Lumbar Intrathecal Injection of Gene Therapy Vectors for Central Nervous System Targeting in Mice and Rats

Our lab develops gene therapies for rare pediatric neurological diseases, using an AV media gene delivery platform combined with direct cerebral spinal fluid injection. We assess therapeutic potential in preclinical models to advance promising candidates to clinical trials. In preclinical studies for rare neurological conditions, including spastic paraplegia 50 and giant axonal neuropathy and RET syndrome, we demonstrated that intrathecal gene delivery is safe and effective at ameliorating disease phenotypes in mice. These treatments are now in phase 1, 2 clinical trials. Our lab will continue to work with patient advocacy groups to develop meaningful treatments for children with rare genetic disorders. In parallel, we are developing new AAV capsids, which we hope will increase the efficacy of these therapies.

[Narrator] To begin, gather the required sterile materials, including a syringe and needle, pipette, and injection solution. Using a microliter pipette, measure the desired volume of the injection solution and transfer it onto a sterile paraffin film. Draw the solution into a microliter syringe fitted with a 30 gauge, 0.5 inch needle. Now, using the dominant hand, hold the conscious mouse by the tail on a paper towel. With the non-dominant hand fold a portion of the paper towel over the head and upper body of the mouse and firmly grasp the pelvic girdle between the thumb and index finger. Gently rotate the base of the tail to align the mouse's spine properly and part the hair approximately two to six millimeters cranial to the iliac crest to visualize the injection site. Palpate the area to feel for the L4, L5, or L5, L6 intervertebral space, located approximately two to six millimeters cranial to the iliac crest. Hold the center of the microliter syringe with the dominant hand and position it perpendicular to the mouse's spine at the L4, L5 or L5, L6 intervertebral space. With the bevel of the needle facing the head of the mouse, puncture the skin over the intervertebral space. Once the needle touches the bone edges of the intervertebral space, reduce the syringe angle to 30 to 45 degrees. After fully depressing the plunger, hold the syringe in place for 15 to 30 seconds to let the solution dissipate and prevent backflow. Then smoothly and slowly withdraw the syringe at the same 30 to 45 degree entry angle. Finally, release the mouse from the restraint and return it to its home cage. Prepare for the procedure as demonstrated earlier. Using the dominant hand, hold the juvenile mouse by the tail on a paper towel. Gently but firmly grasp the pelvic girdle between the non-dominant thumb and index finger and rotate the base of the tail to ensure proper alignment of the spine. Swab the dorsal lumbar region of the animal with a 70% alcohol prep pad to disinfect the area. If applicable, part the hair approximately one to three millimeters cranial to the iliac crest. to help visualize the injection site. Feel for or visualize the L4, L5 or L5, L6 intervertebral space approximately one to three millimeters cranial to the iliac crest. Using the dominant hand, hold the center of the microliter syringe and position it at a 30 to 45 degree angle to the spine of the mouse. With the bevel of the needle facing up, puncture the skin approximately one to three millimeters coddle to the intervertebral space. Inject the solution and finish the procedure as demonstrated earlier, Broad and even distribution of the gene therapy vector within the central nervous system, including the brain, cervical spinal cord, and lumbar spinal cord was observed in successful injections four months post-injection. Concentrated expression in the lumbar spinal cord with absent expression in the brain indicated a failed injection due to intraparenchymal targeting of the spinal cord. Brains from injected mice with dye for practice displayed visible green staining, indicating successful delivery of the injected solution into the central nervous system.