AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta)

Abstract

Many genetic metabolic diseases manifest in infancy, therefore, it is important to develop effective treatments that could be implemented at this time. Adeno-associated virus serotype 8 (AAV8) gene transfer has been studied in neonatal mouse, cat, and dog models and shown some efficacy with a single hepatic injection at birth. AAV8-mediated liver gene transfer has also generated sustained therapeutic effects in feline and canine models of lysosomal storage disorders. In these models, delaying the age of vector treatment increased gene transfer stability. The growth rate of infant nonhuman primates is more similar to the growth trajectory of humans, thus infant monkeys provide an excellent model to study AAV gene transfer efficiency, stability, and safety. In this study, we report for the first time that AAV8-mediated hepatic gene transfer in infant monkeys is safe and efficient but less stable when compared to adolescent animals. Infant monkeys administered AAV8 intravenously at 1 week postnatal age achieved up to 98% transduction of hepatocytes within 7 days of injection; however, there was significant dilution of genomes and loss of transgene expression 35 days postadministration. Delaying the injection to 1 month postnatal age did not improve stability of transduction but decreased the antibody response to AAV8 capsid.

Figure 1

EGFP expression in the livers of infant rhesus monkeys after intravenous administration of 3 × 10¹² GC/kg of AAV2/8.TBG.EGFP. (a) Infant rhesus monkeys were administered vector at 1 week postnatal age and tissues collected 7 days post administration (d7-1w). (b) Infant rhesus monkeys were administered vector at 1 week postnatal age and tissues collected 35 days post administration (d35-1w). (c) Infant rhesus monkeys were administered vector at 1 month postnatal age and tissues collected 45 days post administration (d45-1m). Animal number is shown at the bottom right corner. Representative liver images of each animal are shown. Bar = 100 µm. AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein.

Figure 2

Gene transfer and transduction efficiency in infant rhesus monkeys. Data from previously published adolescent rhesus monkeys (d7-Adole and d35-Adole) administered the same vector at the same dose^, are included for comparison. (a) Vector genome copies in liver. (b) Vector genome copies in spleen. (c) Quantitative morphometric analysis of the transduction efficiency based on percent transduction of liver area. (d) Quantification of EGFP in liver lysates by ELISA. Horizontal lines show the mean levels for each group. EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; GC, genome copies.

Figure 3

Body weight gain for infant rhesus monkeys from birth until tissue harvest. (a) d7-1 w cohort, (b) d35–1 w cohort, (c) d45-1m cohort. Arrow indicates the timepoint when AAV8 vector was administered. Body weights for all animals in this study were within the normative range for infant rhesus monkey based on historical data (A. Tarantal). AAV, adeno-associated virus.

Figure 4

Detection of EGFP in the germinal centers of spleen and development of anti-EGFP antibodies post-AAV8 gene transfer. (a) Detection of EGFP in the germinal centers of spleens at tissue harvest. (b) Anti-EGFP IgG titer in the serum of d35-1w cohort animals at 3, 4, and 5 weeks (W) after vector administration. (c) Anti-EGFP IgG titers in the serum of d45-1m cohort animals at 3, 4, 5, and 6 weeks (W) after vector administration. Bar = 100 µm. AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein.

Figure 5

Development of AAV8 NAb after vector administration. AAV8 NAb titer in the serum of d35-1w cohort (a) and d45-1m cohort (b) animals after vector administration. AAV, adeno-associated virus; NAb, neutralizing antibody.

Figure 6

Detection of AAV8 capsid protein in the germinal centers of infant rhesus monkey spleens. Spleens were collected at 7 (a), 35 (b), or 45 (c) days after vector administration. Immunostaining showed the presence of capsid protein (red) in germinal centers in some animals (#N2, #N3, #N7, #N8). Samples are counterstained with DAPI (shown in blue) to visualize nuclei. Bar = 100 µm. AAV, adeno-associated virus.