AAV Production

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AAV Production

Introduction To AAV

Adeno-associated virus (AAV) is a small single-strand DNA virus that infects humans and certain other primates. At the moment, there is no evidence that AAV causes illness and only generates extremely weak immune responses. As a member of the Parvoviridae family, wild-type AAV requires the help of adenovirus or herpes virus to replicate, thus the name adeno-associated virus. The wild-type AAV2 genome is composed of the viral rep and cap genes (encoding replication and capsid genes, respectively), which are flanked by inverted terminal repeats (ITRs) containing all the essential cis-acting elements for replication and packing. A typical AAV2 genome is approximately 4800bp in length and is composed of two upstream and downstream open read frames (ORFs) sandwiched between two inverted terminal repeats (ITRs) encoding Rep and Cap. ITR is necessary for the production of the complementary DNA strand, whereas Rep and Cap can be translated into a variety of proteins, including the critical AAV viral cycle proteins Rep78, Rep68, Rep52, and Rep40, as well as the enveloped proteins VP1, VP2, and VP3.

Currently, 12 serotypes of AAV have been characterized, each with its own set of characteristics and tropisms. AAV is considered to be the finest gene delivery method for in vivo gene function study due to its high safety, minimal immunogenicity, and long-term expression of foreign genes.



History Of AAV

AAV was discovered in 1969 to have numerous benefits in experimental systems, including a short DNA genome of roughly 5 kb, the packing of plus and minus strands into discrete particles, and, most significantly, the presence of a faulty virus. Its genomic structure, development cycle, and latency were all documented over the first two decades following its discovery. Srivastava and colleagues completed the genomic sequencing of AAV serotype 2 (AAV2) in the early 1980s. By the mid-1980s, this enabled the production of the first recombinant AAV vectors utilizing AAV2. Following that, experiments utilizing AAV for gene transfer in mammalian cell cultures were conducted. Following that, proof of clinical safety prompted researchers to begin using AAV vectors in clinical studies for a variety of hereditary diseases.

How AAV Mechanism Works ?

AAV enters cells mostly by endocytosis via clathrin-coated pits, but other minor processes may be implicated. These potential small processes, however, remain unconfirmed. AAV activates intracellular signaling pathways upon attaching to its cell surface receptors, which in turn increases AAV internalization. This behavior is easily explained by the AAV2 host cell contact mechanisms. Within 5 minutes of AAV2 infection, studies have demonstrated that AAV2 attachment to HSPG and V5 integrin activated Rac1, an intracellular small guanosine triphosphate (GTP)-binding protein, and phosphoinositide 3-kinase (PI3K) in HeLa cells. Furthermore, silencing Notch1, a transmembrane receptor is known to be involved in the activation of Rac1 and PI3K, has been shown to reduce AAV2 cell transduction, suggesting that the Rac1-PI3K pathway is required for AAV2 endocytosis. Direct injection of AAV into the cytoplasm and nucleus of cells resulted in a significantly lower infection rate than cells that are just exposed to the virus, suggesting that endosomal processing of the AAV virion is a key starting step for transduction following endocytosis.

The effectiveness of AAV transduction is highly reliant on the endosomal pH. Increasing the pH of the endosomal compartment to an acidic (pH 4–6) range promotes AAV transduction, but inhibiting acidification during endosomal processing reduces the transduction rate. Additionally, the use of several proteasome inhibitors, such as tripeptidyl aldehydes and N-acetyl-l-leucyl-l-leucyl-l-l-norleucine (LLnL). Additionally, LLnL seems to promote AAV2 capsid ubiquitination, resulting in enhanced gene transfer in a variety of cell types, indicating that the mechanism by which these inhibitors enhance transduction is linked to ubiquitination. Before AAV can translocate to the nucleus, it must escape the endosome. Prior to exiting the endosome, AAV undergoes a conformational shift that exposes the distinctive N-terminal ends of VP1 and VP2, which include a domain of phospholipase A2 (PLA2), an enzyme that breaks the endosomal membrane, allowing for the effective endosomal escape of viral particles. AAV reaches the nucleus intact upon endosomal escape, and uncoating occurs inside the nucleus. However, nuclear transport of AAV is a sluggish process; only about 1–2% of internalized AAV penetrates and expresses in the nucleus, and the entire entrance process takes roughly 2–13 hours. As a result, the majority of viral particles that do not translocate are found outside or distant from the nucleus.

