Our research focuses on the development of gene therapies for diseases that affect the central nervous system. As of December 2023, the FDA has approved AAV-based gene therapies for a handful of genetic disorders, including spinal muscular atrophy, Leber Congenital Amaurosis, Duchenne Muscular Dystrophy, and hemophilia A and B.Many more treatments are currently being investigated in preclinical and clinical trials. Devising safe and effective interventions for genetic diseases requires leveraging a range of approaches, from careful natural history studies in patients to preclinical work in tissue culture and animal models, and gene therapy is really only one of several technologies that are used for this purpose.
Many gene therapy experiments use neonatal or adult animals. Few evaluate treatments in juveniles, even though this represents a significant patient population for genetic disorders. So for older children, body size and the immune system pose significant barriers to intravenous delivery of gene therapies.
Intrathecal delivery may overcome this, but we need appropriate models to test this hypothesis. This procedure that we have developed will allow us to evaluate the efficacy of this approach in juvenile rats. To begin, take the tube containing the AAV9 vector.
Using a tabletop centrifuge, centrifuge the tube briefly. Then gently flick the tube to ensure the solution is well mixed. Next, set up a clean cage on an electric blanket and adjust the blanket's temperature to approximately 37 degrees Celsius.
Using a microwave or a water bath, warm an isothermal pad at 239 degrees Celsius. Then place this pad on the surgical platform and cover it with a clean absorbent bench pad. Begin by positioning the anesthetized animal on the surgical platform.
Apply lubricating eye ointment to each animal's eyes to prevent corneal drying during the surgery. Disinfect the shaved surgical site with three alternating rounds of povidone-iodine and isopropanol. Then position a roll of paper towel or a 1.5 cm diameter tube under the animal just rostral to the hips.
Drape the animal with a fenestrated drape, centering the fenestration over the lumbar spine for surgical access. To verify the depth of anesthesia in the animal, pinch each paw and observe for the absence of any withdrawal response. Then, using a number 11 scalpel blade, make a 3 cm long skin incision down the midline from L2 to L6 vertebrae.
Now insert a sterile curved pair of surgical scissors between the muscle and skin and gently open the tips to separate the skin from the underlying muscle. Remove the fascia covering the spinous processes of L2 to L5 vertebrae. Pipette 25 to 35 microliters of the AAV9 vector into the cap of a sterile microcentrifuge tube.
Draw the entire volume of the vector from the cap into the insulin syringe. Then identify the L5 and L4 spinous processes on the animal. Position the syringe needle to the left of the L5 spinous process, aligning it with the caudal end of the process.
Advance the syringe needle forward about 8 mm over the L5 lamina, and then under the L4 lamina into the lumbar cistern until contact with bone is made. Depress the syringe plunger slowly for approximately five seconds. Then slowly withdraw the needle from the insertion site.
Using a 4-0 suture or surgical staples, start closing the incision at one end of the wound. To evaluate the success of dye injection, after euthanizing the animal, make a 4 cm incision into the muscle parallel to the spine on both sides of the spinous processes. Use fine forceps or scissors to delicately remove the muscle between the spinous processes.
Using a rongeur, extract the spinous processes from the L6 to the lower thoracic spine. Insert the lower tip of the rongeur under the L5 lamina and methodically remove the bone overlying the spinal cord by digging several bites out of it.
