Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
UNIT 13.11
Julius W. Kim,1 J. Robert Kane,1 Jacob S. Young,1 Alan L. Chang,1 Deepak Kanojia,1 Shuo Qian,1 Drew A. Spencer,1 Atique U. Ahmed,1 and Maciej S. Lesniak1 1
The Brain Tumor Center, University of Chicago, Chicago, Illinois
The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials. These clinical trials exploring SC-mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that can provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally-replicative adenoviruses and delivered to the tumor, whereupon they may evoke pronounced therapeutic efficacy. In this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for C 2015 by John SC loading, and evaluate the optimized viral loading on SCs. Wiley & Sons, Inc. Keywords: neural stem cell r mesenchymal stem cell r oncolytic adenovirus r cancer r gene therapy
How to cite this article: Kim, J.W., Kane, J.R., Young, J.S., Chang, A.L., Kanojia, D., Qian, S., Spencer, D.A., Ahmed, A.U., Lesniak, M.S. 2015. Neural stem cell-mediated delivery of oncolytic adenovirus. Curr. Protoc. Hum. Genet. 85:13.11.1-13.11.9. doi: 10.1002/0471142905.hg1311s85
INTRODUCTION As stem cell-mediated delivery of oncolytic adenoviruses has shown promising therapeutic efficacy, it is necessary to standardize the protocol to enhance the efficacy and consistency of the approach. Only when healthy and minimally passaged SCs are loaded with an optimized number of virus particles per cell will these SCs migrate to their destined place(s) around the tumor area during the period of viral progeny production (48 to 72 hr) and then release the virus progenies (104 particles/cell). We describe here how to properly culture and prepare the SCs for oncolytic virus loading. We further describe how to optimize a virus titer for loading on SCs to maximize the oncolytic virus progenies delivered to the target site.
CULTURE OF NEURAL STEM CELLS This protocol describes the growth, splitting, and freezing of stem cells for adenoviral vector loading.
BASIC PROTOCOL 1
Materials Stem cells, e.g., neural stem cells (HB1.F3-CD), or bone marrow- or adipose-derived mesenchymal stem cells Dulbecco’s Modified Eagle’s medium (DMEM; Corning, cat. no. 10-017-CV; see APPENDIX 3G; Phelan, 2006) Current Protocols in Human Genetics 13.11.1-13.11.9, April 2015 Published online April 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg1311s85 C 2015 John Wiley & Sons, Inc. Copyright
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Fetal bovine serum (FBS; Hyclone, cat. no. SH30088.03) 100× Pen/Strep stock (Corning, cat. no. 30-002-CI): penicillin sodium (10,000 units/ml) and streptomycin (10,000 μg/ml) 100× glutamine stock (200 mM; Corning, cat. no. 25-005-CI): only needed if DMEM lacks glutamine 1× PBS (Corning, cat. no. 21-030-CV; also see APPENDIX 2D) 0.25% trypsin-EDTA solution (Corning, cat. no. 25-053-CI) Dimethyl sulfoxide (DMSO; Sigma, cat. no. D2650) T75 cell-culture flasks 15-ml and 50-ml conical tubes Microscope 37°C, 5% CO2 humidified incubator Additional reagents and equipment required for human tissue culture, including subculturing, freezing, thawing, and counting cells (APPENDIX 3G; Phelan, 2006) NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.
Initial culture from a frozen stock of SCs This protocol is tailored for thawing a cryotube containing 2 × 106 cells frozen in 1 ml cell-freezing medium. 1. Add 15 ml cell-growth medium (DMEM containing 10% FBS and 1× Pen/Strep) to a T75 flask. 2. Spread the cell-growth medium throughout the flask by gently tipping the flask in different directions. 3. Thaw the frozen stock of SCs in a 37°C water bath. 4. Transfer the cryotube containing thawed cells to a sterile hood. 5. Slowly add the SCs to the growth media in the T75 flask. Slowly adding the SCs to the culture media minimizes osmotic shock.
6. Gently mix the cells in the media to distribute them evenly across the surface of the T75 flask. 7. Incubate the cells in growth media in a 37°C, 5% CO2 humidified incubator. 8. On the following day, aspirate the media from the T75 flask, and immediately add 15 ml freshly prepared cell-growth medium. This step helps to remove any dead cells and DMSO from the freezing medium.
9. Incubate the cells in a 37°C, 5% CO2 humidified incubator for 3 to 4 days. Please note that it will take approximately 3 to 4 days for SCs to fully recover and propagate efficiently.
Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
Subculture of SCs 10. Aspirate the existing cell-growth medium from the T75 flask. 11. Add 5 ml of 1× PBS and gently tip the flask in several directions to distribute and wash the PBS over the cells. Aspirate the PBS.
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12. Add 1.5 ml of 0.25% trypsin-EDTA solution to the T75 flask, and distribute the trypsin solution over the cells by tipping the flask in several directions. 13. Incubate the cells with trypsin for 5 min or until cells begin to detach. To accelerate cell detachment, the flask can be incubated in a 37°C, 5% CO2 humidified incubator.
