CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAIS LABORATÓRIO NACIONAL DE BIOCIÊNCIAS LABORATÓRIO VETORES VIRAS- LVV 21º PROGRAMA BOLSAS DE VERÃO

Transcription

1 CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAIS LABORATÓRIO NACIONAL DE BIOCIÊNCIAS LABORATÓRIO VETORES VIRAS- LVV 21º PROGRAMA BOLSAS DE VERÃO OPTIMIZATION OF PARAMETERS FOR RAPID AND ACCURATE TITRATION OF LENTIVIRAL VECTORS ORIENTADOR: MARCIO CHAIM BAJGELMAN ANDREA JHOANNA MANRIQUE RINCÓN CAMPINAS-SP 2012

2 It is not the possession of truth, but the success which attends the seeking after it, that enriches the seeker and brings happiness to him. MAX PLANCK 2

3 Acknowledgment I owe my sincere thanks to CNPEM, LNBIO and LVV for the support to this project and also for the wonderful experience in science that it brings to me. I want to express my gratitude to my adviser Marcio Chaim Bajgelman for allowing me to be involved in this research, share with me his knowledge in science and for all his valuable comments that makes me more conscious about the scientific work that I want to do. I thank Anna Carolina Pereira Vieira de Carvalho,MSc for technical assistance and Maria Eugênia Ribeiro de Camargo for the assistance with flow cytometry. I want to thank also to Juan José Builes, Regina Cicarelli, Isabel Ortiz, Juan Pablo Narváez, my family, friends and Professors in University of Antioquia, for their support during this time. Finally to my new friends from this program whose backing was really important for me. 3

4 TABLE OF CONTENTS ACKNOWLEDGMENT 3 ABSTRACT 5 INTRODUCTION 6 RETROVIRUS FEATURES AND GENE TRANSFER 6 LENTIVIRAL VECTORS : IMPROVING GENE TRANSFER EFFICIENCY 7 EVALUATING TRANSDUCTION EFFICIENCY AND CONCENTRATION OF A VIRAL PREPARATION 8 NON FUNCTIONAL METHODS 9 P24 antigen ELISA: 9 Reverse transcriptase activity 9 Determination of the genomic RNA concentration 9 FUNCTIONAL METHODS 9 Lentiviral vector titration from transgene protein expression 9 Flow cytometry 10 Quantitative Polymerase chain reaction 10 OBJECTIVES 12 SPECIFIC AIMS 12 MATERIAL AND METHODS 13 CELL LINES AND CULTURE CONDITIONS 13 VIRUS PRODUCTION 13 VIRUS TRANSDUCTION 13 FLOW CYTOMETRY 14 RESULTS AND DISCUSSION 15 DETERMINATION OF LINEAR RANGE FOR VIRAL DILUTIONS 15 LEVEL OF REPORTER EXPRESSION IS CRUCIAL FOR TITRATION ACCURACY 17 ADDITIONAL EXPERIMENTS 20 BIBLIOGRAPHY 24 4

5 Abstract The use of virus for the introduction of DNA is a technique that has been widely used to the establishment of permanent cell lines, to produce transgenic animals, to study RNAi, to analyze gene function, to produce recombinant proteins and for gene therapy. One of the most commonly used vectors are lentivirus, because of their high efficiency of integration in the host genome of both dividing and non-dividing cells, their low immunogenicity, and the fact that they can be extensively modified to produce recombinant particles. Lentivirus can be transiently produced by co-transfection of plasmids encoding viral genome and packaging plasmids. After transfection, viral particles are produced, released in the supernatant and can be purified. A critical issue regarding virus production is to determine the viability and concentration of viral preparation. A number of methods to evaluate lentiviral titer have been described. Titration can be performed quantifying viral antigens as p24 by Elisa, or using a biological method that allows an accurate detection of functional particles, based on antibiotic selection, detection of reporter genes by flow cytometry or quantifying transducing units using qpcr. All titration methods have advantages and limitations that should be considered. In this work we studied the optimization of a biological titration method based on flow cytometry for detection of a fluorescent reporter gene. We used a lentiviral vector encoding GFP to transduce target cells that were analyzed by flow cytometry, testing parameters as virus dilution, time of incubation to detect reporter gene and efficiency of transduction using different cell lines. Our data revealed some aspects applicable to experimental protocols, to enhance titration accuracy using flow cytometry. 5

