Lumbar Intrathecal Injection of SOD1-ASOs for Precise CNS Targeting and Predictive Efficacy in Human SOD1-G93A ALS Mice

This work focuses on freehand lumbar intrathecal injections to deliver therapeutics directly into the cerebrospinal fluid of mice. This minimally invasive rapid technique offers superior translational relevance compared with more invasive approaches such as intracerebroventricular injections. To begin, apply ophthalmic lubricant to both eyes of the mouse immediately after anesthesia induction to prevent corneal desiccation.

Then position the mouse in ventral recumbent on the heating pad to prevent hypothermia, minimize physiological stress and support normal cardiovascular and respiratory functions throughout the procedure. Inject 0.05 milliliters of buprenorphine per 20 grams of body weight using a sterile syringe with a 25 gauge needle prior to the procedure. After shaving the lumbar region from mid flank to pelvic girdle, disinfect the area using alternating swabs of isopropyl alcohol and Povidone-Iodine.

Repeat three times and allow the site to air dry completely. Stabilize the pelvic girdle by gently grasping the pelvic girdle between the thumb and forefinger of one hand and palpating the dorsal midline with the other hand to locate the target intervertebral space between L4 to L5 or L5 to L6.Align the antisense oligonucleotide containing sterile 0.3 milliliter insulin syringe fitted with a 29 gauge half inch needle perpendicular or at a 70 to 80 degree angle to the dorsal skin surface with the bevel oriented cranially. Advance the needle until slight resistance is encountered as it penetrates the dura mater.

Then continue into the intrathecal space to inject the solution. Note the tail flick reflex which indicates successful intrathecal entry. Evans blue dye injected intrathecally for foundational validation experiments at 20, 30 and 50 microliter volumes showed consistent dorsal to ventral spinal cord staining in all mice with higher volumes yielding more intense rostro-caudal dye saturation.

Mice injected with sterile saline instead of Evans blue dye showed no spinal cord coloration or background staining. At 10 weeks of age, corresponding to five weeks post-treatment, compound muscle action potential amplitude was significantly higher in mice treated with SOD1 targeting antisense oligonucleotide via intrathecal injection or intracerebral ventricular injection compared to mice treated with inactive antisense oligonucleotide. Serum levels of phosphorylated neurofilament heavy chain were significantly reduced in both intrathecal and intracerebroventricular SOD1 antisense oligonucleotide treated mice compared to inactive ASO controls.

Quantitative PCR analysis showed that intrathecal delivery of SOD1 targeting antisense oligonucleotide achieved approximately 60%knockdown of human SOD1 mRNA in spinal cord tissue whereas intracerebral ventricular delivery resulted in approximately 35%knockdown. Both significantly different from the inactive control. The primary procedural challenge is accurately identifying the anatomical entry point to access the lumbar cistern.

Future studies may compare the cell type specific distribution or effects of injected therapeutics between intrathecal delivery and other intra CSF administration methods.