Systemic and Persistent Muscle Gene Expression in Rhesus Monkeys with a Liver De-Targeted Adeno-Associated Virus Vector

Abstract

The liver is a major off-target organ in gene therapy approaches for cardiac and musculoskeletal disorders. Intravenous administration of most of the naturally occurring adeno-associated virus (AAV) strains invariably results in vector genome sequestration within the liver. In the current study, we compared the muscle tropism and transduction efficiency of a liver de-targeted AAV variant to AAV9 following systemic administration in newborn rhesus monkeys. In vivo bioluminescence imaging was performed to monitor transgene expression (firefly luciferase) post administration. Results indicated comparable and sustained levels of systemic firefly luciferase gene expression in skeletal muscle over a period of two years. Quantitation of vector biodistribution in harvested tissues post-administration revealed widespread recovery of vector genomes delivered by AAV9 but markedly decreased levels in major systemic organs from the AAV variant. These studies validate the translational potential and safety of liver de-targeted AAV strains for gene therapy of muscle-related diseases.

Keywords: AAV9; Rhesus monkey; chimeric AAV; liver de-targeting.

Figure 1.

(a) BLI of BALB/c mice that were previously injected intravenously (IV) with AAV2, AAV8, or chimeric AAV2i8 showed that the chimeric AAV2 capsid carrying seven amino acids from AAV8 de-targets the liver and concentrates expression in skeletal muscle and heart (see Asokan et al.). (b) Rhesus monkeys injected IV at birth and imaged using an IVIS®200 system at study time points (1, 3, 6, 12, and 21 months shown).

Figure 2.

Bioluminescence (photons/sec) was quantified for individual animals at all time points. Stable levels of bioluminescence were observed through 21 months as noted. AAV9 showed high, transient expression of firefly luciferase up to 2 months postnatal age. Mean levels of bioluminescence for animals in each group (n = 2 for AAV9, n = 4 for AAV2i8) are shown (standard error bars for AAV2i8).

Figure 3.

Quantitation of vector genome biodistribution with AAV9 or AAV2i8 at 2 years postnatal age. Copies of the firefly luciferase transgene per 5 × 10⁴ copies of globin are shown. The gastrointestinal (GI) tract includes the stomach, duodenum, jejunum, ileum, and colon; lymph nodes include axillary, inguinal, and mesenteric. Mean levels of bioluminescence for animals in each group (n = 2 for AAV9, n = 4 for AAV2i8) (standard error bars for AAV2i8).