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Synthetically Functionalized Retroviruses Produced from the Bioorthogonally Engineered Cell Surface Shirley Wong† and Young Jik Kwon*,†,‡,§,|| Medicinal Chemistry and Pharmacology Program, ‡Department of Pharmaceutical Sciences, §Department of Chemical Engineering and Materials Science, and Department of Biomedical Engineering, University of California, Irvine, California 92697, United States )
†
bS Supporting Information ABSTRACT: Conjugation of desired molecules onto retroviral surfaces through the ease of the bioorthogonal functionalization method was demonstrated. Oxidation of surface sialic acids using periodate and further p-anisidine-catalyzed conjugation with aminooxy-bearing molecules were used to directly label retroviral envelope with a fluorescent dye. The retroviral particles that were produced from a bioorthogonally functionalized virus producing cell surface and further tethered with magnetic nanoparticles were efficiently purified by simple magnetic column separation and capable of magnet-directed transduction.
D
ue to the ability to efficiently deliver DNA or RNA to target cells, viral vectors have been predominantly employed in clinical trials for inherited and acquired genetic abnormalities (e.g., immune deficiencies, cancer, and AIDS) as well as in stem cell-targeted gene transfer.1-5 Retroviruses, including lentiviruses, are one of the most popularly used viral vectors in clinical gene therapy.6 Retroviruses mediate stable integration of the transgene into host chromosomes for sustained expression, with relatively lower immunogenicity than nonenveloped viruses (e.g., adenoviruses and adeno-associated viruses).7-9 Nevertheless, many challenges still exist in employing retroviral vectors for gene therapy. First, the current methods of retroviral production achieve a titer in the range 105-107 viral particles per mL, which requires further concentration to meet the requirements for clinical use.6,10,11 Also, retroviral supernatants collected from the culture of virus producing cells contain various types of impurities which inhibit transduction, lead to cell toxicity, or induce immune responses.6,12 Conventionally used viral purification methods include ultracentrifugation in cesium chloride or sucrose gradients, which often results in significant loss of viral infectivity.6,7,13 Facile introduction of desired functional molecules onto the surface for easy purification and concentration, targeted delivery, and molecular imaging is an indispensable property for ideal gene delivery vectors.14-16 Although successful conjugations of synthetic polymers and polypeptides with adenovirus and adenoassociated virus have been demonstrated,17,18 chemical modifications of retroviruses still present a significant challenge due to low levels of infection after surface modifications, which compromise its utility for gene delivery-based applications.19 For r 2011 American Chemical Society
instance, redirection of ecotropic Moloney murine leukemia retroviruses (MoMLVs) by chemical addition of sugar molecules onto its surface to endow new receptor-binding functions severely reduced transduction.20 In addition, the purification of chemically modified viral particles from unconjugated small molecules using the currently used methods (e.g., dialysis and centrifugation) is cumbersome and yields poor recovery of viral particles.21 Alternatively, incorporating adaptor peptides onto the surfaces of retroviruses by genetically engineering virus producing cells was reported.14 Although it accounts for labeling with an assortment of substrates from the adaptor peptide, it has been shown that the process may disrupt other proteins already on the surfaces that are crucial for viral infectivity.11 Another limitation of this approach includes its lengthy process. Therefore, fast, convenient, versatile, and efficient methodologies for synthetically functionalizing retroviral particles are highly desired. Retroviruses are equipped with the virus producing cellderived envelope during the budding process.22 This implies that chemical and biochemical properties of the producer cell membrane are carried over by the retroviral envelope. Therefore, a new hypothesis for obtaining functionalized retroviral particles by chemically modifying the producer cell membrane surface was explored in this study. Recently, oxime ligation of aminooxyfunctionalized molecules with aldehyde groups of oxidized sialylated glycoproteins by sodium periodate has been reported.23 Because many retroviral producing cells display sialic acids on their surface, Received: November 22, 2010 Revised: January 22, 2011 Published: January 31, 2011 151
