334. Micro-Environmental Effects on Gene Transfer by Pseudotyped Lentiviral Vectors

Abstract

Efforts to target lentivectors to specific tissues in vivo have primarily focused on vector pseudotyping with heterologous envelopes. One barrier to effective gene delivery into the different target organs following intravenous administration of lentivectors may be to find conditions that can bypass the endothelial barrier, which is likely the problem with the use of the conventional vesicular stomatitis virus G protein envelope (VSV-G). Towards this end, we conjectured that micro-environmental factors within specific tissues may also affect lentivector transduction efficiency. In this study, we examined the effect of micro-environmental conditions on lentivectors pseudotyped with two Bunyaviral envelopes. First, the Hantaan (HTNV) envelope was derived from a strain associated with hemorrhagic fever resulting in renal syndrome, and the second, Black Creek Canal Virus (BCCV) was derived from a strain associated with hemorrhagic fever with pulmonary syndrome. These viral envelopes were selected because, although closely related, they display distinct tissue tropisms, i.e., the HTNV strain preferentially infects the kidney, which is our target organ of interest. Preliminary evidence indicates that transduction of HeLa cells by HTNV, but not BCCV, pseudotypes can be significantly improved under acidic conditions and in a dose-dependent fashion in the presence of NaCl (3.6 ± 1.8 × 105 T.U/mL; n = 3) or urea (3.7 ± 2.9 × 105 T.U/mL; n = 3) compared to control conditions (9.3 ± 3.7 × 104 T.U./mL; n = 3). Moreover, decreasing the pH to 6.0 increased viral transduction with HTNV-pseudotyped lentivectors to 1.9 ± 1.6 × 105 T.U./mL from 9.3 × 104 T.U./mL. These conditions are important since they mimic those found within the kidney in vivo. The control VSV-G pseudotypes behaved similarly to BCCV pseudotypes. Enhanced transduction of cells by HTNV pseudotypes under these conditions was shown to occur in Huh7 hepatoma, C166 endothelial and RAG-1 renal carcinoma lines. To investigate the method of viral entry into the cells, we pre-treated the cells with 10 mM NH4Cl to block endosomal acidification. Transduction of cells by HTNV pseudotypes was inhibited by >90% in the presence of NH4Cl indicating that vector entry occurred via an endosomal pathway similar to that of native HTNV. Since we know that electrostatic interactions can affect the binding of many viruses to cell surface heparin sulfate (HS) proteoglycans, and we have investigated some of the conditions that could influence those interactions, we further examined the effect of HS and related molecules on HTNV and BCCV pseudotypes. Transduction by HTNV pseudotypes was significantly inhibited (>50%) in media supplemented with heparin, indicating this particular pseudotype requires HS proteoglycans to establish infection. Conversely, transduction was enhanced by 1–2 orders of magnitude in the presence of dextran sulfate (n = 3). Our findings indicate that micro-environmental conditions mimicking those within the kidney can significantly enhance transduction by HTNV pseudotypes and thus, improve our understanding of basic viral biology and their applications for effective vector design. Funded by NIDDK 69620–01.