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1 Department of Neuroscience, University of Florida McKnight Brain Institute and College of Medicine
2 Department of Physiology and Functional Genomics, University of Florida McKnight Brain Institute and College of Medicine
3 Department of Pharmacodynamics, University of Florida College of Pharmacy, Gainesville, Florida 32610
4 Department of Physiology, University of Bristol School of Medical Sciences, Bristol BS8 1TD, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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cardiomyocytes; in vivo gene delivery; neurons; retina; transfection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The increase in interest in these vectors has given rise to a need for efficient and reproducible methods to produce large quantities of high-titer lentiviral vector. Traditionally, lentiviral vectors are produced by cotransfecting human cell lines with plasmid DNA that encodes the viral components required for packaging. Transient transfection of these cell lines is often accomplished using the conventional calcium phosphate coprecipitation technique (15). Disadvantages of this method include 1) the large amount of plasmid DNA that is required for transfection; 2) the difficulties associated with scaling up the precipitation reaction; and 3) the high degree of variability observed in transfection efficiency and viral production. Recently, several groups have developed packaging cell lines that facilitate the production of lentiviral vectors by reducing the need for multiplasmid transfections (6, 12, 16, 20). Although the use of packaging cell lines has streamlined the packaging procedure, the resulting viral titers have not been significantly higher than those obtained using transient cotransfection methods. In addition, the advantages of these new cell lines are often offset by the need to develop new lines for each generation of improved lentiviral vector.
To achieve large-scale production of high-titer lentiviral vector, it is critical that transfection of the virus-producing cell cultures be both efficient and reproducible; however, little effort has been put forth to optimize this step in vector production. The goal of this study was to develop efficient and reproducible transfection and concentration methods for the production of high-titer lentiviral vector stocks. By combining a transfection method that utilizes the activated dendrimer-based transfection reagent, SuperFect (Qiagen), with a novel vector concentration protocol, we were able to reproducibly generate lentiviral vector stocks with titers greater than 1 x 1010 transducing units per ml (TU/ml) using less than one-third of the total amount of plasmid DNA that is commonly required for production of this vector. The viruses produced using these methods exhibit high transduction efficiencies in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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(EF1) promoter (21). For some experiments, PLAP was replaced with an enhanced green fluorescent protein (eGFP) reporter gene. Transfection-grade DNA was prepared using endotoxin-free DNA mega- or maxiprep kits (Qiagen).
Lentiviral Vector Production, Concentration, and Titers.
Vesicular stomatitis virus G (VSV-G) glycoprotein-pseudo-typed lentiviruses carrying an EF1-PLAP or EF1-eGFP transgene were prepared using the lentiviral vector system illustrated in Fig. 1. The 293T cells (Invitrogen, no. R70007) were seeded in 75-cm2 (T-75) culture flasks at a density of 1 x 107 cells/flask and grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) containing 10% fetal bovine serum and antibiotics (130 U/ml penicillin and 130 µg/ml streptomycin; growth medium). The cultures were maintained at 37°C in 5% CO2 throughout the virus production period. On the following day, when the cultures reached 90–95% confluence, the growth medium was replaced with 5.0 ml of fresh medium. For one large-scale preparation of virus, 20 T-75 flasks of 293T cells were transfected as follows. Transfection mixture for all 20 flasks was prepared by gently mixing 142 µg pNHP, 70 µg pTYF, and 56 µg pHEF.VSVG plasmid DNA and 8.0 ml DMEM in one 50-ml polystyrene tube. After mixing, 560 µl of SuperFect was added to the DNA solution. The contents of the tube were gently mixed and incubated at room temperature for 10 min. Next, 430 µl of the SuperFect-DNA mixture was added drop-wise to the T-75 flask (transfection start point), and the flask was incubated for 4–5 h. Following the incubation period, the medium containing the transfection mixture was replaced with 7.0 ml of fresh growth medium. The next day, the media containing the first batch of virus was harvested from each flask, and 6.5 ml of fresh growth medium was added to the cells. Upon collection, all virus-containing media was filtered through a 0.45-µm low-protein-binding Durapore filter (Millipore) to remove cell debris. To prepare transfection mixture sufficient for one T-75 flask, the amounts of DNA, DMEM, and SuperFect were each divided by 20 to scale the reaction down. We have also found that viral vector can be produced in larger or smaller cell culture flasks or plates by simply scaling cell numbers and the amount of DNA, DMEM, and SuperFect linearly with respect to the cell growth area.
