Published online 12 March 2015 Nucleic Acids Research, 2015, Vol. 43, No. 11 e73 doi: 10.1093/nar/gkv202 Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion Yongxiang Zheng1, Fei Yu1, Yiming Wu1, Longlong Si1, Huan Xu1, Chuanling Zhang1, Qing Xia1, Sulong Xiao1,QiWang1,2, Qiuchen He1, Peng Chen3, Jiangyun Wang4, Kazunari Taira1, Lihe Zhang1 and Demin Zhou1,* 1State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China, 2Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China, 3College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China and 4Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Received July 04, 2014; Revised February 09, 2015; Accepted February 26, 2015 Downloaded from ABSTRACT and nondividing cells and integrate the delivered gene into the host genome (6). Currently the lentiviral vectors have With the aim of broadening the versatility of lentivi- been frequently used by biologists to deliver genetic mate- ral vectors as a tool in nucleic acid research, we ex- rial into a variety of mammalian cells (7). This process can panded the genetic code in the propagation of lentivi- also be performed inside living organisms (8) and has been http://nar.oxfordjournals.org/ ral vectors for site-specific incorporation of chemical explored in clinical trials for gene therapy (9–11). In par- moieties with unique properties. Through systematic ticular, two successful lentiviral vector-based clinical trials exploration of the structure–function relationship of in 2013 for treatment of Wiskott-Aldrich syndrome (WAS) lentiviral VSVg envelope by site-specific mutagene- and Metachromatic leukodystrophy (MLD) have further sis and incorporation of residues displaying azide- strengthened the potential use and impact of the lentiviral and diazirine-moieties, the modifiable sites on the vector, especially for boosting lentiviral gene therapy (10– vector surface were identified, with most at the PH 12). The component that determines the tissue/cell tropism domain that neither affects the expression of en- of the lentiviral vector and the gene delivery efficiency is by guest on August 24, 2015 velope protein nor propagation or infectivity of the the envelope glycoprotein of VSV (VSVg) (Figure 1A), progeny virus. Furthermore, via the incorporation of which mediates host receptor binding, internalization and such chemical moieties, a variety of fluorescence trafficking within the targeted cells (13,14). A deeper un- probes, ligands, PEG and other functional molecules derstanding of the properties of the VSVg envelope via are conjugated, orthogonally and stoichiometrically, structure–function exploration may expand the potential to the lentiviral vector. Using this methodology, a of diverse applications of lentiviral vectors in biomedicine, facile platform is established that is useful for track- bio-technology and even nanotechnology (15). Incorpora- ing virus movement, targeting gene delivery and de- tion of chemical moieties such as an azide group on the tecting virus–host interactions. This study may pro- proper position of the vector surface (Figure 1B) would vide a new direction for rational design of lentivi- facilitate click chemistry-mediated introduction of unique properties that enable single-virus tracking, targeted nucleic ral vectors, with significant impact on both basic re- acid delivery, vector developing for gene therapy and block search and therapeutic applications. building for the construction of nano-structured materi- als. For many of these contemplated applications, the site- INTRODUCTION specific display of chemical moieties may be crucial, since Lentiviral vectors derived from the human immunodefi- random or improper modifications of viruses may abrogate ciency virus (HIV-1) have become major tools for gene de- the propagation and even the infectivity of progeny viruses livery in mammalian cells. They consist of the structural (16,17). and enzymatic components of HIV-1 enveloped by vesicular Here, we report the development of a viable and facile stomatitis virus glycoprotein (VSVg) (1–5). Such lentiviral platform for site-specific incorporation of unnatural amino vectors have been received particular attention among many acids into lentiviral vectors via genetic code expansion. Ge- different viruses due to its ability to infect both dividing netic code expansion is an artificial process that usually uses *To whom correspondence should be