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were treated with sodium periodate and further reacted with 10 μM aminooxy-AF488 in the presence of 10 mM p-anisidine as a conjugation catalyst. Unreacted and remaining reagents were removed by centrifugation, and the fluorescence intensity of aminooxy-AF488-conjugated viral particles suspended in PBS was measured (detailed experimental procedures are described in Supporting Information). Different conditions were used to evaluate the roles of periodate-mediated oxidation of sialic acids and the catalytic activity of p-anisidine in aminooxy-AF488 conjugation to retroviral particles. As shown in Figure 2, the results revealed highly efficient bioorthogonal conjugation of aminooxy-AF488 to retroviral particles. It was also confirmed that both oxidation of viral surface by sodium periodate and use of p-anisidine as a catalyst are required for efficient oxime ligation with aminooxy-AF488. Oxidized viral particles were approximately 70 times more efficiently conjugated with aminooxyAF488 than unoxidized viruses (both with p-anisidine). Addition of p-anisidine to periodate-oxidized viral particles enhanced conjugation efficiency of aminooxy-AF488 approximately 35 times. The supernatants were also quantified for viral particles using reverse transcriptase (RT) activity assay, and the conjugated aminooxy-AF488 was quantified by comparing the fluorescence intensity of the supernatants with a calibration curve obtained by using free dyes at known concentrations. It was estimated that approximately 7.2 108 (1.2 fmol) of aminooxyAF488 was conjugated per retroviral particle after bioorthogonal labeling (Figure 2). The results confirmed that retroviral particles were efficiently labeled with an aminooxy-functionalized fluorescent dye via periodate-mediated oxidation and p-anisidinecatalyzed conjugation. It should be noted that the conjugation was carried out in the supernatant collected from virus producing cell culture without purification, which often results in a significant loss of viral particles and viral activity (i.e., no need for centrifugation and dialysis before conjugation). Efficient and specific labeling of fluorescent dyes (Figure 2) implies that viral particles can be easily purified when they are tagged with separation markers (e.g., biotin and His-tag) even at the level of virus producing cells, as illustrated in Figure 1. First, the optimized time for retroviral collection after functionalization of virus producing cells was explored. Sialic acids on the surface of 293 GPG/eGFP retrovirus producing cells were oxidized by 1 mM sodium periodate, and the resulting aldehyde groups were conjugated with 100 μM aminooxy-biotin in the presence of 10 mM p-anisidine via oxime ligation (detailed experimental procedures in Supporting Information). The oxidation and conjugation were carried out on live cells in the culture medium, and unreacted and unconjugated molecules were easily rinsed away with PBS. Then, supernatants were collected at various post-biotinylation times and mixed with antibiotin magnetic particles at the optimum ratio to viral particles, as determined for ∼90% magnetic purification efficiency (Figure S1 in Supporting Information). The magnetically labeled viral particles were purified by running them through a magnetic column and used to transduce NIH 3T3 cells. The rate of transduced cells was determined by flow cytometry. Experimental details are described in the Supporting Information. Unsuccessfully labeled viral particles, which were not bound but washed out from the magnetic column, were also used to transduce the cells under the same conditions for comparison. Transduction efficiencies of both unbound (unsuccessfully labeled) and bound (successfully conjugated with magnetic particles) viral particles were normalized with the transduction efficiency of the viral supernatant that
Figure 1. Production of functionalized retrovirus by bioorthogonally modifying virus producing cell surface whose sialylated glycoproteins (green) on the phospholipid cell membrane (purple) are oxidized to generate aldehyde groups using sodium periodate. Aminooxy-functionalized molecules (yellow) are conjugated with aldehyde groups via oxime linkages in the presence of p-anisidine (catalyst).