6 Introduction Retrovirus features and gene transfer Retroviruses belong to the large Retroviridae family. The virions are roughly 100 nm in diameter, spherical and the outer layer is a lipid envelope displaying viral glycoproteins. Each particle contains two copies of the linear viral RNA genome (approximately 10 kb), (Figure 1) which contains three essential genes, gag, pol and env. The pol gene encodes three viral enzymes: the protease, reverse transcriptase, and integrase. The gag gene encodes the structural proteins: the capsid, matrix, and nucleocapsid. Proteins are generated by proteolytic cleavage of the gag-pol precursor. The env gene encodes the envelope glycoproteins of the virus. After the retrovirus enters the target cell, the viral genome is converted into the double-stranded DNA form by reverse transcriptase (RT). Proviral genome is then integrated into the genome of the target cell by the integrase. Viral long terminal repeats (LTRs) are important for the initiation of viral DNA synthesis, integration and regulation of viral transcription (Goff.S.P., 2001). Figure 1. Structure of a Retroviral particle Retroviral vectors can be easily engineered to encode an expression cassette. The ability of the retrovirus to integrate and achieve long term expression has made them attractive tools for gene transfer protocols, including functional studies (Taxman 2006), expression of heterologous proteins, silencing of genes (Rubinson 2003, Singer 200 and Sui 2002) and gene therapy (Tolstoshev, 1992). As a safety issue, it is possible to generate defective retroviral particles that wouldn t replicate in the target cell. The genes associated with replication are removed from the viral vector genome creating a defective 6

7 retrovirus which can efficiently transduce a target cell but lack information to replicate (Naviaux et al, 1996; Bajgelman et al., 2003). Considering its utilization, retroviral vectors has been used in the 20% of clinical trials in gene therapy. (Figure 2) Figure 2. Vectors used in gene therapy clinical trials Lentiviral vectors : improving gene transfer efficiency Human lentivirus are members of the retrovirus family and derive from human immunodeficiency virus (HIV). Concerning safety issues, these vectors keep less than 5% of the parental genome, and less than 25% of the genome is incorporated into packaging constructs, which minimizes the possibility of the generation of recombinant replicationcompetent HIV. Biosafety has been further increased by the development of selfinactivating (SIN) vectors that contain deletions of the regulatory elements in the 3 long terminal repeat sequence (LTR), to avoid the possibility of generation of replication competent retrovirus (RCR) particles in the target cell (Thomas, Ehrhardt, & Kay, 2003). Lentiviral vectors usually have a posttranscriptional regulatory element called WPRE that enhanced mrna stability and increase viral expression (Zufferey et al., 1999). With this features they can transduce both dividing and quiescent cells, and allow to establish permanent cell lines (Naldini et al, 1996, Strauss et al., 2006). In contrast with murine retroviral vectors, lentivirus are less susceptible to methylation and are suitable to generate transgenic animals (Tiscornia et al., 2003, Ikawa et al., 2003, Bressan et al, 2011). 7

8 The Lentivirus preparation can be transiently produced by co-transfection of plasmids encoding viral genome and packaging plasmids. After transfection viral particles are released in the supernatant and can be purified (Figure 3). Figure 3 Lentiviral production and transduction of a target cell. The lentivirus can be transiently produced by co-transfection of viral plasmids that encode viral genome, reverse transcriptase, structural proteins and envelope glycoprotein (1-3). Viral particles that are assembled into the cell are released outside the cell and can be harvested in the supernatant (4-6). This viral preparation contain viable particles that lack packaging information and can be used to transduce a target cell, which will express the gene of interest (7-13). Evaluating transduction efficiency and concentration of a viral preparation A critical issue regarding virus production is to determine the infectivity and concentration of viral preparation. A number of methods to evaluate lentiviral titer have been described. Titration can be performed quantifying viral antigens as the p24 by Elisa, or using a biological method that allows an accurate detection of functional particles; based on antibiotic selection, detection of reporter genes by flow cytometry or quantifying 8

