A method to mathematically determine transduction efficiency of lentivirus in HeLa cells Research Article

Gene Therapy and Molecular Biology Vol 15, page 138 Gene Ther Mol Biol Vol 15, 138-146, 2013 A method to mathematically determine transduction efficiency of lentivirus in HeLa cells Research Article Zhipin Liang 1, Bin Liu 1, Xuechao Zhao 1, Chang Liu 1,*, Xiaohong Kong 1,* 1 Laboratory of Medical Molecular Virology, School of Medicine, Nankai University, Weijin Road No.94, Nankai District, Tianjin, P.R. China *Correspondence: Chang Liu and Xiaohong Kong, Laboratory of Medical Molecular Virology, School of Medicine, Nankai University, Weijin Road No.94, Nankai District, Tianjin, P.R. China. Tel: +86-22-23504347; Fax: +86-22-23499505. Email: changliu@nankai.edu.cn; kongxh@nankai.edu.cn. Keywords: 50% Transduction efficiency (TE50), Lentiviral vector, Gene therapy, Flow cytometry Received: 18 December 2013; Revised: 29 December 2013 Accepted: 30 December 2013; electronically published: 31 December 2013 Summary Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are widely applied in gene transfer and gene therapy because of their high transduction efficiency and stable expression. There are various quantification methods for the transduction efficiency (TE) calculation of lentiviral vectors, while most of them usually need serial dilutions and experimental materials costing. So it is required to develop a feasible quantification method for lentiviral vectors' TE calculation. Here, we deduced a math equation between the number of infectious viral particles (v) and the transduction efficiency (TE): v = a ln (1-TE) + b. An HIV-1 based lentiviral vector FG12 encoding the GFP reporter gene was used to evaluate practicability of this method. According to the math equation, TE50 of FG12 was verified in different number of HeLa cells. Our results documented that the math equation was adopted into the TE calculation. Comparing with routine TE50 determination method, this method needed fewer serial dilutions and was more feasible. I. Introduction: Viral vectors are widely applied in gene transfer and gene therapy due to their inherent ability of viruses to introduce genetic material into target cells (Machida 2003; Papayannakos and Daniel 2012; Apolloni et al., 2013). Especially, human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are important tools of gene transduction because of their stable integration in dividing and nondividing cells and allowing transgene longterm expression (Ikeda et al., 2003; Leyva et al., 2011; Gay et al., 2012). Lentiviral vectors biological and safety properties have been improved by multiple genetic modifications (Pauwels K Fau - Gijsbers et al., 2009; Matrai et al., 2010; Persons 2010; Dropulic 2011), 138

Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells and now they have been developed into the third generation (Salmon and Trono 2006). FG12 used in this study is one of the third generation HIV-based lentiviral vectors. Security and transduction efficiency of FG12 have been improved through the following optimizations: 1) deletion of the U3 region to generate self-inactivation (SIN) vectors (Miyoshi et al., 1998), 2) addition of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to increase expression levels, addition a reporter gene of GFP to be observed and quantified easily (Qin et al., 2003), 3) pseudotyping with vesicular stomatitis virus (VSV) glycoproteins extending cell tropism (Lois et al., 2002). Because of FG12 s features described above, it has been successfully applied in the field of immunization and anti-hiv-1 infection (Qin et al., 2003; Yang et al., 2008). All of lentiviral vectors applications require an accurate and reliable method of determination of the transduction efficiency. For this purpose, several methods have been developed (Sanburn and Cornetta 1999; Geraerts et al., 2006; Yamamoto et al., 2006; Leyva et al., 2011). Most of them are focusing on quantification of reporter gene expression or integration and involving a cell transduction step to provide the proportion of infectious particles contained in the vector stocks. The transduction efficiency (TE) is usually evaluated based on the doses of viral vector required to obtain 50% (TE50) of positive cells being transduced (Gay et al., 2012). Quantification methods of TE50 routinely need the dose-response curve of transduced positive cells versus vector input reached the 100% value. They usually need tedious serial dilutions and adequate amount of vector stocks (Salmon and Trono 2006). In our study, HeLa cells infected with the lentiviral vector FG12 were taken as a research model. Based on the simple hypothesis that infections by individual viral particles are individual events (Sigal et al., 2011), a math equation between the infectious viral particle number and the transduction efficiency was deduced. According to this equation, TE50 of FG12 to HeLa cells was easily calculated and also verified in different numbers of HeLa cells. II. Materials and Methods: A. Cell culture The HEK293T cell line, derived from a transformation of HEK293 cell with the SV40 large T gene, is used for virus packaging (Salmon and Trono 2006). HeLa cell line was often used in virus titer assay (Naldini et al., 1996; Salmon and Trono 2006). Both kinds of cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10 % fetal bovine serum (FBS), 100 U/ ml penicillin, and 100 μg/ ml streptomycin at 37 o C with 5 % CO 2. B. Virus packaging and ultracentrifugation Viral vector plasmid pfg12 (25 μg) and helper plasmids (prsvrev 6.25 μg, phcmv-g 12.5 μg and pmdlg/prre 7.5 μg) were cotransfected into the HEK293T cells (1 10 7 cells in 15 cm dish). Total plasmids and polyethylenimine (PEI) were mixed for 10 min in the serum-free DMEM with a ratio of 51.25 μg: 205 μg. Medium was refreshed at 8 hours post transfection. Then 48 hours after transfection, the cultured supernatant was harvested, filtered through a 0.45 μm filter, and concentrated with ultracentrifugation at 4 o C 82,000 g for 1.5 hours. The supernatant was discarded gently after ultracentrifugation, and added 200 µl serum-free DMEM medium into the tube. The pellet was resuspended at 4 o C for overnight (Salmon and Trono 2006; Liang et al., 2012). Next day, medium contained virus was dispensed into 25µl per tube and stored at - 80 o C for further study. 139