it was hypothesized that the novel bioorthogonal cell surface modification would generate chemically functionalized retroviral particles in a simple, fast, and efficient way (Figure 1). This bioorthogonal approach enables labeling at specific sites, rather than introducing conjugates randomly and disrupting envelope membrane and proteins. Applying simple chemistry at the cellular level resolves the problem of implementing difficult and laborious genetic alterations. Unconjugated functional molecules can also be easily removed by simply rinsing them from the producer cells, and viral particles that are labeled with a separation tag (e.g., biotin and His-tag) can be easily purified (i.e., removing the need for inefficient and lengthy purification by ultracentrifugation or dialysis). This method is highly efficient and specific under physiological conditions (e.g., cell culture medium), which allows modifying virus producing cells with various functional molecules at different incubation times. Either completely functionalized retroviral particles or aldehyde-bearing retroviral particles for further conjugation with desired molecules can also be prepared. First, whether the retroviral envelope can be bioorthogonally conjugated with aminooxy-bearing Alexa Fluor 488 (AminooxyAF488) after sodium periodate oxidation to generate aldehyde groups on the viral surface was investigated. eGFP-encoding MoMLVs pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) as envelope proteins, were produced from 293 GPG/ eGFP retrovirus producing cells. The collected viral supernatants 152
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Figure 2. Confirmation and quantification of efficient bioorthogonal conjugation of aminooxy-AF488, in the presence of 10 mM p-anisidine as a catalyst, to periodate-oxidized VSV-G pseudotyped MoMLV particles. The sialic acids of the viral surface glycoproteins were oxidized to bear aldehyde groups by adding 1 mM sodium periodate for 30 min at 4 °C and quenched with 1 mM glycerol. After rinsing twice with PBS by centrifugation at 13 200 rpm for 8 min, the viral pellet was resuspended in 1 mL of PBS with 10 mM p-anisidine and 10 μM aminooxy-AF488 for 90 min at 4 °C. After rinsing three times, fluorescence intensity of the viral pellets resuspended in PBS was measured using a fluorescence microplate reader. Specific fluorescence intensity was determined by dividing its fluorescence intensity with viral particle number measured by RT activity assay. Conjugation efficiency (fmol AF488/viral particle) was determined from the fluorescence intensity of the viral pellets, which was converted to moles of conjugated dye using a calibration standard of known concentration of free aminooxy-AF488 in the same volume of PBS.
at 24 h after biotinylation of virus producing cells showed approximately 12 times higher transduction efficiency than unpurified viral supernatant. Since unbound, bound, and unseparated supernatants were in the same final volume (Supporting Information), theoretically the combined amount of retroviral particles in unbound and bound supernatants cannot be higher than that of the unseparated supernatant. Therefore, the substantially higher transduction efficiency of magnetically purified retroviral supernatant collected 24 h post-biotinylation compared with unseparated supernatant indicates two points: (1) most of the retroviral particles were successfully labeled with magnetic particles, as indicated by minimal transduction in the unbound supernatant, and (2) magnetic purification removed transduction inhibitors6 that were originally rich in the supernatant collected from the cell culture. The results also indicate that it took 24 h for the labeled proteins in the plasma membrane to be incorporated into a budding virus (Figure 3). After the 24 h post-biotinylation time point, enhanced transduction by magnetically purified retroviral particles decreases as hypothesized by the internalization of cell surface biotin for the recycling of glycoproteins.24 The results shown in Figure 3 clearly demonstrated that labeled retroviral particles can be easily obtained by bioorthogonally functionalizing virus producing cell surfaces. The retroviral particles that are produced from functionalized virus producing cells and further tethered with separation tags (e.g., magnetic particles) directly in the cell culture medium can be easily purified for significantly enhanced transduction efficiency. Magnetically labeled retroviral particles can not only be easily purified and concentrated by magnetic column separation, but also used for magnet-directed transduction. The retroviral supernatant collected at 24 h after bioorthogonal biotinylation of virus producing cells (Figure 3) was conjugated with antibiotin magnetic particles at the optimized ratio (Figure S1). The magnetically labeled retroviral particles were incubated with NIH 3T3 cells on a tissue culture dish where a patterned magnet
Figure 3. Optimized virus collection time based on enhanced transduction efficiency by magnetically purified retroviral particles. The surface of virus producing 293 GPG/eGFP cells was oxidized by periodate and aminooxy-biotin was conjugated in the presence of p-anisidine. The supernatants collected from biotinylated cells were mixed with antibiotin magnetic particles in order to prepare magnetically labeled retroviruses. The resulting retroviral particles were purified via magnetic column separation and used to transduce NIH 3T3 cells. For clear presentation, transduction efficiencies of magnetically purified (bound) and unsuccessfully labeled (unbound) retroviruses were normalized to that of the supernatant that was mixed with magnetic particles but not loaded onto the magnetic column. Detailed experimental descriptions are in the Supporting Information.
