Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders - PubMed (original) (raw)

. 2010 Sep;21(9):1093-103.

doi: 10.1089/hum.2010.040.

A P Kells, R M Richardson, P Hadaczek, J Forsayeth, J Bringas, S P Sardi, M A Passini, L S Shihabuddin, S H Cheng, M S Fiandaca, K S Bankiewicz

Affiliations

Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders

E Aguilar Salegio et al. Hum Gene Ther. 2010 Sep.

Abstract

Gene replacement therapy for the neurological deficits caused by lysosomal storage disorders, such as in Niemann-Pick disease type A, will require widespread expression of efficacious levels of acid sphingomyelinase (ASM) in the infant human brain. At present there is no treatment available for this devastating pediatric condition. This is partly because of inherent constraints associated with the efficient delivery of therapeutic agents into the CNS of higher order models. In this study we used an adeno-associated virus type 2 (AAV2) vector encoding human acid sphingomyelinase tagged with a viral hemagglutinin epitope (AAV2-hASM-HA) to transduce highly interconnected CNS regions such as the brainstem and thalamus. On the basis of our data showing global cortical expression of a secreted reporter after thalamic delivery in nonhuman primates (NHPs), we set out to investigate whether such widespread expression could be enhanced after brainstem infusion. To maximize delivery of the therapeutic transgene throughout the CNS, we combined a single brainstem infusion with bilateral thalamic infusions in naive NHPs. We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites.

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Figures

FIG. 1.

FIG. 1.

Intraoperative use of real-time convection-enhanced delivery (CED) in the nonhuman primate (NHP) brainstem and thalamus. Infusion of AAV2-hASM-HA/Gd visualized as a contrast demarcation on MRI indicates cannula tip placement in the targeted region (A and B; white arrows). Note the increase in infusate size as a function of time as demonstrated in sequential MR image acquisitions. In (A), residual contrast signal visible 19 min after initiation of brainstem infusion correlates with diffusion of thalamic signal, completed 30 min before the end of brainstem infusion.

FIG. 2.

FIG. 2.

Coinfusion of AAV2-hASM-HA and gadoteridol (Gd) into the brainstem (open circles) and thalamus (solid circles). Shown are the results of single delivery of various amounts of infusate into the brainstem (open circles, n = 6; mean _V_i:_V_d ratio, 3.3 ± 0.17) and thalamus (solid circles, n = 10; mean _V_i:_V_d ratio, 3.86 ± 0.25). There is a linear relationship between _V_d and _V_i (overall, n = 16, _R_2 = 0.93), with higher _V_i delivered to the brainstem region as compared with the thalamus. No significant difference was found between ratios in these two regions (p > 0.05).

FIG. 3.

FIG. 3.

Comparison of _V_d:_V_i ratios with repeated brainstem and thalamic infusions. Initial _V_d:_V_i delivery parameters during Gd-only infusions (solid squares, n = 5; mean _V_d:_V_i ratio, 3.74 ± 0.25; _R_2 = 0.96) were replicated in later infusions consisting of AAV2-hASM-HA/Gd (open squares, n = 5; mean _V_d:_V_i ratio, 3.72 ± 0.24; _R_2 = 0.98). Note the consistent distribution patterns in consecutive infusions with or without therapeutic agent (overall, n = 10, _R_2 = 0.96). No significant difference was found between primary and secondary infusions (p > 0.05).

FIG. 4.

FIG. 4.

AAV infusion and transduction in brainstem and thalamus. Shown are DICOM MR images representative of brainstem and thalamic infusion (A and E, respectively), and immunostained brain sections anatomically matched to the corresponding MR image (B and F). High-power magnification images demonstrate infusion epicenter with significant neuronal transduction (HA expression) in each targeted region (C and D), as compared with negative controls (G and H). Note that boxes drawn on the MR images are merely representative of the ROIs and are not to scale. Microscopic analysis was performed on NHP tissue perfused 5 weeks after AAV2-hASM-HA infusion. Scale bars: (B and F) 2 cm; (C, D, G, and H) 500 μm.

FIG. 5.

FIG. 5.

High neuronal transduction numbers in targeted regions. (A) Cellular counting of randomized adjacent, histology-processed sections stained against neuronal marker (anti-NeuN) and HA tag epitope (anti-HA). (B) High neuronal transduction levels in the brainstem (82 ± 7.8%, n = 4) and in the thalamus (68 ± 11.3%, n = 7). No significant difference was found between levels of transduction efficiency in these two regions (p > 0.05). Microscopic analysis was performed on NHP tissue perfused 5 weeks after AAV2-hASM-HA infusion. Scale bars: for whole brain mounts, 2 cm; for immunostained sections, 500 μm.

FIG. 6.

FIG. 6.

Efficient neuronal transduction in brainstem. Immunostained brainstem sections indicate neuron-specific transduction (A–C) with no transduction observed in astrocytes (D–F) and/or microglia (G–I) (see high-magnification panels on the right). Note that the total number of colocalized NeuN/HA-positive cells was 61 of 63, representing 97% neuronal specificity. Scale bar: (A–I) 500 μm. Color images available online at

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