AAV Packaging - NHP Grade

In addition to the basic standards, there are also advanced requirements that are not included in the default package and are available upon request with additional cost. These advanced requirements include residual host protein, advanced endotoxin removal, bioburden, mycoplasma contamination, ITR Nanopore sequencing, animal-free production, material archiving, pH (7.5~8.0), residual Kan, USP sterility, dedicated purification resin or filtration membrane, etc.

The combination of these tests allows for a comprehensive assessment of the AAV quality, purity, and safety, ensuring that the AAV produced by PackGene is suitable for use in NHP studies.

Upon receiving the AAV cis plasmid template from our customers, we conduct a double-check of the ITR integrity in addition to verifying the GOI size during clone screening before scaling up the plasmid. After the plasmid preparations are finished, another round of ITR and GOI sequencing is performed to ensure maximum fidelity. You can rest assured that the plasmid we used for NHP grade AAV are of the highest quality and integrity.

You only need to provide 1-4 µg of plasmid. We will handle the plasmid preparation necessary for AAV packaging. You don't need to purchase an additional plasmid prep service unless you wish to receive more plasmid from us. Please note, the timeline in our quote already includes the plasmid preparation.

Research-grade AAV is actually the most common grade used for research and development, while NHP-grade is where we've improved the purification process and more stringent QC test, resulting in higher purity, lower endotoxin, better genome integrity and lower empty capsid rates. Since animal experiments demand higher virus quality and better consistency, we recommend using our NHP-grade AAV for large animal experiments, such as NHP, porcine, canine, etc. Of course, if you're conducting cell experiments and desire higher purity, that's also recommended.

For in vivo imaging, it's generally advised to use vectors with luciferase.

Currently, in vivo imaging primarily utilizes two techniques: bioluminescence and fluorescence. Bioluminescence involves using the luciferase gene to label cells or DNA, while fluorescence employs fluorescent proteins such as GFP, EGFP, RFP, YFP, mCherry, etc., to mark cells or proteins. Bioluminescence offers advantages like straightforward operation, sensitive response, rapid imaging, and clear visualization. However, its drawback lies in its relatively weak signal, necessitating the use of CCD lenses for detection and requiring instruments with high precision. On the contrary, fluorescence allows for the utilization of various proteins for labeling and enables multiplex labeling, making the process relatively straightforward. Nevertheless, nonspecific fluorescence imposes limitations on its sensitivity, necessitating the use of excitation lights of different wavelengths, thereby making precise in vivo quantification challenging. Bioluminescence relies on the interaction with luciferase to emit light, demonstrating high specificity. The red light emitted by luciferase penetrates tissues nearly 100 times more effectively in vivo than the green light emitted by green fluorescent protein, resulting in a higher signal-to-noise ratio. While fluorescent proteins necessitate excitation light to produce reflected light, nonspecific fluorescence from the mouse's fur reduces the signal-to-noise ratio during the detection process. Fluorescent protein detection is more suited to ex vivo detection, whereas luciferase detection is better suited to in vivo detection. Currently, luciferase labeling is more commonly employed. There are two frequently used luciferases: Firefly Luciferase (Fluc) and Renilla Luciferase (Rluc), each utilizing different substrates—D-Luciferin for the former and Coelenterazine for the latter. They emit light of varying colors, with the former emitting light at approximately 560nm and the latter emitting light at approximately 450-480nm. The light emitted by the former penetrates tissues more effectively, while the latter undergoes faster metabolism in vivo compared to the former. Typically, the former is utilized as a reporter gene, although both can be simultaneously employed for dual labeling.

Our AAV products are subjected to standard release testing procedures, including endotoxin assessment using Limulus Amebocyte Lysate (LAL) assay, purity analysis via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and titer determination using quantitative Polymerase Chain Reaction (qPCR) or droplet digital PCR(ddPCR). Moreover, we conduct restriction enzyme digestion for the Gene of Interest (GOI) plasmids utilized in packaging. Different grade AAV may include different QC tests as listed here.

In addition, we offer 40+ analytical tests to measure titer, AAV genome integrity, characterization, purity, aggregation, contamination and safty, including TEM, AUC, TCID50, Nanopore deep sequencing and may others. Please refer to our anlytical tests webpage.

During AAV titer measurement, our instruments are initially calibrated using the AAV standard product ATCC VR-1816™, a globally recognized reference titer verified by 16 laboratories. Subsequently, we employ SYBR Green qPCR methodology to ascertain titers, achieving values of 1E+13GC/ml or higher. This meticulous approach ensures alignment with prevailing academic standards and prevents inaccurate titration results.