14. When obvious cell detachment is observed, add 5 ml fresh cell-growth medium to the T75 flask. 15. Wash and homogenize the cells in the cell-growth media by repeatedly pipetting the cells with a serologic pipet. Be especially gentle while repeatedly pipetting. Strong agitation causes the cells to die.
16. Transfer the trypsinized cells evenly between three new T75 flasks (2.3 ml per flask). 17. Add 12 ml fresh cell-growth medium to each flask, and incubate in a 37°C, 5% CO2 humidified incubator. SCs can grow rapidly for 25 to 30 passages (Aboody et al., 2008; Ahmed et al., 2012). During these passages, carefully check their growth every day. As the media becomes acidic (it will begin to turn yellow), change the media.
Preparation of Frozen Stocks of SCs 18. Wash and trypsinize a highly confluent T75 flask as described in steps 10 to 15. A minimum of 2–3 × 106 cells per 1 ml of freezing media in cryotube is required to restore the cells in a T75 flask. At high confluence, a T75 flask contains approximately 107 cells. Therefore, a single T75 flask will yield three cryotubes of frozen stock.
19. Transfer the trypsinized cells in medium (6.5 ml) to a 50 ml conical tube. 20. Centrifuge the tube at 266 × g for 5 min at 4°C. 21. During the centrifugation, make an appropriate amount of the cell-freezing medium (10% DMSO in FBS). For each T75 flask of cells to be frozen, mix 300 μl DMSO in 2.7 ml FBS. 22. Aspirate the supernatant media, and add 3 ml cell-freezing medium. 23. Homogenize the cells thoroughly, but carefully, and divide between three different cryotubes (1 ml per tube). 24. Place the cryotubes at –80°C overnight, and transfer to liquid nitrogen storage (–180°C) the next day.
OPTIMIZING THE LOADING CAPABILITY OF STEM CELLS The primary receptor of the wild-type human adenovirus serotype 5 (HAd5) is not expressed on the SCs. The Ad5 fiber domain, which dictates the receptor binding, has been genetically modified to achieve the efficient, enhanced, or directed specific infection in most of presently used adenoviral vectors. However, modifying this structural protein (fiber) could in some cases lead to a lower number of viral progeny production (Zheng et al., 2007; Tyler et al., 2009; Kim et al., 2013). Therefore, depending on the modification(s), the infectivity or production of viral progeny can be different in SCs. As such, Basic Protocol 2 is necessary to determine in vitro the optimal virus titer that will yield the maximum number of viral progeny without killing SCs prior to in vivo delivery. In this protocol, we do not include the generation or
BASIC PROTOCOL 2
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Figure 13.11.1 Neural SC plating and virus dilution for cell viability and viral progeny production. To assess the optimal titer of adenovirus for neural SC infection, neural SCs are seeded in 24-well plates at 2 × 104 cells per well, and cells are infected with different virus titers (from 0.1 to 1,000). Virus titers should be evaluated in triplicate to minimize experimental error.
production of adenoviral vectors. Excellent protocols for the design, generation, production, and storage of adenoviral vectors can be found in UNITS 12.4 & 12.14 (He, 2004; Davydova and Yamamoto, 2013).
Materials Stem cells, e.g., neural stem cells (HB1.F3-CD), or bone marrow- or adipose-derived mesenchymal stem cells Adenovirus stock (see UNITS 12.4 & 12.14; He, 2004; Davydova and Yamamoto, 2013) 1× PBS (APPENDIX 2D) DMEM containing 2% FBS and 1× Pen/Strep 0.25% trypsin-EDTA Trypan blue (Sigma, cat. no. T8154) Adeno-XTM Rapid Titer Kit (Clontech, cat. no. 632250) 24-well plates 1.5-ml tubes Hemacytometer NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.
Determine the viability of SCs post virus loading 1. Transfer 2 × 104 cells to each well of a 24-well plate (Fig. 13.11.1), and incubate overnight in a 37°C, 5% CO2 humidified incubator. 2. On the following day, prepare a 100-fold dilution of virus by mixing 10 μl virus stock with 990 μl of 1× PBS. Keep this dilution readily available on ice. With some exception, adenovirus stocks typically contain a high number of virus particles (approximately 1012 virus particles per ml) in 10% to 50% glycerol (UNITS 12.4 & 12.14; He, 2004; Davydova and Yamamoto, 2013). Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
The dissociation factor for adenovirus is very low (Shabram et al., 2002). Therefore, it is highly recommended to prepare a 100-fold dilution of virus stock and use this diluted virus for infections, as the distribution of virus will be more uniform in the infection medium.
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Table 13.11.1 Formula for Diluting Virus for Infection
Virus stock (Vp/ml or Name pfu/ml) A
Cells per well
Viruses per cell (MOI)
B
C
Total wells
Volume (ml) of medium for 4 wells
D
E
Virus titer to use
100-fold dilution virus titer
Volume (μl) of 100-fold diluted virus to add to medium in E
F=B×C×D
G=A/100
H=F/G×1000
Example: Use a 10 virus stock to infect 3 wells of 2 ×10 cells at an MOI of 1000. 12
JK
12
10
2.0 ×104
1000
4
4
2 ml
(1 extra)
(500 μl/well)
8.0 ×107
1010
8 μl
Add 8 μl (H) of 100-fold diluted virus to 2 ml infection medium. Add 500 μl to each well to achieve an MOI of 1000.