Gene Therapy and Molecular Biology Vol 15, page 140 C. Lentiviral transduction and fluorescence determination HeLa cells were seeded in the 24-well plates (1 10 5 cells in each well) the day before infection. A total of 15 wells were needed for the infection at a ratio of 1μl/ 2μl/ 4μl/ 8μl concentrated virus medium and a negative control with three repeats, respectively. At the time of infection, the old medium was discarded and add 500 μl fresh DMEM which contains 40 μg/ ml DEAE-dextran. Different volumes of FG12 viruses were added into the wells with three repeat, respectively. Medium was refreshed at 24 hours post infection. About 48 hours after infection, cells were treated in accordance with the requirements by flow cytometry. Mock infected cells were used as negative controls. Discarded the DMEM and digested for 3 min with 300 μl 0.25 % trypsin each well; terminated the reaction with 300 μl DMEM (10 % serum) and mixed by pipette; pooled the cell suspensions into a 1.5 ml tube; pelleted the cells at 850 g for 5 min; discarded the supernatant and resuspended with 500 μl phosphate buffer solution (PBS); re-pelleted the cell at 850 g for 5 min, discarded the supernatant and resuspended with 500 μl 1 % paraformaldehyde fixed for 30 min. At last, green fluorescent was checked by the flow cytometer (FACSCalibur, BD). D. Statistical analysis Flow cytometry assay data were analyzed by the WinMDI Version 2.9 software provided by Joseph Trotter of the Scripps Research Institute, La Jolla, CA. M1 was determined based on the cells without virus infection, and M1 stands for the GFP negative ratio as shown in Figure 1A. M2 was the GFP positive ratio, and got by M1 minused by 100 % as shown in Figure 1A. Checking time of fluorescence was optimized. Statistical analyses and linear figures (Figure 2, 3) were performed using Prism 5 for Windows version 5.02 (GraphPad Software, Inc., La Jolla, CA). Figure 1: Definition of GFP positive cells and HeLa cellular permissivity to FG12 virus. (A) The above three panels are HeLa cells mock- infected, and M1 was determined based on these cells. Following three panels are 1 10 5 HeLa cells infected with 2 μl FG12 virus, M2 was the ratio of GFP positive cells. Green fluorescence was observed at 48 hours post FG12 infection. Each bar scale = 50 μm in the figure. Peak value of diagram moved to the higher position of FL1- H. (B) Transduction efficiency of HeLa cells by different volumes of FG12 viruses. In the permissivity assay, HeLa cells (1 10 5 ) were infected with increasing doses of FG12 stocks (0.02-40 μl). The percentage of GFP- expressing cells was determined by flow cytometry at 48 hours post infection. All experiments were done in triplicate. 140

Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells Figure 2: TE equation acquiring and testing with flow cytometry results. (A) In the equation acquiring assay, HeLa cells (1 10 5 ) were infected with 1 μl, 2 μl, 4 μl, 8 μl FG12 viruses, mocked infection as a control. Different volumes of FG12 viruses were added into the 24- well plate with 500 μl DMEM which contains 40 μg/ ml DEAE- dextran. Flow cytometry assay was performed at 48 hours post infection. Twenty thousand cells were collected as total events as shown in panel A of the above figure. (B) A linear chart was acquired according to the transduction efficiency of different volumes FG12 infection by linear regression method. Meanwhile, the equation and R- squared value were shown on the chart. All experiments were done in triplicate. III. Results A. Transduction efficiency and HeLa cellular permissivity to FG12 vectors To measure the transduction efficiency of FG12 to HeLa cells, 1 10 5 HeLa cells in one well of 24-well plate were infected or mockinfected with 2 μl concentrated FG12 viral stocks. After HeLa cells were infected by FG12 vectors, the GFP gene was transcribed and expressed in host cells. After 48 hours, infected or mock-infected groups were analysed using flow cytometry for GFP expression (Hawley and Hawley 2004). 141

Gene Therapy and Molecular Biology Vol 15, page 142 Readout of flow cytometry standard (FCS) format files were analyzed with the software WinMDI, and the GFP positive threshold was determined by the marker value of the mockinfected group. As shown in Figure 1A, marker M1 is set around the negative peak of the subclass control on the histogram of mock infection. Marker M2 is set to the right of M1 to designate GFP positive events. M1 in the mock-infected group should be 100%. The percentage of GFP positive cells represented the transduction efficiency of FG12, so the transduction efficiency is equal to M2, the percentage of GFP positive cells, numerically. To explore the permissivity of HeLa cells to the FG12 vector, a dose-response assay of GFP expression processed routinely. HeLa cells were seeded at 1 10 5 cells per well of a 24-wells plate. Then the cells were infected with increasing doses of FG12, and the percentage of GFP-expressing cells was determined by flow cytometry at 48 hours post infection according the method described above. As shown in Figure 1B, the doseresponse curve of GFP positive cells versus vector was drawn. The curve showed that to reach the 100% GFP positive the lowest vector dose was 10-15 μl, and to obtain 50% (TE50) GFP positive cells 2-3 μl FG12 was need. These results suggested that HeLa cells were highly permissive to FG12 vector, with TE50=2-3 μl. B. Transduction efficiency equation deduction The transduction efficiency (TE) equation was deduced based on the hypothesis that HeLa cells infections by individual FG12 vector are individual events (Sigal et al., 2011). When the fraction of infected target cells (I) is low relative to the total target cell population (T), I is a possible product of the total target cell population and the probability (Prob) is that each target cell is infected: I = T Prob (infection per cell) (Sigal et al., 2011) The probability of a single viral infection in a cell is r, and the probability that non-infected cell is 1-r. We suppose that infection probability per viral vector is individual event that is independent of other infections. Given λ, a frequency that viral vector contacting a single cell, the probability that no infection is Prob (no infection by λ viruses) = (1-r) λ. Therefore, the probability of successful infection with λ viruses per cell is 1-(1-r) λ and the total number of infected cell is I = T (1-(1-r) λ ). The transduction efficiency (TE) is the percentage of successful infected cell in total cells, so TE = I/T = 1-(1-r) λ. The following equation could be deduced as ln (1-TE) = λ ln (1-r). We suppose that the probability of a single viral vector infecting a cell (r) is a constant, so ln (1-r) is a constant. There is linear correlation between ln (1-TE) and λ. In a certain viral vector infection, λ is proportional to the volume of FG12 infecting the HeLa cells (v). Finally, v and ln (1-TE) are linear correlation and the TE equation is v = a ln (1-TE) + b. In the equation: v indicated the viral vector volume; TE indicated the transduction efficiency that could be obtained by flow cytometry as above descriptions; a and b are two constants got in the calculation by linear regression method based on value of the v and TE. C. TE equation calculation and verification Theoretically, there are two paired parameters of v and TE needed to determine the value of TE. To test whether the equation could reflect the FG12 infecting HeLa cells in practical application, we took 142

Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells FG12 in a series of volumes (1 μl, 2 μl, 4 μl, 8 μl) respectively, to infect HeLa cells. GFP positives cells were observed using fluorescence microscopic and further quantified by flow cytometry assay at 48 hours post infection of FG12. As shown in Figure 2A, GFP positive cells increased along with the volumes of FG12. Transduction efficiency of FG12 to HeLa cells were calculated according to the definition presented previously. As shown in Table 1, the volume of FG12 (v) and the transduction efficiency (TE) were listed, and ln (1-TE) was also calculated. Table 1: Equation testing in a gradient amount of viruses infection. HeLa cells (1 10 5 ) were infected by a gradient amount of FG12 (1 μl, 2 μl, 4 μl, 8 μl) viruses. At 48 hours post infection, transduction efficiency was determined by flow cytometry assay. v: virus volume; TE: transduction efficiency (GFP positive percentage); ln (1-TE): natural logarithm of negative transduction efficiency. All experiments were done in triplicate. According to the TE equation, v and ln (1-TE) have a linear relationship. As shown in Figure 2B, according to our experimental data and based on the method of least squares which linear curve determination from different points, the TE equation in this study was got v = - 5.92 ln (1-TE) -1.33. The regression coefficient R 2 = 0.97 showed that the strong linear correlation between v and ln (1-TE). D. TE50 calculation and application From the TE equation v = - 5.92 ln (1-TE) - 1.33, TE50 could be easily calculated. As the definition of TE50 described, TE50 should be the vector doses required to achieve 50% positive infected cells (Chen et al., ). In TE equation, supposing TE is equal to 0.5 (50%), and then v was calculated to 2.78μl. Moreover, supposing a serial values of TE, for example, TE= 40%, 50%, 60% transduction efficiency was evaluated. According to the equation, HeLa cells (1 10 5 ) should be infected by 1.69 μl (40%), 2.78 μl (50%), 4.09 μl (60%) FG12, respectively. At 48 hours post infection, GFP expression of HeLa cells were analyzed by flow cytometry and transduction efficiency was calculated. As shown in Figure 3A, the 50% infection efficiency was accurate for a zero error with the equation derived value, experimentally derived values of 40% and 60% infection efficiency were in the vicinity of corresponding equation derived values with 0.01 to 0.03 standard deviations. Consequently the equation could be applied to determine virus transduction efficiency in HeLa cells. During virus infection and fluorescence determination 1 10 5 HeLa cells were seeded in one well of the 24-well plates to validate whether this method is practicable. We next checked the application of this equation in different cells number. A series of cell numbers in 5 10 4, 1 10 5, 1.5 10 5 HeLa cells were respectively passaged in one well of 24- well plates for determination of 50% infection efficiency. We presumed that 50% infection efficiency in 1.5 10 5 HeLa cells was composed of three-fold of 5 10 4 HeLa cells infection, and infection of 1 10 5 HeLa cells was two-fold of 5 10 4 HeLa cells infection. Volumes of FG12 viruses used to infect corresponding amount of cells were 1.39 μl (5 10 4 cells), 2.78 μl (1 10 5 cells), 4.17 μl (1.5 10 5 cells) respectively. An expectable result was got from the results of flow cytometry assay. As shown in Figure 3B, transduction efficiencies of three groups were close to the 50% infection efficiency, and only with a 0 to 0.10 standard deviations with the equation derived values. According to these results, equation can be adapted to different cell numbers within certain errors. 143

