Q1: The injection method of AAV virus includes tail vein injection and local orthotopic injection. How should we choose?
The in vivo injection (administration) of AAV includes systemic administration and local administration. Systemic administration injection sites mainly include the tail vein, jugular vein, orbital vein, and abdominal vena cava; common local administration sites include stereotactic brain injection, intramuscular injection, orthotopic injection of myocardium, intravitreal injection, joint cavity injection, etc. The characteristic of systemic administration is that the volume of virus injection is large, requiring a volume of 100~1000μl. Its operation is relatively simple, the administration is convenient, and the virus spreads in the body in a wide range.
For local administration, the virus injection dose requires less, generally within 100μl, but the titer is higher; because the AAV is directly delivered to the target tissue, the local administration will have better targeting. For clinical gene therapy, the dose of virus injected by local administration is small, so the pathological toxicity will be smaller.
Q2: How long does the AAV take effect after injection? Is there a way to shorten its expression time?
After AAV enters the cell, it undergoes a process from single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA). The time required for the target gene expression to become detected is 1 week or more depending on the target gene, since different genes have different peak expression times. Using Self-complementary Recombinant Adeno-Associated Virus (scAAV) can speed up expression. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, scAAV will modify one of the ITRs so that the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. The caveat of this construct is that instead of the full coding capacity found in rAAV (4.7-6kb), scAAV can only hold about half of that amount (≈2.4kb). Therefore, it can only be used for research with small gene fragments.
Q3: What is the purpose of injecting and purifying AAV in vivo?
If the virus is used to infect cells in vitro, it generally does not need to be purified, but it is necessary to purify any virus used for in vivo injection, whether it is a lentivirus or AAV. The reason is that in the packaging and production process of the virus, the unpurified virus liquid contains a large amount of virus shell, cell (293T) endotoxin, broken particles, and cell debris, etc. If these substances are injected into the animal body with the virus, it may cause a strong immune response, thereby affecting the survival rate of animals. In addition, virus purification is also a concentration process, which is conducive to subsequent dilution and dosing administration.