3. Using Table 13.11.1 and the 24-well plate schematic in Figure 13.11.1, prepare triplicate dilutions of adenovirus in virus infection medium (DMEM containing 2% FBS and 1× Pen/Strep) with multiplicity of infection (MOI) ranging from 1 to 1,000 virus particles (vp) per cell or 0.1 to 100 plaque forming units (pfu) per cell. (Note that 1 pfu is approximately equivalent to 10-100 vp.). 4. Aspirate the medium from each well of the 24-well plate of SCs, and add medium containing the appropriate titer of virus to each well (Figure 13.11.1). 5. Incubate for 2 hr in a 37°C, 5% CO2 humidified incubator, aspirate the viruscontaining medium, wash each well with 0.5 ml of 1× PBS, and aspirate the wash buffer. 6. Add 0.5 ml cell-growth medium, and incubate 72 hr in a 37°C, 5% CO2 humidified incubator. 7. Transfer the culture medium from each well to appropriately labeled 1.5-ml tubes. 8. Add 150 μl of 0.25% trypsin-EDTA to each well, and incubate until the cells detach. 9. Add 300 μl of 1× PBS to each well, and transfer to trypsinized cells to the corresponding 1.5-ml tubes from step 7. Between 48 and 72 hr after infection, viruses will propagate and begin to induce cell detachment. To analyze the cell viability, both adhered and detached cells have to be collected.
10. Homogenize the cells in each tube by repeatedly pipetting. 11. Mix 20 μl each cell suspension with 20 μl trypan blue in 1.5-ml tubes. 12. Quantify the live and dead cells with a hemacytometer. Dead cells will take up trypan blue and appear dark under the microscope. The ideal percentage of viable cells should be at least 80% to 90%. If cell viability is greater than 80% at each MOI (i.e., in every well), then increase the entire range of MOIs. Alternatively, if cell viability is less than 80% at each MOI, then decrease the entire range of MOIs. The goal is to obtain a wide range of cell viability to identify the optimal MOI for loading NSCs.
Analysis of viral progeny production in SC 13. Follow steps 1 to 9 above. 14. Centrifuge at 266 × g for 5 min at 4°C to pellet the cells. 15. Aspirate the supernatant, and suspend in 50 μl of 1× PBS.
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16. Freeze the tubes containing each cell suspension at –80°C, thaw at 37°C, and vortex well. 17. Repeat step 16, three times. Adenovirus particles are relatively stable during the freezing and thawing steps. These steps will, however, rupture the cell membrane and release viral progeny into the supernatant.
18. Check the virus titer with Adeno-XTM Rapid Titer Kit (Clontech). Around 104 virus particles can be produced in one cell. Therefore 106 SCs loaded with an optimized viral titer (between 10 and 100 infectious units) is expected to produce and release approximately 108 to 1010 virus particles in 72 hr. BASIC PROTOCOL 3
LOADING OF ADENOVIRUS INTO STEM CELLS This protocol describes the final loading of adenovirus based on optimal adenovirus titers determined in Basic Protocol 2 (Yong et al., 2009; Bolontrade et al., 2012; Ahmed et al., 2013). Virus-loaded SCs are intended for use shortly after preparation.
Materials Stem cells, e.g., neural stem cells (HB1.F3-CD), or bone marrow- or adipose-derived mesenchymal stem cells 1× PBS (APPENDIX 2D) Adenovirus stock (UNITS 12.4 & 12.14; He, 2004; Davydova and Yamamoto, 2013): the optimal virus titer should have been determined (see Basic Protocol 2) T75 flasks 15- and 50-ml conical tubes Hemocytometer NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol. NOTE: Virus-loaded SCs should be prepared immediately prior to use. 1. Wash and trypsinize a confluent T75 flask (minimum 80% to 90% confluency) of SCs as described in steps 10 to 15 of Basic Protocol 1. 2. Transfer the cells to a 50-ml conical tube, and pellet the cells by centrifugation at 266 × g for 5 min at 4°C. 3. Aspirate the growth-medium supernatant, and add 5 ml of 1× PBS. 4. Quantify the cell number using a hemocytometer. 5. Transfer approximately 106 cells to a new 15-ml tube, add 5 ml of 1× PBS for a second wash, and pellet the cells by centrifugation at 266 × g for 5 min at 4°C. 6. During the centrifugation, prepare optimal virus titers (see Basic Protocol 2) in 100 μl of 1× PBS. Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
7. Aspirate the supernatant from the pelleted cells, and add the 100 μl of optimal virus titer to the cells. 8. Mix gently by pipetting, and incubate 2 hr at room temperature.
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9. Add 0.5 ml of 1× PBS, and pellet the cells by centrifugation at 266 × g for 5 min at 4°C. 10. Aspirate the supernatant, gently suspend in 0.5 ml of 1× PBS, and pellet the cells by centrifugation at 1,200 rpm for 5 min. 11. Repeat step 10 three times. 12. After washing, suspend the cells in a desired amount of 1× PBS (Yong et al., 2009; Bolontrade et al., 2012; Ahmed et al., 2013). The amount of 1× PBS needed will vary depending on the application. For example, 5 to 10 μl per injection would be the maximum volume for intracranial injection, but other applications might require 100 μl.
