Viral Vector Packaging Services

Obtain reliable and consistent results with AAV packaging services anchored in quality and technical expertise. Using techniques such as qPCR for accurate physical (genome) titer measurement, we can ensure precise and targeted research outcomes. For projects of certain scales, we can deliver within 2-3 weeks and have the capability and capacity to scale our viral vector services for preclinical and clinical AAV manufacturing and packaging. Our highly experienced team of scientists can handle complex projects, including clones with high GC content or highly repetitive sequences. Beyond the standard viral vector packaging, we also offer a range of custom AAV cloning services, making us a comprehensive, one-stop solution for your gene delivery needs.

There are several steps involved in AAV manufacturing, including:

• Plasmid development and production involves cloning the gene of interest (GOI) into an AAV plasmid vector and producing helper plasmids that contain the AAV viral packaging proteins, rep and cap.
• Cell expansion is where the production cells, Human Embryonic Kidney (HEK)293, are cultured and expanded to achieve the necessary cell density for transfection and AAV production.
• Plasmid transfection is the process of introducing plasmid DNA into the host cells, enabling expression of the encoded genes and producing the desired viral vectors.
• Viral vector production involves the entire process of growing host cells, introducing the necessary genetic material, and allowing the cells to produce, assemble, and release viral vectors, which are engineered viruses used to deliver the therapeutic genetic sequences into target cells.
• Viral vector purification is the process of isolating the produced viral vectors from the host cells, cellular debris, and other contaminants to obtain a pure and concentrated viral vector preparation for research or therapeutic applications.
• Viral vector formulation, fill, and finish is typically the last step during AAV manufacturing where the AAV vectors are dispensed into vials or other delivery containers, sealed, labeled, and packaged before QC testing and shipment.
• Quality testing is done when the concentration of AAV particles is determined to ensure the correct available dosage for research or therapeutic applications. This is followed by quality control testing of the final AAV product to confirm safety, titer, and purity.

In viral vector production, a packaging vector serves as the vehicle that actively delivers the necessary genes required for assembling and packaging viral vectors. When producing vectors such as adeno-associated virus (AAV) or lentivirus, a packaging vector acts as plasmid DNA, harboring the vital viral genes that are indispensable for creating viral particles. These embedded genes play pivotal roles in the processes of replication, assembly, and encapsulation of the viral genome into capsids, leading to the formation of infectious viral particles. In the case of AAV, the replication (rep) and capsid (cap) genes are contained within the packaging vector and are fundamental for replication and capsid formation. By providing these essential components, the packaging vector actively facilitates the generation of functional viral vectors for various research and therapeutic applications.

The AAV packaging process begins with the triple transfection method. This method involves co-transfecting the packaging cell line, usually HEK293T, with a three-plasmid system:

• Recombinant AAV plasmid containing the gene of interest
• Plasmid carrying the essential rep and cap genes
• An adenovirus-derived helper plasmid that supplies the necessary genes for replication

This is followed by purifying the AAV particles using IDX gradient ultracentrifugation or chromatography purification. Next, the viral titer is determined using qPCR with quantification based on the inverted terminal repeats (ITRs) present in the viral genome. Finally, after any additional QC tests that might be required, the AAV particles are shipped overnight. Upon arrival, they are ready for immediate use in in vitro or in vivo research.

Viral vectors are delivered by in vivo or ex vivo methods. 

In vivo delivery occurs directly within the organism and involves administering the viral vectors to the target tissues or circulating systems of a living body. This can either be done by localized gene delivery to specific tissues like muscle, tumor, eye, or brain, or intravenously through intravenous (IV) injection.

Ex vivo delivery refers to the process where cells are modified outside the organism before being reintroduced. In this approach, target cells are harvested from the patient or a donor and exposed to the viral vector in a controlled laboratory environment. This exposure allows the vector to enter the cells and deliver the therapeutic gene. Once modified, the cells are expanded in culture and reinfused into the patient. 

The total packaging limit of AAVs is approximately 4.7 kilobases (kb), but the DNA packaging size that can be delivered is about 4.4 kb. This is because the DNA that can be introduced is sandwiched between a pair of symmetrical inverted terminal repeats (ITRs), situated at the genome termini, which are about 0.2-0.3 kb.

If you are using self-complementary AAV, the packaging capacity can be further reduced to ~2.5 kb. This can be especially limiting when working with a large gene of interest. To overcome this packaging constraint, we developed a Cre-dependent trans-splicing approach that allows for reconstituted gene expression from two independent recombinant AAV vectors. However, trans-splicing has an expression efficiency of ~20% when compared to the use of a single recombinant AAV vector.