Abstract
BackgroundAdeno-associated virus type 2 (AAV-2) attachment and internalization is thought to be mediated by host cell membrane-associated heparan sulfate proteoglycans (HSPG). Lack of HSPG on the apical membrane of respiratory epithelial cells has been identified as a reason for inefficient rAAV-2 infection in pulmonary applications in-vivo. The aim of this investigation was to determine the necessity of cell membrane HSPG for efficient infection by rAAV-2.ResultsRates of transduction with rAAV2-CMV-EGFP3 in several different immortalized airway epithelial cell lines were determined at different multiplicities of infection (MOI) before and after removal of membrane HSPG by heparinase III. Removal of HSPG decreased the efficacy of infection with rAAV2 by only 30–35% at MOI ≤ 100 for all of respiratory cell lines tested, and had even less effect at an MOI of 1000. Studies in mutant Chinese Hamster Ovary cell lines known to be completely deficient in surface HSPG also demonstrated only moderate effect of absence of HSPG on rAAV-2 infection efficacy. However, mutant CHO cells lacking all membrane proteoglycans demonstrated dramatic reduction in susceptibility to rAAV-2 infection, suggesting a role of membrane glycosaminoglycans other than HSPG in mediating rAAV-2 infection.ConclusionLack of cell membrane HSPG in pulmonary epithelia and other cell lines results in only moderate decrease in susceptibility to rAAV-2 infection, and this decrease may be less important at high MOIs. Other cell membrane glycosaminoglycans can play a role in permitting attachment and subsequent rAAV-2 internalization. Targeting alternative membrane glycosaminoglycans may aid in improving the efficacy of rAAV-2 for pulmonary applications.
Highlights
Adeno-associated virus type 2 (AAV-2) is a non-enveloped parvovirus that has demonstrated efficacy as a gene replacement vector in numerous tissues [1]
Heparinase III treatment efficiently removes heparan sulfate proteoglycans (HSPG) from epithelial cell membranes To study the effects of HSPG removal on rAAV-2 infection efficacy and to ensure maximal removal of HSPG from cell membranes, human tracheal epithelial HTE cell line, fetal human tracheal epithelial FHTE cell line, and cystic fibrosis IB3-1 cell line were treated with increasing amounts of heparinase III until a plateau in effect was noted
Chinese Hamster Ovary (CHO) mutant cell line pgsD-677 completely deficient in surface HSPG demonstrates only moderately decreased susceptibility to r-AAV2 infection, while CHO mutant cell line pgsA-745 deficient for all glycosaminoglycans demonstrates dramatic decrease in susceptibility To determine if the observed moderate effect of absence of surface HSPG on rAAV-2 infection efficacy was seen in cell lines besides respiratory epithelia, we studied mutant CHO cell lines pgsD-677, previously demonstrated to be completely deficient in HSPG [23,24], and pgsA-745, deficient for all surface GAG
Summary
Adeno-associated virus type 2 (AAV-2) is a non-enveloped parvovirus that has demonstrated efficacy as a gene replacement vector in numerous tissues [1]. In 1998 Summerford and Samulski first identified heparan sulfate proteoglycans (HSPG) present on cell membrane surfaces as a receptor for AAV-2 infection [2]. HfFrioegmpuarrreiensa1psieraItIIotrryeaetpmitheneltiaelffciecilel nmtelymrbermanoevses heparan sulfate Heparinase III treatment efficiently removes heparan sulfate from respiratory epithelial cell membranes. Adeno-associated virus type 2 (AAV-2) attachment and internalization is thought to be mediated by host cell membrane-associated heparan sulfate proteoglycans (HSPG). Lack of HSPG on the apical membrane of respiratory epithelial cells has been identified as a reason for inefficient rAAV-2 infection in pulmonary applications in-vivo. The aim of this investigation was to determine the necessity of cell membrane HSPG for efficient infection by rAAV-2
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