13. Keep the virus-loaded SCs on ice until use. It is recommended to use the virus-loaded SCs as soon as possible, but they can be stored on ice for up to an hour.
COMMENTARY Background Information Initially isolated from human adenoids as infectious particles in the mid-20th century, adenoviruses may now be engineered for use as delivery vehicles for gene therapy and for their oncolytic activity against cancer (Nandi and Lesniak, 2009). More specifically, adenovirus-mediated cancer therapy has sparked particular interest in treating glioma (Nandi and Lesniak, 2009). This is due to the fact that these viruses function particularly well as delivery vehicles, offering specific tropism to the engineered site so desired (Zheng et al., 2007; Tyler et al., 2009; Kim et al., 2013). However, despite many advantages of adenoviral vectors, there have been major challenges for their use, such as pre-existing anti-adenoviral immunity within the population, and minimal penetration and spreading of viruses in the tumor mass (Hendrickx et al., 2014). The use of a stem cell carrier can overcome these challenges and enhance the oncolytic activity of adenovirus. Since the discovery of stem cells (SCs), their biological classification, function, and potential for therapeutic use has been investigated in a litany of preclinical and translational studies. The self-renewal and regenerative capabilities of SCs make them attractive agents for treatment of neurodegenerative disorders. However, it is their tropism to brain tumors that motivated the study of their potential as therapeutic delivery vehicles (Aboody et al., 2008). This remarkably intrinsic tumor tropism of neural stem cells allows them to specifically deliver a therapeutic agent, such as an oncolytic adenovirus, to a tumor, drastically reducing the off-target side effects all too
commonly experienced. Using SCs to deliver oncolytic viruses can enhance viral distribution throughout the tumor bed and simultaneously shield the viruses from the immune system (Ahmed et al., 2010). Furthermore, SCs can deliver oncolytic viruses across the blood– brain barrier to a tumor, making multiple, lessinvasive intravenous doses of therapy possible (Aboody et al., 2000). The groundbreaking use of SCs as a carrier for therapeutic agents has already progressed to early clinical trials, and hopefully, clinical trials exploring stem cell-mediated delivery of oncolytic adenoviruses will commence in the near future. Preclinical studies have demonstrated that SCs can successfully be loaded with conditionally replicative viruses, deliver them to a tumor, and provide a therapeutic benefit in animal models (Ahmed et al., 2011; Ahmed et al., 2012; Thaci et al., 2012; Tobias et al., 2013). The innate limitations and obstacles facing oncolytic virotherapy can be overcome by delivering them with stem cell carriers like neural SCs and mesenchymal SCs, and the future is bright for this promising combination of molecular agents against currently devastating malignancies.
Critical Parameters and Troubleshooting Several steps are critical for the efficient loading of adenoviruses on SCs. The initial recovery of cells from the frozen stock takes about 3 days. At this point, SCs grow slowly. However, SCs grow exponentially after 2 to 3 passages. Therefore, during the initial passages, extra careful observation is absolutely required.
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SCs can be cultured for 25 to 30 passages, after which SCs start to grow extremely slow. Cells should not be cultured or used for any experiments after 30 passages. Moreover, it is highly recommended to make frozen stocks at the earliest time point. In addition, SCs are highly sensitive to pH change. When SCs are confluent and left to themselves for too long, the pH of the media acidifies, causing SCs to die. Observe the SCs on daily, and change the media if any media acidification is observed. Optimization of virus titer to maximize viral progeny production and minimize cytopathic effects on SCs is the most important step for a stem cell-mediated adenovirus delivery approach. Depending on how well it is optimized, it will completely change the therapeutic effect from the SC-mediated adenovirus delivery approach. As mentioned above, within 72 hr the viral progeny production should reach the maximum without killing the SCs. At least 80% to 90% of virus loaded SCs should be healthy but detached from the bottom of the well, which should generate approximately 104 virus particles per cell. For optimized virus titer, it is recommended to dilute an original virus stock to 100-fold in 1× PBS and use it for infection. Keep in mind that the dissociation factor of adenovirus particles is low and the sample must be thoroughly mixed to achieve homogeneity of virus particles in the solution and minimize variation between experiments.
Anticipated Results It takes approximately 48 to 72 hr for adenovirus to produce the maximum number of virus progeny (104 virus particles) in a host cell and to be released from the host cell. When healthy and minimally passaged, SCs will be loaded with optimized virus particles/cell and these SCs will migrate to areas around the tumor during this progeny production period and then release the virus progeny. When 106 SCs are loaded with adenoviruses at an optimal MOI (between 10 and 100 infectious units), they are expected to produce and release approximately 108 to 1010 virus particles at their destination.