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120 ml) was divided into two 60-ml aliquots and centrifuged through Centricon-80 ultrafiltration columns (Millipore) for 1 h in 4°C at 2,500 g. The retentate was retrieved by centrifuging the inverted column for 1 min in 4°C at 990 g and was stored at 4°C until further processing. On the following day, the virus-containing retentate was added to the 120 ml of virus-containing media collected at 45 h posttransfection. Four 30-ml conical-bottom tubes (polyallomer Konical tubes; Beckman), each containing a 220 µl cushion of 60% iodixanol solution (used directly from the Optiprep stock solution obtained from Axis-Shield), were prepared. Iodixanol was used because of its demonstrated safety in human clinical trials (11). Media containing virus (30 ml) was gently pipetted into each tube, taking care not to disturb the iodixanol, and the samples were centrifuged at 50,000 g for 2.5 h at 4°C using a Beckman SW-28 swinging bucket rotor. The media just above the media/iodixanol interface was carefully removed from each tube and discarded, leaving 750 µl of the solution in each tube (220 µl of iodixanol plus 500 µl of media). The residual media containing virus and the iodixanol were mixed gently by shaking at 200 rpm for 2–3 h at 4°C. The resulting mixtures were pooled into one 3-ml conical-bottom tube (polyallomer Konical tubes; Beckman) and centrifuged at 6,100 g for 22–24 h at 4°C using a Beckman SW-50.1 swinging bucket rotor. The resulting supernatant was removed and discarded, and the remaining pellet was resuspended in 50 µl of PBS or artificial cerebrospinal fluid by incubating the virus at 4°C for 10–14 h. The final viral vector was gently mixed by pipetting, then aliquoted and stored at -80°C until use.
Infectious titers of the TYF.EF1.PLAP virus were determined by incubating 1.75 x 105 TE671 cells seeded in 12-well plates with limiting dilutions of the viral stock (1/10, 1/100, and 1/1,000) in the presence of 8 µg/ml Polybrene. After a 4–5 h incubation period, fresh medium was added directly to the cells and, after 48 h, cultures were fixed, rinsed in PBS, heated in PBS at 65°C for 30 min and stained for PLAP activity using previously reported methods (7). The number of transducing units (TU; defined as an infectious particle) was determined by estimating the number of PLAP-positive cells per well, and final infectious titers were expressed as "TU/ml."
Delivery of EF1-PLAP Vector to Chicken Neural Tube.
The neural tube injections and preparation of retinal flat mounts were carried out using previously described methods (4). The brains of injected embryos were fixed overnight in 4% paraformaldehyde at 4°C. The next day, the tissues were rinsed thoroughly in PBS, and 100-µm thick sections were cut using a Vibratome. Floating brain sections and retinal flat mounts were subsequently processed for routine PLAP histochemistry as described above. All tissues were collected on embryonic day 7 (E7), 5 days after injections. Digital images of retinal flat mounts were captured with a Nikon Coolpix 995 camera fitted to a Zeiss Stemi V6 microscope. The percent area of retina transduced by the vector was determined as follows: TIFF images at a resolution of 1,024 x 768 pixels were reduced by 35%, converted to grayscale using Adobe Photoshop, and imported into the Scion Image program (available at http://www.scioncorp.com). The density slice setting was used to select all of the pixels within the area of the flat mount that represented PLAP-positive areas, and these were expressed as a percent of the total retinal area. Three to seven retinas were analyzed for each dose of vector.
Delivery of EF1-eGFP Vector to Brain Nuclei.