9 transducing units using qpcr (Geraerts et al, 2006). Titration methods have advantages and limitations that should be considered. Next we are going to review some methods commonly used for lenviral titration: Non functional methods P24 antigen ELISA: This test is directed toward the capsid protein p24. The approach is generally used to determine the number of physical particles (pp), And is based on the estimation that a HIV core particle is composed of two thousands capsid proteins, (Farson, 2001). In this way 1 fg of p24 represents around 12 pp. From this calculation, it is possible to estimate viral concentration for any HIV-1-based lentiviral preparation. The estimation is routinely employed for quality control validation of the different batches being produced. Nevertheless, this technique can overestimate the functional vector titer because the p24 protein pool that is quantified includes a variable amount of free p24 that is originated from non-functional vector particles. Reverse transcriptase activity Assessment of viral titer can be estimated performing a quantitative assay to determine reverse transcriptase activity using an Elisa method. As seen for the p24 assay, this non functional assay can mislead viral titer (Bukrinsky, et al.1993). Determination of the genomic RNA concentration Determination of the genomic RNA concentration in vector preparations can be done by semi-quantitative northern blotting or dot blot analysis, however these techniques lack accuracy to quantify the viral RNA content and viral titer. Moreover, RNA titers will also assess defective particles that can mislead viral titer (Forghani 1991). Functional methods Lentiviral vector titration from transgene protein expression A more accurate, functional titer is determined by transduction of cells following limiting dilution of viral preparations and subsequent evaluation of reporter protein activity, (e.g. betagalactosidase positive cells) or by assessment of the number of colony forming units following antibiotic selection. Depending on the type of reporter used, lentiviral 9

10 transducing units are defined in several ways. Colony forming units are generally used for drug selection genes. (Metharom 2000). The term of transducing unit used in the context of lentiviral vector gene transfer has been routinely employed, especially for the expression of living color and lacz genes. (D Costa 2003) Flow cytometry Flow cytometry (FC) allows quantifying cell populations based on fluorescence. A viral vector encoding a fluorescent reporter gene, as GFP, can be titrated against non transduced cells to estimate the number of transduced cells using a known volume of viral preparation. Therefore is possible to extrapolate viral concentration to calculate viral titer. As described in this work, flow cytometry may have some issues regarding viral dilution, time of viral incubation before titration and the cell line that is chosen to the assay. Another limitation of this technique is associated to the reporter gene, which should be a fluorescent reporter gene (as GFP, dsred, etc.), or even another gene which product could be labeled by a fluorescent antibody. Quantitative Polymerase chain reaction An accurate method to quantify functional particles can be accomplished by determination of the integrated proviral DNA copies per cell by Quantitative Polymerase chain reaction (qpcr). The qpcr is a real time PCR that allows monitoring reaction as it progresses. The principle of detection is binding of a reporter molecule to double stranded amplicon, or hybridization of a fluorescent-labeled probe to one amplicon strand. The reaction requires minimal amounts of nucleic acid as template, there is no needing for the post-pcr processing and the quantization of the end product is accurate. These advantages of the fluorescence-based qpcr technique have completely revolutionized the approach to PCR-based quantification of DNA and RNA. Real-time assays are now easy to perform, have high sensitivity, more specificity, and provide scope for automation. (Delenda, C., Gaillard, 2005). 10

11 Titration analyzing gene expression may provide the most useful data for predicting the performance of vector supernatants in various cell types; however, two studies have shown that this method likely underestimates the number of vector genomes transferred into target cells, because variability in gene expression will lead some marked cells to be counted as unmarked (Sastry et al., 2002; Lizée et al., 2003). Consequently, a more accurate determination of the total number of infectious units may be achieved by analyzing gene transfer by quantitative polymerase chain reaction (PCR). (Logan et al., 2004). 11

12 Objectives The main target of this work is to standardize a functional assay to titrate virus using flow cytometry. Specific aims 1. Determine a linear range for viral dilutions to avoid multiple integrations underestimating titer, and also to establish a cut-off for the lower percentage of transduction labeling to avoid overestimate titer. 2. Investigate the importance of incubation time after transduction before submitting cells to flow analysis. 3. Evaluating titration using two different cell lines. 12

13 Material and methods Cell lines and culture conditions The murine cell line derived from fibroblasts NIH3T3 and the human HT 1080 derived from lung tumor fibrosarcom cell were maintained in DMEM (Gibco-BRL, Grand Island, NY, USA) plus 10% Fetal Bovine Serum (HyClone USA), at 37 C, 5% CO2. Virus production Virus was transiently produced by Viral vector laboratory LNBio, co-transfecting packaging cell line with a viral plasmid encoding the GFP reporter and packaging plasmids encoding structural proteins and vesicular stomatitis vírus glycoprotein (VSV-G) envelope. The lentiviral platform used was a second generation, in which the packaging information is split in two different plasmids. The viral plasmid is self-inactivating (SIN) and also has a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to increase virus expression. The virus produced is defective because it lacks information to replicate in the target cell, and can t be packed by other wild type virus due the SIN-LTR. Virus transduction Virus transduction: Cells were seeded and transduced the next day with viral supernatant diluted 1:100 in complete media plus 8ug/ml of polybrene. The cells were then incubated for 24h, harvested or were changed media and harvested next day. To harvesting for flow citometry, cells were trypsinized and resuspended in 1ml of 1X phosphate buffer saline (PBS). 13