addressed. Tel: +86 10 8280 5857; Fax: +86 10 8280 5857; Email: [email protected] Present address: Demin Zhou, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] e73 Nucleic Acids Research, 2015, Vol. 43, No. 11 PAGE 2 OF 12 barkeri MS pyrrolysyl tRNA synthetase/tRNACUA pair (MbPylRS/tRNACUA) and pSupAR-Mb-DiZPK-RS for site-specific incorporation of UAAs were developed in- house as previously reported (23,24), The mutant plas- mids (pCMV-VSVG-TAG) containing an amber codon within the open reading frame were obtained from the wild-type pCMV-VSVG via site-directed mutagenesis (Ag- ilent Technologies) and confirmed by gene sequencing (BGI Beijing). All plasmids used for transfection were amplified using a Maxiprep kit (Promega), according to the manufacturer’s instructions. Antibodies used included a mouse monoclonal anti-VSVg (Sigma), rabbit mono- clonal anti-GAPDH (Cell Signaling Technology), goat anti- mouse IgG-HRP or goat anti-rabbit IgG-HRP (ZSGB- BIO), monoclonal anti-integrin ␣v␤3 (Millipore), normal rabbit IgG (Santacruz), normal mouse IgG (Santacruz), Alexa 488-labeled secondary antibody (Cell Signaling Tech- nology) and Alexa 594 Donkey Anti-Mouse IgG (Invitro- gen). Downloaded from N⑀-2-azidoethyloxycarbonyl-L-lysine (NAEK) and ([3-(3-methyl-3H-diazirine-3-yl)-propamino] carbonyl)- N⑀–lysine (DiZPK) were synthesized as previously reported (23,24) and characterized by mass spectroscopy (Supple- mentary Figure S1). Dibenzocyclooxtyne -arginine-glycine- aspartic acid (DIBO-RGD) and Dibenzocyclooxtyne- http://nar.oxfordjournals.org/ arginine-alanine -aspartic acid (DIBO-RAD) were Figure 1. Genetic and site-specific display of unnatural amino acids purchased from GL Biochem (Shanghai) Ltd and dis- (UAAs) on the envelope of lentiviral vectors. (A) The structure of lentivi- solved in phosphate buffered saline (PBS) to a desired ral VSVg protein. Total 33 residues, marked in red, within VSVg were mu- concentration. DIBO-Alexa 488, 1, 1-dioctadecyl-3,3,3,3 tated in this study. (B) Schematic representative of lentiviral vector engi- -tetramethylindocarbocyanine perchlorate (DiI) and 4,6- neering via genetic code expansion for site-specific incorporation of UAAs (NAEK or DiZPK) on the vector surface. (C) Schematic representation of diamidino-2-phenylindole (DAPI) was purchased from genetic code expansion for site-specific incorporation of UAAs. The UAAs Life Technologies; Dibenzocyclooxtyne- Polyethylene were incorporated into VSVg protein via UAG-code and an orthogonal glycol (DIBO-PEG) was synthesized in-house. by guest on August 24, 2015 aaRS/tRNA. a stop codon to encode an unnatural amino acid (UAA) into a protein of interest (18–22)(Figure1C). The high fi- delity of genetic code expansion plus the facile technique Cell culture of mutagenesis allows precise control of the placement of the chemical moiety-containing UAA and the following or- Cell lines Human Embryonic Kidney 293T (HEK-293T), thogonal modification into the lentiviral vector, providing HeLa, MDA-MB-435S, U-87 MG (U87) and Michigan a foundation for the systematic structure–function explo- Cancer Foundation-7 (MCF-7) were obtained from the ration of such a vehicle for delivery of a variety of func- National Platform of Experimental Cell Resources for tional nucleic acid molecules. Via the display of azide- and SCI-Tech (Beijing, China). HEK-293T, HeLa and MCF- diazirine-moieties, modifiable sites on the surface of the 7 cells were maintained in Dulbecco’s Modified Eagle lentiviral vector were identified that have a minimal ef- Medium (DMEM, Macgene), Roswell Park Memorial In- fect on propagation and infectivity, even upon tailing with stitute medium 1640 (RPMI1640, Macgene) medium and Modified Eagle Medium (MEM; Macgene), respectively, proper ligands. Using this approach, site-specific and stoi- / chiometric conjugation of a variety of biophysical probes, were supplemented with 10% (v v) fetal bovine serum (FBS; Gibco), 100-IU/ml penicillin and 100-␮g/mL streptomycin tags and novel functional molecules on the envelope of ◦ lentiviral vectors is realized, which has a significant impact (Macgene). These cells were cultured at 37 C in a humidi- fied incubator under 5%2 CO and passaged every 2 days at on single-virus tracking, targeted gene delivery and other ∼ biological applications. 80–90% confluency. U87 cells were maintained in MEM supplemented with 10% (v/v) FBS, 100-IU/ml penicillin and 100-␮g/ml streptomycin.