AAV Packaging Frequently Asked Questions
BioInnovatise Viral Vector Team
Updated December 23, 2024
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As a viral vector, AAV is in very high demand for numerous reasons. The packaging process for AAV is always customized and executed according to specific research application requirements.
Our viral vector team has successfully produced hundreds of AAV packaging productions for researchers in cell and gene therapy, immunotherapy, and oncology, and along the way, we have addressed many technical questions about AAV packaging. Through these collaborations, we’ve developed extensive expertise in optimizing AAV packaging for diverse experimental needs and therapeutic applications. Here are some of the most common questions we receive from researchers. If you have any questions about your AAV project, please don’t hesitate to contact our viral vector team.
What Is The Best Bacteria For Amplifying Plasmid For AAV Packaging?
Our viral vector team, regardless of the particular vector (adenovirus, AAV, lentivirus, or retrovirus) highly recommends E. coli for amplifying plasmids. Even for mutagenesis and molecular cloning productions that do not require viral vectors, by default we choose E. coli.
The most commonly used E. coli strains for AAV plasmid production are:
DH5α : This strain has low endonuclease activity, prevents recombination, provides high quality plasmid yield, and possess high transformation efficiency.
Stbl3: This strain is particularly important for plasmids containing ITRs because it reduces DNA recombination rates, maintains ITR Stability better than other E. coli strains, and is suitable for AAV helper and packaging plasmid amplification.
Can An AAV Package Micro-Dystrophin Constructs?
Yes! Micro-dystrophin represents the most promising therapeutic approach for Duchenne muscular dystrophy (DMD) using AAV vectors. These engineered constructs typically range from 3.5-4.0 kb and contain the most critical functional domains of dystrophin while fitting within AAV’s packaging capacity. Micro-dystrophin constructs retain essential N-terminal and C-terminal domains, along with carefully selected spectrin-like repeats and the crucial cysteine-rich domain. Several micro-dystrophin gene therapies are currently in clinical trials, demonstrating that while we cannot package the full dystrophin gene, these smaller functional versions can provide therapeutic benefit for DMD patients.
Why Is Stbl3 Specifically Important for ITR-Containing Plasmids?
ITR (Inverted Terminal Repeat) sequences are prone to recombination due to their palindromic nature and secondary structure formation. Standard E. coli strains like DH5α can experience recombination events that delete or rearrange ITR sequences, rendering AAV plasmids non-functional. Stbl3 E. coli strain contains mutations in recA and endA genes that significantly reduce homologous recombination and endonuclease activity. This makes Stbl3 particularly valuable for maintaining the integrity of AAV vector plasmids, helper plasmids, and packaging plasmids during amplification. The strain’s enhanced stability ensures that ITR sequences remain intact and functional throughout the plasmid preparation process, which is critical for successful AAV packaging and vector performance.
Can You Preform AAV Packaging Without WPRE?
Sure! Many AAV vectors have been successfully assembled and effectively and efficiently delivered genetic payloads to target cells. For those who don’t know, WPRE (Woodchuck Hepatitis Post-transcriptional Regulatory Element) is a regulatory element on an AAV transgene that aims to improve the stability and processing of mRNA in the nucleus and increases the efficiency of protein production from the transgene. Altogether these boost transgene expression without taking up more amount of space in the limit cargo capacity of the AAV vector and useful in gene therapy applications with high protein expression is desired.
If you are interested in removing the WPRE element or adding the WPRE element to your AAV transgene, our molecular cloning team can assist.
Can An AAV Package The Entire Dystrophin cDNA?
No, the current AAV packaging capacity (~4.7 kb) is way too limited for the full length cDNA genome which is 14 kb. The two leading workarounds for packaging cDNA in AAV vectors is using fragmented cDNA versions that contain vital cDNA sequences in the packaged vector or using a dual-vector approach which contains larger amounts (yet not the full cDNA sequence) in two AAV packaged vectors.
Should I Outsource AAV Packaging?
Many researchers consider outsourcing AAV packaging instead of using a kit because specialized service providers like our viral vector team offer higher yields, improved purity, and consistent viral titers through optimized protocols and equipment that are difficult to replicate in standard lab settings. Outsourcing also saves valuable time and labor, allowing researchers to focus on experimental design and data analysis rather than the technically demanding and time-consuming steps of viral production and purification. Learn more about choosing an AAV packaging service rather than a kit.
If you have additional AAV packaging questions that were not answered, don’t hesitate to reach out to our viral vector team before getting started on your AAV production.
Learn about our quick turnaround AAV packaging service.
Want to learn more about the latest in AAV based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News are always collecting and publishing the latest information on AAV based research.
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