is necessary to detect the purity of the capsid protein after the detection of rAAV genome. Commercial protein extraction kits are recommended for rAAV capsid protein extraction, followed by sdS-PAGE staining/silver staining assay for rAAV capsid protein purity. The molecular weights of the capsids VP1, VP2, and VP3 of high-quality rAAV were 87KD, 72KD, and 62KD, respectively, and the number ratio was about 1:1:10.
Taking Cyagen’s data as an example, we packaged multiple rAAV8 and rAAV9 containing different target sequences. After extraction of different combinations of rAAV capsid proteins, coomassie blue staining and silver staining were performed, respectively. It could be seen from the results that the sizes of rAAV capsid proteins in different combinations were in line with expectations, and the band brightness of VP1, VP2, and VP3 also conformed to the 1:1:10 ratio. In addition, there is no hetero-band, so this result can indicate that the purity of the packaged rAAV capsid protein is relatively high, which indirectly indicates that each group of rAAV should have higher infectious activity.
Figure 3. Detection of rAAV capsid protein. (Source: Cyagen)
SDS-PAGE Coomassie Blue Staining: Lane1: rAAV9-CMV-EGFP, 3E11vg; Lane2: rAAV9-CMV-Luc, 3E11vg; Lane3: rAAV8-CMV-EGFP, 3E11vg; Lane4: rAAV8-CMV-Luc, 3E11vg; Lane5: rAAV9-CMV-EGFP, 1.5E11vg.
SDS-PAGE Silver Staining: Lane1: rAAV2-IRES-hrGFP, 9E10vg; Lane2: rAAV2-IRES-hrGFP, 4.5E10vg; Lane3: rAAV2-IRES-hrGFP, 1.5E10vg.
Biological activity detection refers to using rAAV to infect cells such as 293T to detect the infection efficiency of rAAV. The commonly used detection method is to package a fluorescent protein in rAAV and directly indicate the infection efficiency of rAAV by fluorescence brightness after transfection of 293T cells. In addition, more rigorous data can be obtained through Flow Cytometry（FC）analysis.
Again taking Cyagen’s data as an example, we infected 293T cells with different serotypes and concentrations of rAAV, and evaluated the infection efficiency of rAAV by observing fluorescence and FCM. In the observation of fluorescence intensity, rAAV2, rAAV8 and rAAV9 infected 293T cells with MOI=1E5, and a strong fluorescence signal could be observed 48h later.
Figure 4. Biological activity of 293T cells infected with rAAV for 48h. (Source: Cyagen)
Flow cytometry showed that rAAV2, rAAV8 and rAAV9 infected 293T cells with MOI=1E5, 1E4, 1E3 and 1E2, respectively. The GFP positive rate of raAV2-MOI=1E5 reached 99.66%, and other combinations also had a higher GFP positive rate. The results showed that the rAAV had strong infectivity.
Figure 5. Flow cytometry of 293T cells infected with rAAV. (Source: Cyagen)
Why Choose Cyagen?
The consistent quality of rAAV has a great impact on all subsequent research. In order to ensure that the follow-up experiments can be conducted smoothly, it is necessary to strictly control the quality inspection of rAAV production for high-quality research results.
As a comprehensive solution provider, Cyagen has established a fully-integrated innovative CRO platform providing viral vector design and model development services for gene therapy research. In the past ten years, we have accumulated a large amount of genetic information. The continuous in-depth cultivation of model animals has also allowed us to be at the forefront of the industry in gene editing technology. Combined with Cyagen’s exploration in artificial intelligence (AI), we can provide efficient one-stop solutions for gene function analysis and gene therapy, including: design and packaging of viral vectors such as AAV, LV, and ADV, target screening and functional studies, animal model construction, phenotypic analysis, and more.