Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates

Abstract

Injection of adeno-associated virus (AAV) into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4 and AAV5) in rodents, we report that a naturally occurring capsid (AAV9) and rationally engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ∼2 × 10(12) vector genome (vg) per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the central nervous system (CNS). AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared with intravascular delivery, and the presence of circulating anti-AAV-neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases.

Figure 1. AAV2.5 can cross the ependymal cell barrier and transduce neurons after ventricular administration

AAV2.5/GFP (10 uL, 6.6×10¹⁰ vg) was injected into the anterior portion of the right lateral ventricle of adult rats, and after 2 weeks gene expression was assessed by anti-GFP IHC. (A) Transduction of cells with neuronal morphology in the hypothalamus along the third ventricle. (B) GFP-positive cells with neuronal morphology in the dorsal central gray. Scale bar is 50 microns.

Figure 2. Comparison of AAV9 and AAV2.5 transduction after intracisternal administration in NHPs, versus AAV9 intrathecal administration

scAAV9 or scAAV2.5 vectors were injected into the cisterna magna (IC) or lumbar intrathecal space (IT) of NHPs, and the animals were sacrificed 4 weeks later. (A) Five to seven micron sections from the brain, spinal cord, and DRG were subjected to IHC against GFP, then quantified by histomorphometry. For the location of brain samples 1-6, see supplemental figure 5. (B) Total DNA was purified from the samples, and the copies of GFP relative to the NHP GAPDH locus were ascertained by qPCR. C = cervical, T = thoracic, L = lumbar, ND = no data. The legend for both panels is provided in (A).

Figure 3. Intrathecal AAV9 injection in NHPs provides widespread CNS transduction with reduced peripheral biodistribution and evasion of NAbs

NHPs received a single intrathecal injection of 1.83×10¹² vg (low dose) or 5.5×10¹² vg (high dose) scAAV9/CBh-GFP. Four weeks later, the animals were sacrificed and the indicated samples from the CNS and peripheral organs were processed. (A and B) Samples were sectioned at 5-7 microns and subjected to IHC against GFP with histomorphometric quantification of GFP expression. (C) Total DNA was purified from the samples, and the copies of GFP relative to the NHP GAPDH locus were ascertained by qPCR. All brain measurements are the average of all quadrants per block analyzed. For the location of brain samples 1-5 and individual data points, see supplemental figure 6 and supplemental table 2. Brain IHC for animals 303 and 304 examined slightly different brain areas, so this data is portrayed only in Figure 2. C = cervical, T = thoracic, L = lumbar. Asterisks indicate tissues where a sample from only 1 animal was tested. NAb+ = NAb titer >1:2, and NAb− = NAb titer <1:2. All error bars are S.E.M. The legend for all panels is provided in (A).

Figure 4. The NHP spinal cord and DRG are efficiently transduced following intrathecal AAV9 vector administration

Four weeks following intrathecal injection of scAAV9/CBh-GFP vector, 5-7 micron sections from the spinal cord and DRG were subjected to IHC against GFP. Shown are sample images from those used for histomorphetric quantitation in Figure 3, and more information is available in Supplemental Figure 6 and Supplemental Table 2. These images are from NHP 203 and the specific areas are as follows, with spinal cord in panels A-D and DRG in panels E-G: (A) cervical, ventral gray matter, (B) thoracic, ventral white matter, (C) lumbar, dorsal gray matter, (D) lumbar, dorsal white matter, (E) cervical, (F) thoracic, and (G) lumbar. For each panel, the right image is a 4x enlargement of the area indicated by an arrow, and the total width of the right field is 55 microns. Slides are counterstained with cresyl violet (blue) to show nuclei, and brown (DAB) indicates GFP expression. Scale bar in (A) is the same for all panels and is 55 microns.

Figure 5. The NHP brain is transduced following intrathecal AAV9 vector administration

Four weeks following intrathecal injection of scAAV9/CBh-GFP vector, 5-7 micron sections from the spinal cord and DRG were subjected to IHC against GFP. Shown are sample images from those used for histomorphetric quantitation in Figure 3A, and more information is available in Supplemental Figure 6 and Supplemental Table 2. These images are from NHP 203 brain and the specific areas are as follows: (A) brain region 1C, (B) brain region 2B, (C) brain region 3C, (D) brain region 4, (E) brain region 5B, (F) liver, and (G) spleen. For each panel, the right image is a 4x enlargement of the area indicated by an arrow, and the total width of the right field is 55 microns. Slides are counterstained with cresyl violet (blue) to show nuclei, and brown (DAB) indicates GFP expression. Scale bar in (A) is the same for all panels and is 55 microns.

Figure 6. AAV9 strongly transduces the choroid plexus after intrathecal administration in NHPs

Four weeks following intrathecal injection of scAAV9/CBh-GFP vector, 5-7 micron sections were collected and subjected to IHC against GFP. The image is from NHP #206, block 2A (see supplemental Figure 6), with the lateral ventricle and surrounding parenchyma shown. Dense GFP staining is observed in virtually all of the ependymal cells of the choroid plexus (open arrow), no GFP seen in the ependymal cells lining the ventricles (closed arrow), and light GFP staining is seen in some cells within the surrounding parenchyma. The scale bar is 100 microns, and the inset is a 2x magnification of the area highlighted by the arrows.