Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates - PubMed (original) (raw)

Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates

Steven J Gray et al. Mol Ther. 2011 Jun.

Abstract

Other labs have previously reported the ability of adeno-associated virus serotype 9 (AAV9) to cross the blood-brain barrier (BBB). In this report, we carefully characterized variables that might affect AAV9's efficiency for central nervous system (CNS) transduction in adult mice, including dose, vehicle composition, mannitol coadministration, and use of single-stranded versus self-complementary AAV. We report that AAV9 is able to transduce approximately twice as many neurons as astrocytes across the entire extent of the adult rodent CNS at doses of 1.25 × 10¹², 1 × 10¹³, and 8 × 10¹³ vg/kg. Vehicle composition or mannitol coadministration had only modest effects on CNS transduction, suggesting AAV9 crosses the BBB by an active transport mechanism. Self-complementary vectors were greater than tenfold more efficient than single-stranded vectors. When this approach was applied to juvenile nonhuman primates (NHPs) at the middle dose (9-9.5 × 10¹² vg/kg) tested in mice, a reduction in peripheral organ and brain transduction was observed compared to mice, along with a clear shift toward mostly glial transduction. Moreover, the presence of low levels of pre-existing neutralizing antibodies (NAbs) mostly occluded CNS and peripheral transduction using this delivery approach. Our results indicate that high peripheral tropism, limited neuronal transduction in NHPs, and pre-existing NAbs represent significant barriers to human translation of intravascular AAV9 delivery.

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Figures

Figure 1

Figure 1

Dose response of mouse central nervous system (CNS) transduction and biodistribution of adeno-associated virus serotype 9 (AAV9). (a) Mice were injected with scAAV9/CBh-GFP at the indicated doses, then killed 4 weeks later for quantitative PCR biodistribution and immunohistochemistry to detect green fluorescent protein (GFP) in the (b) hippocampus, (c) striatum, (d) cerebellum, and (e) spinal cord. In b–e, the left panel shows 2.5 × 1010 vg (1.25 × 1012 vg/kg), the middle panel shows 2 × 1011 vg (1 × 1013 vg/kg), and the right panel shows 1.6 × 1012 vg (8 × 1013 vg/kg). All 3,3′-diaminobenzidine tetrachloride (DAB) staining reactions were done in parallel for the same amount of time. In a, error bars indicate standard deviation. (b–e) Bar = 200 µm. Negative control tissue, magnified images, and longer DAB exposures for the high dose are provided as Supplementary Figures S2–S8. Fold expression values and corresponding P values of organs relative to the brain and spinal cord can be found in Supplementary Table S1.

Figure 2

Figure 2

Efficient transduction of neurons and glia in the mouse hippocampus. Enlarged image of Figure 1b, right panel. Bar = 200 µm. Arrowheads indicate examples of cells with neuronal (black) or glial (white) morphology.

Figure 3

Figure 3

Broad distribution of neuronal and glial transduction across the mouse central nervous system (CNS). Representative images showing immunohistochemistry using an anti-green fluorescent protein (GFP) antibody on coronal brain and spinal cord sections. These were taken from the same set of mice depicted in Figure 1, at the 2 × 1011 vg dose (scAAV9/CBh-GFP injected i.v. in adult mice). (a–i) Filled scale bars are 100 µm, and (j,k) open scale bars are 1 mm. Filled black arrowheads indicate examples of cells with neuronal morphology, and open (white) arrowheads indicate examples of cells with astrocytic morphology. (a) Primary motor cortex, (b) piriform cortex, (c) hypothalamus, (d) thalamus, (e) medulla, (f) arcuate nucleus and median eminence, (g) amygdala, (h) caudal hippocampus, (i) ventral horn of the cervical spinal cord, (j) coronal section, bregma −1.9 mm, (k) coronal section, bregma −6.2 mm.

Figure 4

Figure 4

Adeno-associated virus serotype 9 (AAV9) transduces a variety of cell types after i.v. delivery to the mouse central nervous system (CNS). (a–f) Alternate serial sections described in Figure 1 were subjected to co-immunofluorescence with the indicated antibodies. For each set of three panels, the left panel shows green fluorescent protein (GFP) expression, the middle shows labeling with the indicated antibody, and the right panel shows a merged image. The inset in the right panel highlights co-localization observed, and the arrow points to the cells magnified. All images are four merged confocal Z-stacks of samples from mice injected with 2 × 1011 vg (1 × 1013 vg/kg), except for panel f which shows the 1.6 × 1012 vg (8 × 1013 vg/kg) dose. Note that with panel e, native fluorescence was used to visualize GFP expression. Scale bars for all panels are shown in the right panels. Bar = 200 µm.