Time Considerations Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
Initial cell recovery time from a frozen stock can vary depending on the condition of the cell stock and personal skills, but subculturing of SCs should be done within 72 hr of plating to keep them healthy. When the SCs do not grow well at the initial recovery or during
the subculture period, aspirate and discard the old media, and replace with fresh cell-growth medium. All of the procedures, from the preparation of SCs and adenovirus infection (loading) to delivery, should be done in 3 to 4 hr to maximize the therapeutic efficacy of SC-mediated adenovirus delivery.
Acknowledgements This work was supported by NIH/NINDS grant U01NS069997 to MSL.
Literature Cited Aboody, K.S., Najbauer, J., and Danks, M.K. 2008. Stem and progenitor cell-mediated tumor selective gene therapy. Gene. Ther. 15:739-752. Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., and Snyder, E.Y. 2000. Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U.S.A. 97:12846-12851. Ahmed, A.U., Alexiades, N.G., and Lesniak, M.S. 2010. The use of neural stem cells in cancer gene therapy: Predicting the path to the clinic. Curr. Opin. Mol. Ther. 12:546-552. Ahmed, A.U., Ulasov, I.V., Mercer, R.W., and Lesniak, M.S. 2012. Maintaining and loading neural stem cells for delivery of oncolytic adenovirus to brain tumors. Methods Mol. Biol. 797:97109. Ahmed, A.U., Thaci, B., Alexiades, N.G., Han, Y., Qian, S., Liu, F., Balyasnikova, I.V., Ulasov, I.Y., Aboody, K.S., and Lesniak, M.S. 2011. Neural stem cell-based cell carriers enhance therapeutic efficacy of an oncolytic adenovirus in an orthotopic mouse model of human glioblastoma. Mol. Ther. 19:1714-1726. Ahmed, A.U., Thaci, B., Tobias, A.L., Auffinger, B., Zhang, L., Cheng, Y., Kim, C.K., Yunis, C., Han, Y., Alexiades, N.G., Fan, X., Aboody, K.S., and Lesniak, M.S. 2013. A preclinical evaluation of neural stem cell-based cell carrier for targeted antiglioma oncolytic virotherapy. J. Natl. Cancer Inst. 105:968-977. Bolontrade, M.F., Sganga, L., Piaggio, E., Viale, D.L., Sorrentino, M.A., Robinson, A., Sevlever, G., Garcia, M.G., Mazzolini, G., and Podhajcer, O.L. 2012. A specific subpopulation of mesenchymal stromal cell carriers overrides melanoma resistance to an oncolytic adenovirus. Stem Cells Dev. 21:2689-2702. Davydova, J. and Yamamoto, M. 2013. Oncolytic adenoviruses: Design, generation, and experimental procedures. Curr. Protoc. Hum. Genet. 78:12.14:12.14.1-12.14.21. He, T.-C. 2004. Adenoviral vectors. Curr. Protoc. Hum. Genet. 40:12.4:12.4.1-12.4.25. Hendrickx, R., Stichling, N., Koelen, J., Kuryk, L., Lipiec, A., and Greber, U.F. 2014. Innate immunity to adenovirus. Hum. Gene Ther. 25:265284.
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Kim, J.W., Glasgow, J.N., Nakayama, M., Ak, F., Ugai, H., and Curiel, D.T. 2013. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer. PLoS One 8:e55533. Nandi, S. and Lesniak, M.S. 2009. Adenoviral virotherapy for malignant brain tumors. Expert. Opin. Biol. Ther. 9:737-747. Phelan, M. C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Hum. Genet. 51:A.3G.1-A.3G.18. Shabram, P., Vellekamp, G., and Scandella, C. 2002. Purification of Adenovirus, Elsevier. Thaci, B., Ahmed, A.U., Ulasov, I.V., Tobias, A.L., Han, Y., Aboody, K.S., and Lesniak, M.S. 2012. Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: Targeted delivery of the therapeutic payload in an orthotopic brain tumor model. Cancer Gene Ther. 19:431-442. Tobias, A.L., Thaci, B., Auffinger, B., Rincon, E., Balyasnikova, I.V., Kim, C.K., Han, Y., Zhang,
L., Aboody, K.S., Ahmed, A.U., and Lesniak, M.S. 2013. The timing of neural stem cellbased virotherapy is critical for optimal therapeutic efficacy when applied with radiation and chemotherapy for the treatment of glioblastoma. Stem Cells Transl. Med. 2:655-666. Tyler, M.A., Ulasov, I.V., Sonabend, A.M., Nandi, S., Han, Y., Marler, S., Roth, J., and Lesniak, M.S. 2009. Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo. Gene Ther. 16:262-278. Yong, R.L., Shinojima, N., Fueyo, J., Gumin, J., Vecil, G.G., Marini, F.C., Bogler, O., Andreeff, M., and Lang, F.F. 2009. Human bone marrowderived mesenchymal stem cells for intravascular delivery of oncolytic adenovirus Delta24RGD to human gliomas. Cancer Res. 69:89328940. Zheng, S., Ulasov, I.V., Han, Y., Tyler, M.A., Zhu, Z.B., and Lesniak, M.S. 2007. Fiber-knob modifications enhance adenoviral tropism and gene transfer in malignant glioma. J. Gene Med. 9:151-160.