was mixed with magnetic particles but not loaded onto the magnetic column. This normalization is void of variance in daily viral concentrations and clearly presents the effects of the magnetic purification. The results shown in Figure 3 demonstrated that retroviral particles in the supernatants collected until 12 h after biotinylation of the virus producing cells were not efficiently conjugated with magnetic particles (no biotinylation). However, the purified retroviruses from the supernatant collected 153
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’ ASSOCIATED CONTENT
bS
Supporting Information. Cell culture and virus production, viral titer and viral particle quantification, Alexa Fluor 488 dye conjugation onto viral particles, preparation of magnetic particle-conjugated retroviral particles, optimized supernatant collection time after biotinylation of virus producing cell surface, magnet-directed transduction by magnetic particle-conjugated viruses. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Figure 4. Magnet-directed transduction of magnetically labeled retroviral particles that were produced from bioorthogonally engineered cell surface. NIH 3T3 cells plated at 7.5 104 cells in a tissue culture dish (6 cm in diameter) were incubated with magnetically labeled retroviral particles with a patterned magnet placed underneath for 2 h. Magnetically labeled retroviral particles were prepared using approximately 12 000 biotinylated retroviruses (determined by RT activity assay) with 50 μL of antibiotin MPs for 30 min. After transduction, virus-containing media were replaced with fresh media, and the cells were further incubated for another 18 h, followed by incubation in G418-containing media (1 mg/mL) for selection of transduced (neomycin-resistant) cells. After one week of incubation with G418, selected cells were stained with 0.1% methylene blue in methanol for visualization.
Corresponding Author
*Corresponding author. 916 Engineering Tower Irvine, CA 92697-2575. Tel: þ949 824 8714. Fax: þ949 824 2545. E-mail address:
[email protected] (Y.J. Kwon).
’ REFERENCES (1) Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., Casanova, J. L., Bousso, P., Le Deist, F., and Fischer, A. (2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669–672. (2) Sterman, D. H., Treat, J., Litzky, L. A., Amin, K. M., Coonrod, L., Molnar-Kimber, K., Recio, A., Knox, L., Wilson, J. M., Albelda, S. M., and Kaiser, L. R. (1998) Adenovirus-mediated herpes simplex virus thymidine kinase gene delivery in patients with localized malignancy: Results of a phase 1 clinical trial in malignant mesothelioma. Hum. Gene Ther. 7, 1083–1092. (3) Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 1–12. (4) Edelstein, M. L., Abedi, M. R., and Wixon, J. (2007) Gene therapy clinical trials worldwide to 2007 - an update. J Gene Med. 9, 833–842. (5) Kohn, D. B., Bauer, G., Rice, R., Rothschild, J. C., Carbonaro, D. A., Valdez, P., Hao, Q. H., Zhou, C., Bahner, I., Kearns, K., Brody, K., Fox, S., Haden, E., Wilson, K., Salata, C., Dolan, C., Wetter, C., AguilarCordova, E., and Church, J. (1999) A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34þ cells from the bone marrow of human immunodeficiency virus-1-infected children. Blood 94, 368–371. (6) Rodrigues, T., Carvalho, A., Roldao, A., Carrondo, M. J. T., Alves, P. M., and Cruz, P. E. (2006) Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors. J. Chromatogr., B 837, 59–68. (7) Baekelandt, V., Eggermont, K., Michiels, M., Nuttin, B., and Debyser, A. (2003) Optimized lentiviral vector production and purification procedures prevents immune response after transduction of mouse brain. Gene Ther. 10, 1933–1940. (8) Yang, Y., and Wilson, J. M. (1995) Clearance of adenovirusinfected hepatocytes by MHC class I-restricted CD4þ CTLs in vivo. J. Immunol. 155, 2564–2570. (9) Recchia, A., Parks, R. J., Lamartina, S., Toniatti, C., Pieroni, L., Palombo, F., Ciliberto, G., Graham, F. L., Cortese, R., La Monica, N., and Colloca, S. (1999) Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector. Proc. Natl. Acad. Sci. U.S.A. 96, 2615–2620. (10) Saha, K., and Wong, P. K. Y. (1994) A simple method for obtaining highly viable virus from culture supernatant. J. Virol. Methods 46, 349–352. (11) Lei, Y., Joo, K., Zarzar, J., Wong, C., and Wang, P. (2010) Targeting lentiviral vector to specific cell types through surface displayed single chain antibody and fusogenic molecule. Virol. J. 7, 1–12.