Figure 5

Figure 5

Mannitol and vehicle can modestly affect adeno-associated virus serotype 9 (AAV9) mouse central nervous system (CNS) delivery. In each case, mice received a tail vein injection of 1×1011 vg (5×1012 vg/kg) of scAAV9/GFP. After 4 weeks, tissues were harvested, and extracted DNA tested by quantitative PCR as described in the methods. (a) Mice received an intravenous (i.v.) injection of mannitol 8 minutes prior to i.v. vector injection. (b) A virus stock was diluted to create the vehicle compositions shown, then intravenously injected into mice. Error bars indicate standard deviation. n.d., no data. *P < 0.05 compared to 167 mmol/l phosphate-buffered saline + 0.7% sorbitol (Student's _T_-test: two-tailed, unpaired, equal variance).

Figure 6

Figure 6

AAV9/GFP, but not AAV9/GAN, vectors show a transient liver toxicity and reduction in vector genomes. Mice were injected with either ssAAV9/CBA-GFP (n = 6) or ssAAV9/CBA-GAN (n = 8). (a) Just after the 4-week time point postinjection, a group of mice (n = 3 green fluorescent protein (GFP), n = 4 giant axonal neuropathy (GAN)) was killed to assess biodistribution by quantitative PCR. In panels b–e, the arrow denotes the time when these mice were removed from the cohorts. (b) Weights were taken at the indicated time points and expressed relative to the starting weight. Serum was collected at the indicated time points and tested for levels of (c) albumin, (d) blood urea nitrogen (BUN), and (e) alanine amino transferase (ALT). All error bars indicate standard deviation. *Significantly higher value with P < 0.05 (Student's _t_-test: two-tailed, unpaired, equal variance). AAV9, adeno-associated virus serotype 9.

Figure 7

Figure 7

Intravascular injection of adeno-associated virus serotype 9 (AAV9) in nonhuman primates (NHPs) results in broad central nervous system central nervous system (CNS) transduction that is limited by an anti-AAV9 immune response. NHPs were injected with scAAV9/CBh-GFP via the carotid artery (i.c., animals 26149 and 26706) or the saphenous vein (intravenous, animals 26945 and 26165). Green fluorescent protein (GFP) biodistribution was assessed by immunohistochemistry 4 weeks after injection. Examples of GFP expression detected by 3,3′-diaminobenzidine tetrachloride staining in the cortex, hippocampus, cerebellum, dorsal (D) and ventral (V) spinal cord are shown for each animal. Arrows point to GFP-positive cells highlighted in the magnified insets with neuronal (short arrow) or glial (long arrow) morphology. An asterisk (*) denotes that NHP 26706 had detectable pre-existing neutralizing antibodies (see Table 1). Bar = 100 µm.

Figure 8

Figure 8

Intravascular adeno-associated virus serotype 9 (AAV9) delivery in nonhuman primates (NHPs) transduces mostly glia in the central nervous system (CNS). Representative sections from the NHPs showing higher levels of transduction (left panels: i.c., 26149; right panels: i.v., 26945) were selected and subjected to co-immunofluorescence with the indicated antibodies. For each picture, green fluorescent protein-expressing cells are green and (a–f) glial fibrillary acidic protein or (g–l) NeuN-positive cells are red. Control sections in which no primary antibodies were used are depicted in (m) green and (n) merged green and red. The insets highlight observed co-localization, arrows pointing to the magnified cells. Scale bars for all panels are on the bottom panels. Bar = 100 µm.

Figure 9

Figure 9

Nonhuman primate (NHP) biodistribution and expression in peripheral organs. At the time of euthanasia, various peripheral organs were rapidly dissected and frozen in liquid nitrogen for quantitative PCR (qPCR) analysis of vector genome biodistribution (top panel) or postfixed in 4% paraformaldehyde and processed for immunohistochemical detection of green fluorescent protein (GFP) by 3,3′-diaminobenzidine tetrachloride staining (bottom panel). For the qPCR biodistribution studies, the “brain” is the average value of three samples and the “spinal cord” is the average of cervical and lumbar samples. Bottom: animals with no pre-existing antibodies (i.c., 26149; i.v., 26945) showed high levels of transduction in the liver, heart, and adrenals whereas the kidney, spleen, and testes showed lower levels of GFP expression. The animal with pre-existing neutralizing antibodies (NAbs) (i.c., 26706) had no observable GFP expression except in the spleen. The fourth NHP showed rising levels of NAbs throughout the 4 weeks postadministration and had high expression of GFP in the liver, moderate in the heart and adrenals, and low in the kidney, spleen and testes. Scale bar for each organ is shown in the picture of the control sample. Bar = 100 µm.

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