The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice

Abstract

Improved delivery of adeno-associated virus (AAV) vectors to the CNS will greatly enhance their clinical utility. Selection of AAV9 variants in a mouse model led to the isolation of a capsid called PHP.B, which resulted in remarkable transduction of the CNS following intravenous infusion. However, we now show here that this enhanced CNS tropism is restricted to the model in which it was selected, i.e., a Cre transgenic mouse in a C57BL/6J background, and was not found in nonhuman primates or the other commonly used mouse strain BALB/cJ. We also report the potential for serious acute toxicity in NHP after systemic administration of high dose of AAV.

Figure 1

Comparison of AAV9- and PHP.B-Mediated Gene Transfer in Mice (A) Direct GFP fluorescence in the brain, liver, and gastrocnemius muscle. Scale bars, 2 mm (brain) and 100 μm (liver, muscle). (B) GFP quantification of whole-slide scans. (C) Vector biodistribution in tissues. After systemic administration of 5E13 GC/kg to adult mice, PHP.B’s ability to target the brain with high efficiency is limited to the C57BL/6J strain. In BALB/cJ adult mice, AAV9 and PHP.B are equivalent and display low blood-brain-barrier crossing. PHP.B-mediated brain transduction is intermediate in CB6F1 hybrids. *p < 0.05; **p < 0.01; ****p < 0.0001. Error bars represent SD.

Figure 2

Comparison of AAV9- and PHP.B-Mediated Gene Transfer in Nonhuman Primates (A) Direct GFP fluorescence in the cortex, spinal cord, dorsal root ganglia (DRG), liver, and quadriceps muscle. Insets show GFP immunohistochemistry in high-dose animals. *Euthanized 5 days post injection; all others were euthanized 21 days post injection. Scale bars, 100 μm. (B) GFP quantification showing values for whole-slide scans (cortex, liver, muscle) or averages and SD from multiple images taken with a microscope (10 images for DRGs, 2 images for lumbar spinal cord). (C) Vector biodistribution in tissues. After systemic administration of 2E13 GC/kg (low dose) or 7.5E13 GC/kg (high dose) to adult rhesus macaques, both AAV9 and PHP.B vectors are equivalent with low to mild CNS targeting. Organs unprotected by the blood-brain barrier, such as dorsal root ganglia, liver, and muscles, are efficiently transduced. Note that GFP expression is not directly comparable in the PHP.B high-dose monkey that was euthanized 5 days post injection due to vector-related clinical condition. Vector biodistribution is similar for dose-matched AAV9 and PHP.B vectors, showing that both vectors share the same tropism and properties in NHP.

Figure 3

Toxicity Event in the High-Dose-Treated PHP.B NHP Platelet counts, total bilirubin, transaminase levels, and gross picture of the cutaneous petechiae and bruises on the thorax and abdomen of the NHP treated with 7.5E13 GC/kg of PHP.B-GFP vector. The toxicity event was characterized by thrombocytopenia, hyperbilirubinemia, and increased transaminases on day 3 and day 5 post vector administration. Massive subcutaneous and internal bleeding led to humane euthanasia of the animal on day 5.