This study explores the use of neural stem cells (NSCs) loaded with conditionally replicating oncolytic viruses for targeting brain tumors. The method involves preconditioning NSCs under low oxygen conditions to enhance their proliferation and tumor-targeting capabilities.
To begin, subject a culture of neural stem cells or NSCs to low oxygen conditions. This preconditioning facilitates enhanced NSC proliferation and tumor-targeting abilities. Next, harvest the NSCs and treat with the desired concentration of conditionally replicating oncolytic virus. Incubate to load the viruses into the cells.
Now, centrifuge the culture to remove unbound viruses. Resuspend the virus-loaded NSCs in a suitable buffer. Meanwhile, position an anesthetized brain tumor-bearing mouse in the supine position for easier access to nostrils. Carefully administer the virus-loaded NSCs into each nostril of the mouse, repeating at short-time intervals.
Allow the mouse to recover. The intranasally administered NSCs in the nasal cavity bypass the blood-brain barrier to enter the brain. Inside the brain, the virus replicates within these preconditioned cells and migrates to the tumor site, protecting itself from the immune system. Over time, the viral particles released from the NSCs interact and bind to specific cell surface receptors overexpressed on tumor cells.
Following virus internalization, these conditionally replicating viruses preferentially replicate in the tumor cells and cause cell lysis. The lysed tumor cells release viral particles, which infect the surrounding tumor cells, leading to a cascade of infection and tumor cell lysis. The cell lysis exposes the tumor antigens, activating an immune response against tumor cells.
To load human neural stem cells with oncolytic virus, infect the stem cells with 50 conditional replicate of adenovirus viral particles per cell according to standard adenovirus transfection protocols for 2 hours at room temperature with periodic tapping. Then, wash the cells three times with fresh medium to remove any unbound viral particles and suspend the cells in sterile saline for intranasal delivery.
When the cells have been modified as experimentally appropriate, place the anesthetized tumor-bearing animals in the supine position on a clean drape over a heat pad and place a padded pillow under the heads to maintain the nostrils at an upright angle. The pillow material should be either alcohol-wipeable plastic or disposable rolled-up paper towels to avoid cross animal contamination.
When the mice are in position, use a micropipette to dispense 2 microliters of the experimental human neural stem cell population of interest into each nostril of the first mouse, visually confirming aspiration of the droplet before applying the next volume of cells. Repeat the delivery to each mouse in turn. Then, wait 5 minutes and apply the next round of cells. When all four rounds of cells have been administered, place the animals in a recovery chamber with monitoring until full recovery.
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