Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer

Abstract

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.

FIG. 1

AAV5-based cytomegalovirus (CMV) lacZ was derived from plasmid pAAV5 Rn (6), while the AAV5 packaging construct pack 5 was derived from viral AAV5 DNA (3). The hybrid packaging construct pack 2/5 was created by exchanging AAV2 cap from the AAV2 packaging construct p600 trans (2) with AAV5 cap. The AAV2-based AAV CMV lacZ construct has been described previously (2). (A) Transduction of murine muscle with AAV5 CMV lacZ. The right anterior tibialis of C57BL/6 mice was injected with 10¹⁰ genome copies of AAV5 CMV lacZ and harvested 28 days postinjection. (B) Higher magnification of panel A. (C and D) Transduction of murine muscle with AAV2/5 CMV lacZ (C) and AAV2 CMV lacZ (D). The right anterior tibialis muscle of C57BL/6 mice was injected with 4 × 10¹⁰ genome copies of AAV2/5 CMV lacZ and AAV2 CMV lacZ, respectively. Muscles were harvested 60 days postinjection. (E) Transduction of murine smooth muscle with AAV2/5 CMV lacZ. We injected 10¹⁰ genome copies of AAV2/5 CMV lacZ subcutaneously into C57BL/6 mice. Expression of β-gal was assessed 60 days after vector administration. (F) Higher magnification of panel E. (G and H) Apical transduction of primary human epithelial airway cells with AAV2/5 (G) and AAV2CMV (H). Primary human airway epithelial cells were infected apically with 5 × 10¹⁰ genomic particles of the corresponding virus (liquid-air transwell system). Cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) 7 days postinfection.

FIG. 2

(A) Influence of neutralizing antibodies to AAV2 on vector readministration. Immunocompetent mice (C57BL/6) were injected with 3 × 10¹⁰ genome copies of AAV2 CMV eGFP in the right anterior tibialis muscle (11) and injected 28 days later with 3 × 10¹⁰ genome copies of either AAV2 CMV lacZ or AAV2/5 CMV lacZ in the left anterior tibialis muscle. Expression of β-gal was determined 14 days after the second injection by ELISA (Boehringer GMbH, Mannheim, Germany) in accordance with the manufacturer's instructions. Mean values of six animals per group are shown. (B) Influence of neutralizing antibodies to AAV2/5 on vector readministration. Immunocompetent C57BL/6 mice were injected with 4 × 10¹⁰ genome copies of AAV2/5 CMV eGFP in the right anterior tibialis muscle and injected 28 days later with 10¹⁰ genome copies of either AAV2 CMV lacZ or AAV2/5 CMV lacZ in the left anterior tibialis muscle. (Note that the AAV2/5 CMV lacZ virus preparation in this experiment is different from those used for Fig. 1 and 2A.) Expression of β-gal was determined 14 days after the second injection by ELISA in accordance with the manufacturer's (Boehringer) instructions. Mean values of eight animals per group are shown. PBS, phosphate-buffered saline control.

FIG. 3

Prevalence of neutralizing antibodies (NAB) to AAV in a human population. Neutralizing antibody titers were determined as previously described (1, 4, 9), except that AAV5 CMV eGFP3 was used instead of an AAV2 eGFP virus for the measurement of AAV5 neutralizing antibodies. Sera of 85 human volunteers were tested for the presence of neutralizing antibodies to AAV1, AAV2, and AAV5. All of the samples tested were negative for AAV5 neutralizing antibodies. Even sera with high AAV1- or AAV2- neutralizing activity did not inhibit AAV5 infectivity.