AAV requires importin-β, a nuclear import protein that has been demonstrated to play a critical role in enabling viral particle attachment to host nuclei in other viral infectious routes. Upon entrance into the nucleus, the AAV genome's single-stranded DNA (ssDNA) is converted to double-stranded DNA (dsDNA) by the target cell's nuclear machinery for transcription of the transgene. The synthesis of the second DNA strand has been suggested to be a rate-limiting step in the initiation and effectiveness of transgene expression in ssAAV vectors. As a result, second-generation AAV vectors containing dsDNA often referred to as self-complementary AAV (scAAV) vectors, were created to increase transduction and transcription efficiency. Several research published over the last decade have demonstrated that novel scAAV vectors enable safe, reliable, and organ-specific transduction in vitro and in vivo. This indicates that the limitations of ssAAV-mediated cell transduction can be addressed by the use of scAAV vectors in gene therapy.

AAV Production Protocol

Step 1

The first step is to clone the desired gene into a suitable plasmid carrier. In the majority of cases, the desired cDNA is cloned into one of the ITR/MCS-containing vectors. These vectors contain all of the cis-acting components required for AAV replication and packing via inverted terminal repeat (ITR) sequences.

Step 2

Co-transfection of the recombinant expression plasmid into AAV-293 cells with pHelper (carrying adenovirus-derived genes) and pAAV-RC (containing AAV2 replication and capsid genes) provides all of the trans-acting components necessary for AAV replication and packing in AAV-293 cells. Recombinant AAV viral particles are generated from infected AAV-293 cells and then utilized to infect a variety of mammalian cells.

Step 3

When a single-stranded virus infects a host cell, it must be transformed to a double-stranded virus in order to produce its genes. The AAV virus synthesizes the complementary strand by using cellular replication components. . Typically, the AAV genome forms high-molecular-weight concatemers that are responsible for cell-specific gene expression.

AAV Production Stepwise Protocol

  • 1. Propagate HEK293 cells in PriGrow III Medium (Cat# TM003) with 10% FBS (Cat# TM999) and 1% Pen/strep (Cat# G255). The day before transfection, plate the cells in a 15cm dish such that the cells reach 70-80% confluency the next day.

  • 2. On the day of transfection, set up the 3-plasmid co-transfection as follow:

    Solution A: dilute 12 µg of helper plasmid, 12 µg of rep/cap plasmid and 10 µg of transgene plasmid in 2.5 ml of serum-free, antibiotic-free medium.

    Solution B: dilute 250 µl of DNAfectinTM2100 (Cat#G2100) in 2.5 ml of serum-free, antibiotic-free medium.

  • 3.Combine the solutions, mix gently to ensure uniform distribution, and incubate for 20 minutes at room temperature.
  • 4. After 20 minutes, add 10.0 ml of serum-free, antibiotic-free medium to the DNAfectinTM 2100-DNA complex. Mix solution gently.
  • 5. Remove the growth medium from the HEK293 cells and add the DNAfectinTM2100-DNA complex solution to the cells.
  • 6. After 5-8 hours, remove the transfection solution and add a complete growth medium to the cells. Incubate the cells at 37°C in a CO2 incubator for another 48 hours.
  • 7. Around 48 hours after transfection, harvest the cells from the 15cm plate with a cell scraper.
  • 8. Spin the cells at 1,500 x g for 5 minutes to collect the cell pellet. Resuspend the cell pellet in 0.5ml lysis buffer (10 mM Tris-HCl (pH8.5), 150 mM NaCl).
  • 9. Freeze/thaw the cell pellet 3 times by rotating through a dry ice/ethanol bath and a 37°C water bath to obtain the ‘crude lysate’.
  • 10.Freeze/thaw the cell pellet 3 times by rotating through a dry ice/ethanol bath and a 37°C water bath to obtain the ‘crude lysate’.

