The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice
Christie L. Bell, Luk H. Vandenberghe, Peter Bell, Maria P. Limberis, Guang-Ping Gao, Kim Van Vliet, Mavis Agbandje-McKenna, James M. Wilson
Christie L. Bell, Luk H. Vandenberghe, Peter Bell, Maria P. Limberis, Guang-Ping Gao, Kim Van Vliet, Mavis Agbandje-McKenna, James M. Wilson
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Research Article Genetics

The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice

Abstract

Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction. This was confirmed in mutant CHO Pro-5 cells deficient in the enzymes involved in glycoprotein biogenesis, as well as lectin interference studies. Binding of AAV9 to glycans with terminal galactose was demonstrated via glycan binding assays. Co-instillation of AAV9 vector with neuraminidase into mouse lung resulted in exposure of terminal galactose on the apical surface of conducting airway epithelial cells, as shown by lectin binding and increased transduction of these cells, demonstrating the possible utility of this vector in lung-directed gene transfer. Increasing the abundance of the receptor on target cells and improving vector efficacy may improve delivery of AAV vectors to their therapeutic targets.

Authors

Christie L. Bell, Luk H. Vandenberghe, Peter Bell, Maria P. Limberis, Guang-Ping Gao, Kim Van Vliet, Mavis Agbandje-McKenna, James M. Wilson

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Figure 2

AAV9 dependence on galactose for binding and transduction of CHO cells.

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AAV9 dependence on galactose for binding and transduction of CHO cells. ...
(A and B) AAV2, AAV6, or AAV9 expressing ffLuc was added to Pro-5, Lec-2, or Lec-8 cells and incubated at 4°C for 1 hour. (A) Total DNA was isolated to determine bound vector genome copies by qPCR or (B) cells were incubated at 37°C for 48 hours and analyzed for ffLuc expression. (C and D) AAV2 and AAV9 were applied to NA-treated Pro-5 cells in the presence of various lectins to compete for AAV binding (C) or transduction (D). RCA was not used in transduction studies because of its toxicity to the cells. (E and F) AAV2 and AAV9 were added to Pro-5 cells that were treated with either NA or both NA and β-gal to assess AAV binding (E) and transduction (F). *P < 0.001. For C and D, statistical significance was determined compared with the no lectin control.