14 Flow cytometry The cell suspension was analyzed using a FACScalibur (Becton-Dickenson, CA, USA) flow cytometer. The percentage of EGFP-positive cells is used to extrapolate the number of infected cells. The dilution factor is then applied to arrive at the number of green fluorescence units per milliliter (gfu ml -1 ) of virus supernatant. According to the equation: #!" 100 P: percentage of GFP(+) cells, Negative: background of negative control, #cells: number of cells on the transduction day, vol: volume of virus (ul), dilution of virus stock. 14

15 Results and discussion All the experiments were performed using aliquotes of purified FUGW virus from the same batch, lot#071211, produced by Viral vector lab - LNBio. Transduction was done as described in material and methods, altering some parameters as necessary, and indicated in the experiments. Determination of linear range for viral dilutions Is common sense that using more than one particle of virus per cell should underestimate viral titer, because flow cytometry will detect the percentage of transduced cells. On the other hand, it is not clear what should be the minimal amount of viral particles to assure transducing cells with one copy of virus per cell. To investigate the relationship between transduction and amount of virus we designed an experiment of titration, using experiment number (3), with cell line NIH3T3, number of cells 4x104 and incubation time of 48 hours, adding different volumes of virus per cell. As seen in figure 4, we have found linearity considering percentage of transduction and viral volume from 1 to 25 ul of viral preparation. Using 25ul yielded 46% of transduced cells, however using twice this volume (50ul) the transduction efficiency did not follow the same pattern. In addition, using 50ul the intensity of signal increased, suggesting the possibility of multiple viral integration. According our experience and experimental data, we also notice that we could have an error up to 20% between duplicates. Because of this, to increase precision we considered to work with percentages of GFP positive cells, higher than 10%. So, it was possible to determine a linear range for the titration curve, choosing 5 ul of our 1:100 diluted viral stock (lot#071211) as a standard, to compare titration between different experiments. This because 5ul is the minimal virus volume that allows a good percentage of GFP positive cells close to one particle of virus per cell. 15

16 Figure 4 Determining linear range for flow cytometry-based titration using experiment number (3), with cell line NIH3T3, number of cells 4x10 4 and incubation time of 48 hours. (A) Raw data: Percentage of GFP positive cells and intensity obtained by flow cytometry. (B) Graph representing the percentage of transduction versus viral volumes and (C) table summarizing experimental condition, transduction efficiency and calculated titer 16

17 Level of reporter expression is crucial for titration accuracy In this assay we investigated the sensitivity of our titration method regarding detection of the GFP reporter gene. The experiment consisted in evaluate detection of GFP in two different experiments using the same amount of virus. The first experiment was incubated 24h after transduction, and the second experiment was incubated 48h after transduction. As seen in Figure 5, the viral titer is underestimated 90% when incubation time was 24h versus 48h. 24 hs incubation time NIH3t3 48 hs incubation time NIH3t3 Figure 5- Titration accuracy depends on gene reporter expression. The figure shows two titration experiments that were performed using the same batch of virus. As seen above, incubating 24h yields only 10% of positive cells that could be detected by flow, compared with 48h. 17

18 Evaluating titration using two different cell lines. The last parameter analyzed was to investigate the influence of cell line transduction efficiency in viral titration. Is very well known that, there are cell lines more susceptible to viral transduction and other cell lines that are more difficult to transduce. In this way we compared the viral titer estimated using a very well described standard cell line NIH 3T3 versus the HT1080 that is easily transduced with viruses. The first cell line is a murine cell line derived from fibroblasts called NIH 3T3, and the second cell line is the human HT 1080 derived from lung tumor fibrosarcom. The transduction was performed using different viral dilution per cell line, and incubating 48h. As seen in Figure 6 the cell line NIH 3T3 has shown a better transduction efficiency, and this cell line also yielded a better titer. It is important to observe both cell lines yielded titers in the same magnitude, what is consistent for these cell lines that are easily transduced with virus from ours system. Figure number 6 NIH 3t3 (48 Hs INCUBATION) Ht1080 (48 Hs INCUBATION) Figure 6 - Comparison transduction efficiency by flow citometry. NIH 3T3 and HT1080 were transduced using the indiceted amount of virus from the same batch. Cells were incubated 48h for transduction. 18