was applied underneath. Transduced cells (neomycin-resistant cells) survived and were selected to proliferate for one week before visualization with methylene blue staining. Supporting Information provides the detailed description of the experiment. The results clearly showed that significantly more cells were transduced in the areas where magnetic forces were applied than other areas without magnetic forces (Figure 4). This confirms that a majority of magnetically labeled retroviral particles were directed to transduce the cells where the external magnetic force was applied. Therefore, magnetically labeled retroviral particles can be used for targeted gene delivery by applying magnetic forces in desired target tissues for localization of significant quantities of viral particles. Magnetic particles are also potent contrast agents for magnetic resonance imaging (MRI). Magnetically labeling retroviral particles by bioorthogonally functionalizing virus producing cell surfaces is a promising approach to prepare novel viral vectors for gene delivery in combination with in vivo imaging of vector biodistribution by MRI.25,26 In conclusion, in order to address the limited chemical modifications of retroviral particles, the surfaces of virus producing cells were bioorthogonally functionalized after being oxidized by periodate and conjugated with aminooxy-functionalized molecules in the presence of p-anisidine. The retroviral particles carrying a functionalized envelope, which was derived from the virus producing cell membrane, were further conjugated with versatile molecules such as magnetic nanoparticles for highly convenient and efficient purification and effective magnet-directed transduction. It was also demonstrated that the viral envelope can also be directly modified using the bioorthogonal approach. Further optimization of the bioorthogonal conjugation method, molecular characterization and quantification of bioorthogonally functionalized retroviral particles and virus producing cells, and conjugation of other types of desired molecules for targeted delivery and stimuli-responsive transduction are currently under investigation. The novel strategy developed in this study can be utilized to overcome several key challenges in retroviral gene delivery. 154
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(12) Tuschong, L., Soenen, S. L., Blaese, R. M., Candotti, F., and Muul, L. M. (2002) Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy. Hum. Gene Ther. 13, 1605–1010. (13) Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. U.S.A. 90, 8033–8037. (14) Morizono, K., Xie, Y., Helguera, G., Daniels, T. R., Lane, T. F., Penichet, M. L., and Chen, I. S. Y. (2009) A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide. J. Gene Med. 11, 655–663. (15) Pariente, N., Mao, S. H., Morizono, K., and Chen, I. S. Y. (2008) Efficient targeted transduction of primary human endothelial cells with dual-targeted lentiviral vectors. J. Gene Med. 10, 242–248. (16) Steinmetz, N. F. (2010) Viral nanoparticles as platforms for next-generation therapeutics and imaging devices. Nanomedicine 6, 1–10. (17) O’Riordan, C. R., Lachapelle, A., Delgado, C., Parkes, V., Wadsworth, S. C., Smith, A. E., and Francis, G. E. (1999) PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Hum. Gene Ther. 10, 1349–1358. (18) Zhong, Q., Koll, J. K., and Schwarzenberger, P. (2001) A novel, high transduction efficiency, potentially safety-improved, gene transfer system. J. Biol. Chem. 276, 24601–24607. (19) Boerger, A. L., Snitkovsky, S., and Young, J. A. T. (1999) Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types. Proc. Natl. Acad. Sci. U.S.A. 96, 9867– 9872. (20) Benedict, C. A., Tun, R. Y. M., Rubinstein, D. B., Guillaume, T., Cannon, P. M., and Anderson, W. F. (1999) Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus-cell fusion. Hum. Gene Ther. 10, 545–557. (21) Williams, S. L., Nesbeth, D., Darling, D. C., Farzaneh, F., and Slater, N. K. H. (2005) Affinity recovery of Moloney Murine Leukemia Virus. J. Chromatogr., B 820, 111–119. (22) Ory, D. S., Neugeboren, B. A., and Mulligan, R. C. (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. U.S.A. 93, 11400–11406. (23) Zeng, Y., Ramya, T. N. C., Dirksen, A., Dawson, P. E., and Paulson, J. C. (2009) High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6, 207–209. (24) Volz, B., Orberger, G., Porwoll, S., Hauri, H. P., and Tauber, R. (1995) Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells. J. Cell Biol. 130, 537–551. (25) Singh, R., and Kostarelos, K. (2009) Designer adenoviruses for nanomedicine and nanodiagnostics. Cell 27, 220–229. (26) Raty, J. K., Liimatainen, T., Wirth, T., Airenne, K. J., Ihalainen, T. O., Huhtala, T., Hamerlynck, E., Vihinen-Ranta, M., Narvanen, A., Yla-Herttuala, S., and Hakumaki, J. M. (2006) Magnetic resonance imaging of viral particle biodistribution in vivo. Gene Ther. 13, 1440– 1446.
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