Gene Therapy and Molecular Biology Vol 15, page 144 Figure 3: Equation application and a series of tests of transduction efficiency. (A) Different transduction efficiency can be got in a certain amount of cells by virus quantities. HeLa cells (1 10 5 cells) were infected by 1.69 μl, 2.78 μl, 4.09 μl FG12 viruses to get a series of 40 %, 50 %, 60 % transduction efficiency. (B) In order to check the application in different cell numbers, a series of 5 10 4, 1 10 5 and 1.5 10 5 HeLa cells were checked for a 50 % transduction efficiency with 1.39 μl (5 10 4 cells), 2.78 μl (1 10 5 cells) and 4.17 μl (1.5 10 5 cells) FG12 viruses infection. Experimentally derived values got from the flow cytometry results were compared with the equation derived values. Blue bars stand for the equation derived value, red bars stand for the experimentally derived value got from the flow cytometry assay. All experiments were done in triplicate. IV. Discussion and conclusion Lentivirus can permanently integrate into a chromosome of an infected cell and subsequently express viral genes in that cell and its progeny, thereby lentiviral vector was widely used in gene delivery and gene therapy (Buchschacher and Wong-Staal 2000; Federico 2003; Campbell and Hope 2005). To facilitate their applications in gene therapy, it is required to develop a feasible quantification method for lentiviral vectors' transduction efficiency determination. Many methods were based on the characters induced by viruses or expression of viral DNA or RNA (Sanburn and Cornetta 1999; Pourianfar et al., 2012). Transduction efficiency of fluorescence virus is determined according to the expression level of fluorescence gene in target cells. The fluorescence intensity can be checked by flow cytometry, which is easy and convenient for fluorescence virus quantification. Additionally, the dose of lentiviral vectors or the cells transduced by lentiviral vector is very important in clinical application. So it s necessary to quantify a virtual transduction efficiency of lentiviral vector before used in the clinical gene therapy. This study provided a feasible method of quantification virtual TE50 of HIV-based lentiviral vector by flow cytometry. In this work, an HIV-1 based lentiviral vector FG12 was used to evaluate the TE50 calculating method. According to our method, an accurate TE50 of variable amount of cells could be obtained without any tedious serial dilutions. Such as TE50 of HeLa cells from 5 10 4 to 1.5 10 5 in 24-well plate was acquired easily and exactly in our study. In conclusion, our method was based on the hypothesis that HeLa cells infections by individual FG12 are individual events. A formula v = a ln (1-TE) + b could be obtained in the range of FG12 permissivity to HeLa cells. This equation was calculated by linear regression method based on the measured value of the v and TE. According to the calculated equation, different transduction efficiency and TE50 of different amount of cells were verified. Experimentally derived values were consistent with equation derived values. What s more, virus infection didn t need serial dilutions in TE calculation. This makes TE50 calculation of 144

Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells predetermined viral transduction efficiencies became feasible. Theoretically, it can be straight forwardly modified to imply that, this method can be used in TE calculation of any replication defective viral vectors with a fluorescent reporter gene. Acknowledgements This work was supported by the National Natural Science Foundation of China (81101245 and 81371820), the Fundamental Research Funds for the Central University (65011871), Natural Science Foundation of Tianjin Municipal Science and Technology Commission (13JCQNJC09800), the Ministry of Education of Talents in the New Century (NCET-11-0250) and Doctoral Fund of Ministry of Education (20110031110037). Authors' contributions CL and XK made great contributions to equation derivation and design in this study. ZL, BL and XZ carried out all the experiments and statistical analysis. ZL and CL drafted the manuscript. CL and XK revised the manuscript. All the authors have read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. References: Apolloni A., Sivakumaran H., Lin M.H., Li D., Kershaw M.H., Harrich D. (2013). A mutant Tat protein provides strong protection from HIV-1 infection in human CD4+ T cells. Hum Gene Ther 24, 1-13. Buchschacher G.L., Jr., and Wong-Staal F. (2000). Development of lentiviral vectors for gene therapy for human diseases. Blood 95, 2499-504. Campbell E.M., and Hope T.J. (2005). Gene therapy progress and prospects: viral trafficking during infection. Gene Ther 12, 1353-9. Chen C., Akerstrom V., Baus J., Lan M.S., Breslin M.B. Comparative analysis of the transduction efficiency of five adeno associated virus serotypes and VSV-G pseudotype lentiviral vector in lung cancer cells. Virol J 10, 86. Dropulic B. (2011). Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Human gene therapy 22, 649-657. Federico M. (2003). Lentivirus Gene Engineering Protocols. In Methods in Molecular Biology Gay V., Moreau K., Hong S.-S., Ronfort C. (2012). Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity. Arch Virol 157, 217-223. Geraerts M., Willems S., Baekelandt V., Debyser Z., Gijsbers R. (2006). Comparison of lentiviral vector titration methods. BMC Biotechnol 6, 34. Hawley T.S., and Hawley R.G. (2004). Flow Cytometry Protocols. Humana Press, Totowa, N.J. Ikeda Y., Takeuchi Y., Martin F., Cosset F.L., Mitrophanous K., Collins M. (2003). Continuous high-titer HIV-1 vector production. Nat Biotechnol 21, 569-572. Leyva F.J., Anzinger J.J., McCoy J.P., Jr., Kruth H.S. (2011). Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors. BMC Biotechnol 11, 13. Liang Z., Guo Z., Wang X., Kong X., Liu C. (2012). Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells. 145

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