19 Conclusions The infectivity and concentration of a lentivirus preparation can be evaluated using a suitable titration method. Here in we optimized a flow cytometry titration protocol which requires a GFP reporter gene cloned in the viral vector. This method is based on detection of transduced cells that are expressing the reporter gene. The analysis of our experimental data indicated some aspects that have to be considered to optimize titration: 1. There is a linear range for flow cytometry detection. Flow cytometry can detect percentage of transduced cells and it is important to analyze several dilutions to verify a linearity between volume of viral preparation and percentage of transduced cells. In our data we found that 5ul of volume virus is the best amount of virus to have a good percentage of GFP positive cells avoiding multiple viral integrations. 2. The time of incubation is crucial for GFP detection. Considering a 100% GFPexpressing cells population, only 10% of GFP- transduced cells are detected within 24h. 3. Tranduction efficiency depends on the susceptibility of the cell line, and can interfere in viral titer. We compared titration of a standard cell line, NIH 3T3, versus HT1080, using the same batch of virus and same incubation time (The NIH 3T3 is being used by LVV to titrate viral preparations). Both cell lines exhibited a good level of reporter gene expression in the same magnitude, however, the NIH 3T3 cell line shows a better transduction efficiency, and also yielded a better titer. 19

20 Additional experiments During the period I worked in the Viral Vector Lab - LNBio, besides performing experiments with flow cytometry I had the opportunity to be introduced to other activities that are routine in this lab. I spend a few weeks being trained in molecular biology. I prepared plasmids, performed restriction maps and started cloning vectors that will be used in the lab. Also I had the opportunity to start the standardization of another titration method based on qpcr. This method has the advantage of detecting virus that lacks a reporter gene for flow cytometry. Next I summarize some data produced during this period that would be used as preliminary data for qpcr titration. Preliminary assays to Standardize of qpcr titration Objective The main purpose of this experiment was to validate oligos designed for titration of targets, as the WPRE and Neo cdna. Material and Methods The qpcr was done using the syber green reagent (Applied Biosystems USA) and custom primers developed in the lab. The standard curves were done using plasmid dilutions, encoding target cdnas, as template. Conditions of qpcr for Neomycin resistance gene and WPRE element qpcr reactions were prepared in 96-wells provided by applied Biosystems, with 10µl of syber green, 0,08 µl of primers (100um) (forward and reverse) for neomycin and WPRE element, and 4,92 µl of miliq water, DNA amplification was carried out using an Applied Biosystems 7500 Real-Time PCR Sequence detection system using cycling conditions of 95 C 10 minutes, 40 times: 95 C 15 se conds and 60 C 1 minute, 95 C 15 seconds 60 C 20 seconds and 95 C 15 seconds. 20

21 Results Standard curve for WPRE For the standard curve were prepared serial dilutions that were made in triplicates, starting from 10 6 to 10 0 copies of plasmid template encoding desired target. The results obtained show a R 2 of , which indicates a good correlation between the different dilution points (Figure 7). Figure 7 Standard curve for WPRE with dilutions ( ) Standard curve for Neomycin For the standard curve were prepared serial dilutions that were made made in triplicates, starting from 10 6 to 10 0 copies of plasmid template encoding desired target. The results obtained show a R 2 of , which indicates a good correlation between different dilutions. (Figure 8). 21

22 Figure 8 Standard curve of Neomycin Testing titration of transduced cells The aim of this assay was to perform a quantitative detection of virus from transduced cells. The first step was to establish a cell line expressing an internal control that could be used to normalize the absolute quantization. Therefore we transduced NIH- 3T3 with a diluted viral preparation of pcl virus which encodes neomycin resistance gene. After transduction cells were selected with G418, establishing a pool of resistant cells that should have one copy of neomycin gene per cell. Next step was to transduce the neomycin resistant cells with a FUGW virus, which encodes GFP and also has the WPRE element. In this way we performed two different transductions: sample#1 was transduced with 50 ul of virus and sample#2 was transduced with 25 ul of virus. Cells were analysed by flow cytometry to confirm the expected virus expression and then the genomic DNA (gdna) was isolated, quantified and then we used 100ng as template for qpcr. As seen in Figure 9, our internal control (neomycin) was detected around 294 copies/100ng of gdna for sample#1 and a very similar level was detected for sample#2: 224 copies/100ng of gdna. This data is consistent because samples 1 and 2 derives from the same batch of neomycin expressing cells and should have the same copy number of neo gene. On the other hand, quantitation of WPRE wasn t consistent, and should be repeated for further experiments (Figure 9) 22

23 Figure 9. Comparison of the qpcr results between Neomycin and WPRE Conclusion of qpcr titration assay The qpcr should provide an accurate method for viral titration. In this experiment we checked the feasibility of the method and more experiments should be performed to optimize conditions. 23

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