Besides qPCR, we have several other methods available for AAV titer determination:

1. 1. Genome titer detection by ddPCR.
   2. Capsid titer detection by ELISA technology.
   3. Infectious titer detection by TCID50.

Adeno-associated viruses (AAV) are single-stranded DNA viruses (ssAAV) that must first undergo a transition from single-stranded genome to transcriptionally active double-stranded form before expression can begin. This process limits gene transduction mediated by AAV vectors and directly affects gene expression efficiency. Self-complementary double-stranded DNA adeno-associated viruses (scAAV), on the other hand, mutate the 3' ITR trs site, forming double-stranded DNA packaged into AAV. They do not require the transition from single-stranded to double-stranded form. In other words, after entering cells, scAAV viruses can express directly and more rapidly, with higher expression levels. The drawback of scAAV is its smaller packaging capacity and the potential to enhance immunogenicity. It is suitable for research requiring faster expression of target genes or stronger expression of genes smaller than 2.2kb.

As of now, nine naturally occuring serotypes of human AAV have been discovered (AAV1/2/3/4/5/6/7/8/9) and widely applied in scientific research. AAV10 and AAV11 were first discovered in non-human primates in 2004, and no cross-reactivity was observed between AAV10, AAV11, and AAV2, making them promising candidate vectors. Subsequently, researchers isolated AAV12 and AAV13 from simian adenovirus, with limited research on these serotypes currently. Based on these wild-type AAVs, researchers have developed many AAV mutants, such as AAV-DJ and the PHP series, through various modification strategies.

Due to differences in the spatial structure of capsid proteins among AAV serotypes, there are significant variations in their recognition and binding to cell surface receptors, leading to tropism of different AAV serotypes for different tissues. When selecting serotypes, experimental purposes can refer to AAV serotypes used in peer-reviewed literature. For example, AAV1 and AAV9 are more commonly used in brain research than other wild-type AAV serotypes, while AAV6 exhibits higher lymphocyte selectivity.

There are also many engineered serotypes that have been modified or engineered to enhance specific properties for gene therapy applications. These modifications can include alterations to the capsid proteins to change tissue tropism, improve transduction efficiency, evade immune responses, or increase payload capacity. Engineered AAV serotypes have been developed through various strategies such as directed evolution, rational design, or hybridization of existing serotypes. These engineered serotypes offer enhanced performance and versatility, making them valuable tools for targeted gene delivery in biomedical research and therapeutic applications.

PackGene offers nearly 100 serotypes for our packaging service to assist your research work.

Additionally, the development of AAV mutant serotypes with more tissue specificity and stronger infectivity is crucial for innovation in AAV-mediated gene delivery. PackGene provides comprehensive AAV serotype engineeringg services to offer you a one-stop solution.

However, despite the tissue tropism of wild-type AAVs to some extent, the infection of non-target tissues cannot be completely avoided. In such cases, combining tissue- or cell-specific promoters with serotypes can greatly enhance AAV specificity. PackGene offers various tissue-specific promoters, such as the muscle-specific promoter MHCK7-2 and the liver-specific promoter TBG669. Our piVector Design embed in our online ordering system offers various promoters including universal and tissue specific promoters. You may easily build your vector into our AAV backbones that have been rigorously verified for effective viral packaging.

AAV vectors stand out for their safety, low immunogenicity, ability to transduce non-dividing cells, and potential for long-term gene expression without integrating into the host genome. These features make them particularly attractive for gene therapy applications targeting diseases where long-term expression and safety are paramount.

However, the limited packaging capacity is a constraint when delivering larger genes. In contrast, vectors like adenovirus and HSV can carry larger genetic payloads but come with higher immunogenicity and safety concerns. Lentiviral and retroviral vectors offer stable, long-term expression through genome integration but carry risks associated with insertional mutagenesis.

By leveraging the unique advantages of AAV, such as tissue-specific targeting through various serotypes and a favorable safety profile, therapies can be designed for a range of genetic disorders with minimized risks. These characteristics contribute to the growing preference for AAV vectors in both research and clinical gene therapy programs.

Serotype selection: If you are unsure which AAV serotype is most suitable for your experiments, we advise that you test the infection rates of 3 or more different serotypes in your experimental system with our rAAV fluorescent reporter constructs.

Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments.

Experimental control: We advise the use of a GFP positive control vector of the same serotype and promoter as your experimental vector.