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UNIT 13.11
Julius W. Kim,1 J. Robert Kane,1 Jacob S. Young,1 Alan L. Chang,1 Deepak Kanojia,1 Shuo Qian,1 Drew A. Spencer,1 Atique U. Ahmed,1 and Maciej S. Lesniak1 1
The Brain Tumor Center, University of Chicago, Chicago, Illinois
The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials. These clinical trials exploring SC-mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that can provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally-replicative adenoviruses and delivered to the tumor, whereupon they may evoke pronounced therapeutic efficacy. In this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for C 2015 by John SC loading, and evaluate the optimized viral loading on SCs. Wiley & Sons, Inc. Keywords: neural stem cell r mesenchymal stem cell r oncolytic adenovirus r cancer r gene therapy
How to cite this article: Kim, J.W., Kane, J.R., Young, J.S., Chang, A.L., Kanojia, D., Qian, S., Spencer, D.A., Ahmed, A.U., Lesniak, M.S. 2015. Neural stem cell-mediated delivery of oncolytic adenovirus. Curr. Protoc. Hum. Genet. 85:13.11.1-13.11.9. doi: 10.1002/0471142905.hg1311s85
INTRODUCTION As stem cell-mediated delivery of oncolytic adenoviruses has shown promising therapeutic efficacy, it is necessary to standardize the protocol to enhance the efficacy and consistency of the approach. Only when healthy and minimally passaged SCs are loaded with an optimized number of virus particles per cell will these SCs migrate to their destined place(s) around the tumor area during the period of viral progeny production (48 to 72 hr) and then release the virus progenies (104 particles/cell). We describe here how to properly culture and prepare the SCs for oncolytic virus loading. We further describe how to optimize a virus titer for loading on SCs to maximize the oncolytic virus progenies delivered to the target site.
CULTURE OF NEURAL STEM CELLS This protocol describes the growth, splitting, and freezing of stem cells for adenoviral vector loading.
BASIC PROTOCOL 1
Materials Stem cells, e.g., neural stem cells (HB1.F3-CD), or bone marrow- or adipose-derived mesenchymal stem cells Dulbecco’s Modified Eagle’s medium (DMEM; Corning, cat. no. 10-017-CV; see APPENDIX 3G; Phelan, 2006) Current Protocols in Human Genetics 13.11.1-13.11.9, April 2015 Published online April 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg1311s85 C 2015 John Wiley & Sons, Inc. Copyright
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Fetal bovine serum (FBS; Hyclone, cat. no. SH30088.03) 100× Pen/Strep stock (Corning, cat. no. 30-002-CI): penicillin sodium (10,000 units/ml) and streptomycin (10,000 μg/ml) 100× glutamine stock (200 mM; Corning, cat. no. 25-005-CI): only needed if DMEM lacks glutamine 1× PBS (Corning, cat. no. 21-030-CV; also see APPENDIX 2D) 0.25% trypsin-EDTA solution (Corning, cat. no. 25-053-CI) Dimethyl sulfoxide (DMSO; Sigma, cat. no. D2650) T75 cell-culture flasks 15-ml and 50-ml conical tubes Microscope 37°C, 5% CO2 humidified incubator Additional reagents and equipment required for human tissue culture, including subculturing, freezing, thawing, and counting cells (APPENDIX 3G; Phelan, 2006) NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.
Initial culture from a frozen stock of SCs This protocol is tailored for thawing a cryotube containing 2 × 106 cells frozen in 1 ml cell-freezing medium. 1. Add 15 ml cell-growth medium (DMEM containing 10% FBS and 1× Pen/Strep) to a T75 flask. 2. Spread the cell-growth medium throughout the flask by gently tipping the flask in different directions. 3. Thaw the frozen stock of SCs in a 37°C water bath. 4. Transfer the cryotube containing thawed cells to a sterile hood. 5. Slowly add the SCs to the growth media in the T75 flask. Slowly adding the SCs to the culture media minimizes osmotic shock.
6. Gently mix the cells in the media to distribute them evenly across the surface of the T75 flask. 7. Incubate the cells in growth media in a 37°C, 5% CO2 humidified incubator. 8. On the following day, aspirate the media from the T75 flask, and immediately add 15 ml freshly prepared cell-growth medium. This step helps to remove any dead cells and DMSO from the freezing medium.
9. Incubate the cells in a 37°C, 5% CO2 humidified incubator for 3 to 4 days. Please note that it will take approximately 3 to 4 days for SCs to fully recover and propagate efficiently.
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Subculture of SCs 10. Aspirate the existing cell-growth medium from the T75 flask. 11. Add 5 ml of 1× PBS and gently tip the flask in several directions to distribute and wash the PBS over the cells. Aspirate the PBS.
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12. Add 1.5 ml of 0.25% trypsin-EDTA solution to the T75 flask, and distribute the trypsin solution over the cells by tipping the flask in several directions. 13. Incubate the cells with trypsin for 5 min or until cells begin to detach. To accelerate cell detachment, the flask can be incubated in a 37°C, 5% CO2 humidified incubator.
14. When obvious cell detachment is observed, add 5 ml fresh cell-growth medium to the T75 flask. 15. Wash and homogenize the cells in the cell-growth media by repeatedly pipetting the cells with a serologic pipet. Be especially gentle while repeatedly pipetting. Strong agitation causes the cells to die.