Male Wistar rats were anesthetized with a mixture of ketamine (60 mg/kg) and medetomidine (250 µg/kg) and placed in a stereotaxic frame. Vector delivered to the brain was suspended in artificial cerebrospinal fluid. For the paraventricular nucleus (PVN) injections, 275-g rats were used, and the head of the animal was flexed 5 mm below the interaural line. The microinjection pipette was angled 10 degrees relative to the midline to avoid the midsagittal sinus. A slow injection of 500 nl (5 x 105 TU) of virus was performed at the following coordinates: 1.8 mm lateral, 1.8 mm caudal to the bregma, and 7.5 mm below the surface. The caudal nucleus of the solitary tract (NTS) was also injected bilaterally with three injections per side for a total of 300 nl (3 x 105 TU). Injections were made at the level of calamus scriptorius and up to 500 µm caudal to it, 350–700 µm from midline, and 500–600 µm below the dorsal surface of the medulla. The head of the animal was flexed 10 mm below the interaural line. The animals were killed either 7 days (PVN, n = 2) or 30 days (NTS, n = 2) following the injections and fixed by intracardial perfusion with 4% paraformaldehyde in PBS. Brains were removed and cryoprotected in 30% sucrose, then 60-µm-thick brain sections were cut on a cryostat, and confocal microscopy (Leica SP) was used to visualize GFP fluorescence.
Systemic Delivery of EF1-PLAP Vector to Neonatal Rat.
Twelve, 5-day-old Sprague-Dawley rats (Charles River, Wilmington, MA) were divided into two groups. Six rats in the control group received injections of artificial cerebrospinal fluid (viral suspension buffer), while the remaining six animals received 2.5 x 108 TU of TYF.EF1.PLAP virus. A single bolus of vehicle or virus was administered in a total volume of 25 µl into the chamber of the left ventricle of the heart (9). Animals were killed at 30 (n = 4 per group) and 120 (n = 2 per group) days postinjection. Tissues were processed for routine PLAP histochemistry as described above. Digital images were obtained using the methods described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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3.0 ml, resulting in a 33-fold increase in titer and an average recovery of 84%. Further concentration of the virus stock by low-speed centrifugation (Fig. 3, steps 5c and 6) yielded 1.40 ± 0.44 x 1010 TU/ml, a 958-fold increase over the average starting titer. The average overall percent recovery of the virus was 40%.
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0.5 µl of TYF.EF1.PLAP virus (1 x 1010 TU/ml) into the chicken neural tube resulted in efficient transduction of large numbers of neural progenitor cells (Fig. 4). Cross sections of stained retinas revealed numerous PLAP-positive cell columns (data not shown). Columns of PLAP-positive cells were also observed throughout the developing brain (Fig. 4E). We also examined the relationship between viral dose and the percent of the retina transduced by the virus and determined that the transduction efficiency of the virus in developing retina was dose dependent (Fig. 4, A–C). The percent of total retinal area exhibiting PLAP expression was estimated to be 5%, 63%, and 85% in embryos receiving injections of 108, 109, and 1010 TU/ml vector, respectively (Fig. 4D).
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-eGFP transgene was delivered into the PVN and the NTS of the adult rat brain. Examination of transverse sections cut from the brains of animals either 7 days (PVN) or 30 days (NTS) after injection revealed that the vector transduced a high proportion of cells in both nuclei as evidenced by the presence GFP-positive cells (Fig. 5). Many GFP-positive cells exhibited a neuronal phenotype (Fig. 5C), and in many cases fluorescent axons could be seen hundreds of microns away from the area of concentration of fluorescent cell bodies.
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.PLAP virus transduced 30% of the heart tissue (Fig. 6E) and 40% of the liver tissue (Fig. 6G). PLAP-positive cells in the heart were distributed throughout both the atria and ventricles; morphological examination revealed that 90–95% of the stained cells in the ventricles were cardiomyocytes (Fig. 6E, inset). Similar staining patterns and transduction efficiencies were observed at 120 days postinjection. Limited, but significant expression of PLAP was observed in the testes of 120-day-old rats. Several spermatogonium-like cells situated near the perimeter of the testicular tubules exhibited PLAP expression, indicating that administration of vector into the systemic circulation of neonates can result in transduction of germ cells (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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120 ml) with starting titers averaging >1.0 x 107 TU/ml, titers that were comparable to vector stocks prepared using other transfection reagents. Use of SuperFect greatly simplified the transfection protocol and significantly reduced the amount of plasmid DNA required for the procedure. The viral concentration protocol that we developed consistently increased the titers of the viruses by 1,000-fold (1 x 1010 TU/ml). Furthermore, all vectors that we produced using these methods exhibited high transduction efficiencies in vivo. Three different in vivo paradigms were used to examine the transduction efficiency of the viral particles produced using this protocol. In the first paradigm, we delivered lentiviral vector into the neural tube of the developing chicken embryo. The injected virus transduced several populations of neural progenitor cells, including those fated to become the neural retina (Fig. 4). A majority of cells exposed to virus during this stage of development are mitotic and have not yet differentiated (18). By varying the concentration of the virus injected, we found that the percent of retina transduced could be controlled in a linear fashion using doses between 108 and 109 TU/ml. Injections of virus at a concentration of 1010 TU/ml produced maximal levels of retinal transduction. In a previous study, we showed that it is possible to specifically target lentiviral vector-mediated expression of transgenes to retinal photoreceptor cells by selecting appropriate promoter fragments (4). Together, these results illustrate the effectiveness of our vector to transduce cells within the developing nervous system and illustrate the potential use of this vector as a tool for studies of mechanisms regulating gene expression in vivo.