AAV Production Troubleshooting guide

1. Prior to initiating rAAV production, critical aspects such as plasmid purity and RNAase contaminants should be considered.
2.To guarantee that your materials are free of contamination and as pure as possible, they can be pre-screened for plasmid integrity using specific digests.
3. When plating your HEK293 / AAV-293 cells, please ensure that they are between 70% and 80% confluent and in good health at the time of transfection.
4. Concentration and purification can be done using iodixanol gradient ultracentrifugation.
5.Always avoid bubbles during purification.
6.Iodixanol is difficult to eliminate. Add more formulation buffers and viral purifications and pipet back and forth many times to include the iodixanol that has collected at the bottom or wall of the column into the solution following each centrifugation.
7. It is advised to concentrate AAV on a minimum of 500µl. If the volume of the concentrate is less than 500µl, increase the volume with formulation buffer.
8.Using chimeric capsids in rAAV vectors expands the range of cell types that can be transfected and increase the efficiency of transduction.
9.Mosaic capsid’s goal is to expand the tropism of the AAV vector to a more broad range of cells.

What is AAV Gene Therapy?

Gene therapy is a method of delivering therapeutic genes to cells or tissues using modified viruses or other technologies in order to address genetic disorders at their source. The understanding of how the adeno-associated virus (AAV) may be utilized as an efficient delivery mechanism for therapeutic genetic material into living tissue is one of the most exciting breakthroughs in contemporary medicine. AAV gene therapy has the potential to be used to treat a wide variety of illnesses.

AAV is a virus that has not been shown to cause illness in humans.AAV cannot reproduce itself without external assistance, and so can not multiply in the body in the same way as conventional viruses do. This enables scientists to carefully regulate the amount of AAV administered.

AAV has surpassed all other forms of gene therapy in treating genetic disorders. AAV is at the forefront of therapeutic advancements today, having been employed in the development of the only two FDA-approved gene treatments now accessible.

To increase the efficiency and specificity of AAV infection of target tissues, researchers must genetically modify the viral capsid and generate mosaic vectors to create chimeric AAV by swapping domains or amino acids between serotypes. This may enable researchers to specifically target cells with certain serotypes in order to effectively transduce and express genes in a localized area.

Pros and cons of AAV gene therapy

ProsCons
Nonpathogenic Smaller sizes limit the number of foreign genes that can be inserted.
Broad host and Cell type Tropism rangeSmall onset of gene expression
Transduce both dividing and non-dividing cells
Maintain a high level of gene expression over a long period of time in vivo

Challenges in AAV Gene Therapy

1. Each batch of rAAV must achieve a specific vector concentration in order for the therapy to be successful and safe. Typically, conventional upstream bioprocesses produce a low concentration.

2. Delivering the required genetic sequence into the rAAV capsid is inefficient, frequently resulting in a large number of empty viral vectors. These empty vectors can account for up to 90% of the batch, reducing the efficacy of the therapy and necessitating a larger dosage for the patient.

3. If the cell lines utilized to generate the rAAV vectors include oncogenic DNA sequences, these sequences may be transferred to the final gene therapy product.

4. rAAVs are less immunogenic than other viral vectors because of the absence of modified lipids or other chemical components that might elicit an immune response. However, the capsid protein itself may provoke a reaction that diminishes the therapy's efficacy.

5. Purification of rAAV from its host cells can be challenging, resulting in a longer and more expensive manufacturing procedure. In certain situations, rAAV capsids stay in the host cell, requiring gene therapy developers to damage the cell in order to extract the capsid for purification. Disrupting cells might result in the release of more contaminants.