16. Transfer the trypsinized cells evenly between three new T75 flasks (2.3 ml per flask). 17. Add 12 ml fresh cell-growth medium to each flask, and incubate in a 37°C, 5% CO2 humidified incubator. SCs can grow rapidly for 25 to 30 passages (Aboody et al., 2008; Ahmed et al., 2012). During these passages, carefully check their growth every day. As the media becomes acidic (it will begin to turn yellow), change the media.
Preparation of Frozen Stocks of SCs 18. Wash and trypsinize a highly confluent T75 flask as described in steps 10 to 15. A minimum of 2–3 × 106 cells per 1 ml of freezing media in cryotube is required to restore the cells in a T75 flask. At high confluence, a T75 flask contains approximately 107 cells. Therefore, a single T75 flask will yield three cryotubes of frozen stock.
19. Transfer the trypsinized cells in medium (6.5 ml) to a 50 ml conical tube. 20. Centrifuge the tube at 266 × g for 5 min at 4°C. 21. During the centrifugation, make an appropriate amount of the cell-freezing medium (10% DMSO in FBS). For each T75 flask of cells to be frozen, mix 300 μl DMSO in 2.7 ml FBS. 22. Aspirate the supernatant media, and add 3 ml cell-freezing medium. 23. Homogenize the cells thoroughly, but carefully, and divide between three different cryotubes (1 ml per tube). 24. Place the cryotubes at –80°C overnight, and transfer to liquid nitrogen storage (–180°C) the next day.
OPTIMIZING THE LOADING CAPABILITY OF STEM CELLS The primary receptor of the wild-type human adenovirus serotype 5 (HAd5) is not expressed on the SCs. The Ad5 fiber domain, which dictates the receptor binding, has been genetically modified to achieve the efficient, enhanced, or directed specific infection in most of presently used adenoviral vectors. However, modifying this structural protein (fiber) could in some cases lead to a lower number of viral progeny production (Zheng et al., 2007; Tyler et al., 2009; Kim et al., 2013). Therefore, depending on the modification(s), the infectivity or production of viral progeny can be different in SCs. As such, Basic Protocol 2 is necessary to determine in vitro the optimal virus titer that will yield the maximum number of viral progeny without killing SCs prior to in vivo delivery. In this protocol, we do not include the generation or
BASIC PROTOCOL 2
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Figure 13.11.1 Neural SC plating and virus dilution for cell viability and viral progeny production. To assess the optimal titer of adenovirus for neural SC infection, neural SCs are seeded in 24-well plates at 2 × 104 cells per well, and cells are infected with different virus titers (from 0.1 to 1,000). Virus titers should be evaluated in triplicate to minimize experimental error.
production of adenoviral vectors. Excellent protocols for the design, generation, production, and storage of adenoviral vectors can be found in UNITS 12.4 & 12.14 (He, 2004; Davydova and Yamamoto, 2013).
Materials Stem cells, e.g., neural stem cells (HB1.F3-CD), or bone marrow- or adipose-derived mesenchymal stem cells Adenovirus stock (see UNITS 12.4 & 12.14; He, 2004; Davydova and Yamamoto, 2013) 1× PBS (APPENDIX 2D) DMEM containing 2% FBS and 1× Pen/Strep 0.25% trypsin-EDTA Trypan blue (Sigma, cat. no. T8154) Adeno-XTM Rapid Titer Kit (Clontech, cat. no. 632250) 24-well plates 1.5-ml tubes Hemacytometer NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.
Determine the viability of SCs post virus loading 1. Transfer 2 × 104 cells to each well of a 24-well plate (Fig. 13.11.1), and incubate overnight in a 37°C, 5% CO2 humidified incubator. 2. On the following day, prepare a 100-fold dilution of virus by mixing 10 μl virus stock with 990 μl of 1× PBS. Keep this dilution readily available on ice. With some exception, adenovirus stocks typically contain a high number of virus particles (approximately 1012 virus particles per ml) in 10% to 50% glycerol (UNITS 12.4 & 12.14; He, 2004; Davydova and Yamamoto, 2013). Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
The dissociation factor for adenovirus is very low (Shabram et al., 2002). Therefore, it is highly recommended to prepare a 100-fold dilution of virus stock and use this diluted virus for infections, as the distribution of virus will be more uniform in the infection medium.
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Table 13.11.1 Formula for Diluting Virus for Infection
Virus stock (Vp/ml or Name pfu/ml) A
Cells per well
Viruses per cell (MOI)
B
C
Total wells
Volume (ml) of medium for 4 wells
D
E
Virus titer to use
100-fold dilution virus titer
Volume (μl) of 100-fold diluted virus to add to medium in E
F=B×C×D
G=A/100
H=F/G×1000
Example: Use a 10 virus stock to infect 3 wells of 2 ×10 cells at an MOI of 1000. 12
JK
12
10
2.0 ×104
1000
4
4
2 ml
(1 extra)
(500 μl/well)
8.0 ×107
1010
8 μl
Add 8 μl (H) of 100-fold diluted virus to 2 ml infection medium. Add 500 μl to each well to achieve an MOI of 1000.