In the second paradigm, lentiviral vector carrying an EF1-eGFP transgene was injected into specific nuclei within adult rat brain. Analyses of the brains of these animals revealed that we were able to effectively target the virus to cells, including neurons, within the PVN and NTS (Fig. 5). Furthermore, the expression of GFP was robust and persisted for at least 30 days postinjection. Our ability to transduce neurons in brain nuclei involved in cardiovascular homeostasis will allow us to study both the acute and chronic physiological impact of the expression of relevant genes without generating transgenic and/or knockout animals.
Finally, we show that lentiviral vector delivered systemically can transduce several different tissues (Fig. 6) and that the transgenes carried by these vectors exhibit long-term expression (120 days, duration of the experiment). Expression of the PLAP reporter gene was highest in the liver and cardiomyocytes. Other organs, such as lung, kidney, and adrenal glands were also transduced by the vector but exhibited only limited PLAP expression. Surprisingly, our studies also showed that the systemically delivered lentiviral vector transduced germ cells in the male rat (Fig. 7). To our knowledge, this is the first example of viral vector-mediated germ cell transduction in which transgene expression was detected using histochemical methods rather than PCR-based detection methods (1, 17). Expression of PLAP was seen within the testicular tubule spermatogonia and included PLAP-positive spermatocyte, spermatid, and mature spermatozoa.
Our observation of transduced germ cells in male rats, while intriguing, must be interpreted with caution with regard to its potential impact on the use of lentiviral vectors for gene therapy. We believe that the transduction we observed could be attributed to the poorly developed blood-testicular barrier that is present in 5-day-old rats (19). It is our hypothesis that injections of lentivirus after this barrier has matured will not result in transduction of germ cells. It would be interesting to determine whether our hypothesis is correct by conducting these experiments in adult animals, an experiment that is now possible using our new viral packaging protocol.
In summary, the transfection and concentration protocols outlined here allow efficient, reproducible production of high-titer lentiviral vectors that exhibit robust transduction properties in vivo. The transfection protocol itself is simple and can be easily implemented by investigators interested in producing lentiviral vector in their laboratories. Furthermore, the methods can be easily adapted to large-scale lentiviral production protocols that are currently being developed for use in large animal studies or for possible use in clinical studies.
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ACKNOWLEDGMENTS |
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The work in the United States was supported by National Institutes of Health Grants HL-33610, HL-56921, EY-11388, Training Grant EY-07132, and Core Grant EY-08571. In the United Kingdom, J. F. R. Paton is supported by the British Heart Foundation Grant BS/93003. The financial support of the Biotechnology and Biological Sciences Research Council and Royal Society is acknowledged.
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FOOTNOTES |
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Addresses for reprint requests and other correspondence: S. L. Semple-Rowland, UF McKnight Brain Institute, Dept. of Neuroscience, 100 Newell Dr., Bldg 59, Rm L1-100, Gainesville, FL 32610-0255 (E-mail: rowland{at}mbi.ufl.edu); and M. K. Raizada, UF McKnight Brain Institute, Dept. of Physiology and Functional Genomics, Box 100274, Gainesville, FL 32610-0255 (E-mail: mraizada{at}phys.med.ufl.edu).
10.1152/physiolgenomics.00135.2002.
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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