Comparison of high-dose intracisterna magna and lumbar puncture intrathecal delivery of AAV9 in mice to treat neuropathies

Abstract

Gene therapy clinical trials for neurological disorders are ongoing using intrathecal injection of adeno-associated virus (AAV) vector directly into the cerebral spinal fluid. Preliminary findings from these trials and results from extensive animal studies provides compelling data supporting the safety and benefit of intrathecal delivery of AAV vectors for inherited neurological disorders. Intrathecal delivery can be achieved by a lumbar puncture (LP) or intracisterna magna (ICM) injection, although ICM is not commonly used in clinical practice due to increased procedural risk. Few studies directly compared these delivery methods and there are limited reports on transduction of the PNS. To further test the utility of ICM or LP delivery for neuropathies, we performed a head to head comparison of AAV serotype 9 (AAV9) vectors expressing GFP injected into the cisterna magna or lumbar subarachnoid space in mice. We report that an intrathecal gene delivery of AAV9 in mice leads to stable transduction of neurons and glia in the brain and spinal cord and has a widespread distribution that includes components of the PNS. Vector expression was notably higher in select brain and PNS regions following ICM injection, while higher amounts of vector was found in the lower spinal cord and peripheral organs following LP injection. These findings support that intrathecal AAV9 delivery is a translationally relevant delivery method for inherited neuropathies.

Keywords: Adeno-associated virus; Intracisterna Magna; Intrathecal; Lumbar Puncture; Neuropathy; Peripheral Nervous System.

Figure 1.

Biodistribution of AAV9/GFP following intrathecal delivery in mice. AAV9 was administered to C57BL/6 mice at a dose of 4.15×10¹¹ vg by injection into the cisterna magna (ICM) or the lumbar subarachnoid space (LP). All animals were sacrificed 4 weeks post-injection and vector genomes were quantified in tissue samples by qPCR. Vg copies are normalized to mouse lamin B as an endogenous housekeeping gene and are represented as vg copies per cell. Error bars represent SEM (n=4 per group). Each tissue group was analyzed by an unpaired t test. *p<0.5, **p<0.01. DRG, dorsal root ganglia.

Figure 2.

CNS transduction profile of AAV9 following intrathecal delivery in mice. (A, B) Transduction profiles 4 weeks after intrathecal delivery of AAV9 packaging a scCBh-GFP transgene at a dose of 4.15×10¹¹ vg via injection into the cisterna magna (ICM, top) or lumbar subarachnoid (LP, bottom) spaces across the (A) brain and (B) spinal cord. (A) Representative coronal sections from the whole mouse brain progressing from forebrain to the hindbrain (left to right). (B) Spinal cord sections from cervical (left), thoracic (middle) and lumbar (right) regions.

Figure 3.

Transduction throughout the brain following ICM and LP AAV9 delivery. (A-L) Expression profile 4 weeks after intrathecal delivery of scAA9/CBh-GFP vector at a dose of 4.15×10¹¹ vg. (A) motor cortex, (B) somatosensory cortex, (C) CA1 of hippocampus, (D) dentate gyrus of hippocampus, (E) striatum, (F) thalamus, (G) amygdala, (H) hypothalamus, (I) midbrain, (J) pons, (K) medulla, and (L) cerebellum. Scale bars, 100 μm.

Figure 4.

Intrathecal AAV9 transduces neurons and glia in mice. (A) Cortical brain sections and (B) medulla brain sections from mice injected by ICM or LP routes were imaged for GFP expression. Sections are 5 μm thick. Images were overlaid with NeuN immunostaining for neurons or GFAP immunostaining for astrocytes. Counterstain is DAPI. Scale bar = 100 μm.

Figure 5.

Intrathecal delivery of scAAV9 vector results in peripheral nervous system expression. Tissues were collected 4 weeks after ICM or LP delivery of scAA9/CBh-GFP vector at a dose of 4.15×10¹¹ vg in mice. Sections are 5 μm thick, stained with anti-GFP and counterstained with hematoxylin. Arrows, Myenteric plexus; arrow heads, submucosal plexus. Scale bars, 100 μm.