3. Using Table 13.11.1 and the 24-well plate schematic in Figure 13.11.1, prepare triplicate dilutions of adenovirus in virus infection medium (DMEM containing 2% FBS and 1× Pen/Strep) with multiplicity of infection (MOI) ranging from 1 to 1,000 virus particles (vp) per cell or 0.1 to 100 plaque forming units (pfu) per cell. (Note that 1 pfu is approximately equivalent to 10-100 vp.). 4. Aspirate the medium from each well of the 24-well plate of SCs, and add medium containing the appropriate titer of virus to each well (Figure 13.11.1). 5. Incubate for 2 hr in a 37°C, 5% CO2 humidified incubator, aspirate the viruscontaining medium, wash each well with 0.5 ml of 1× PBS, and aspirate the wash buffer. 6. Add 0.5 ml cell-growth medium, and incubate 72 hr in a 37°C, 5% CO2 humidified incubator. 7. Transfer the culture medium from each well to appropriately labeled 1.5-ml tubes. 8. Add 150 μl of 0.25% trypsin-EDTA to each well, and incubate until the cells detach. 9. Add 300 μl of 1× PBS to each well, and transfer to trypsinized cells to the corresponding 1.5-ml tubes from step 7. Between 48 and 72 hr after infection, viruses will propagate and begin to induce cell detachment. To analyze the cell viability, both adhered and detached cells have to be collected.
10. Homogenize the cells in each tube by repeatedly pipetting. 11. Mix 20 μl each cell suspension with 20 μl trypan blue in 1.5-ml tubes. 12. Quantify the live and dead cells with a hemacytometer. Dead cells will take up trypan blue and appear dark under the microscope. The ideal percentage of viable cells should be at least 80% to 90%. If cell viability is greater than 80% at each MOI (i.e., in every well), then increase the entire range of MOIs. Alternatively, if cell viability is less than 80% at each MOI, then decrease the entire range of MOIs. The goal is to obtain a wide range of cell viability to identify the optimal MOI for loading NSCs.
Analysis of viral progeny production in SC 13. Follow steps 1 to 9 above. 14. Centrifuge at 266 × g for 5 min at 4°C to pellet the cells. 15. Aspirate the supernatant, and suspend in 50 μl of 1× PBS.
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16. Freeze the tubes containing each cell suspension at –80°C, thaw at 37°C, and vortex well. 17. Repeat step 16, three times. Adenovirus particles are relatively stable during the freezing and thawing steps. These steps will, however, rupture the cell membrane and release viral progeny into the supernatant.
18. Check the virus titer with Adeno-XTM Rapid Titer Kit (Clontech). Around 104 virus particles can be produced in one cell. Therefore 106 SCs loaded with an optimized viral titer (between 10 and 100 infectious units) is expected to produce and release approximately 108 to 1010 virus particles in 72 hr. BASIC PROTOCOL 3
LOADING OF ADENOVIRUS INTO STEM CELLS This protocol describes the final loading of adenovirus based on optimal adenovirus titers determined in Basic Protocol 2 (Yong et al., 2009; Bolontrade et al., 2012; Ahmed et al., 2013). Virus-loaded SCs are intended for use shortly after preparation.
Materials Stem cells, e.g., neural stem cells (HB1.F3-CD), or bone marrow- or adipose-derived mesenchymal stem cells 1× PBS (APPENDIX 2D) Adenovirus stock (UNITS 12.4 & 12.14; He, 2004; Davydova and Yamamoto, 2013): the optimal virus titer should have been determined (see Basic Protocol 2) T75 flasks 15- and 50-ml conical tubes Hemocytometer NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol. NOTE: Virus-loaded SCs should be prepared immediately prior to use. 1. Wash and trypsinize a confluent T75 flask (minimum 80% to 90% confluency) of SCs as described in steps 10 to 15 of Basic Protocol 1. 2. Transfer the cells to a 50-ml conical tube, and pellet the cells by centrifugation at 266 × g for 5 min at 4°C. 3. Aspirate the growth-medium supernatant, and add 5 ml of 1× PBS. 4. Quantify the cell number using a hemocytometer. 5. Transfer approximately 106 cells to a new 15-ml tube, add 5 ml of 1× PBS for a second wash, and pellet the cells by centrifugation at 266 × g for 5 min at 4°C. 6. During the centrifugation, prepare optimal virus titers (see Basic Protocol 2) in 100 μl of 1× PBS. Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
7. Aspirate the supernatant from the pelleted cells, and add the 100 μl of optimal virus titer to the cells. 8. Mix gently by pipetting, and incubate 2 hr at room temperature.
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9. Add 0.5 ml of 1× PBS, and pellet the cells by centrifugation at 266 × g for 5 min at 4°C. 10. Aspirate the supernatant, gently suspend in 0.5 ml of 1× PBS, and pellet the cells by centrifugation at 1,200 rpm for 5 min. 11. Repeat step 10 three times. 12. After washing, suspend the cells in a desired amount of 1× PBS (Yong et al., 2009; Bolontrade et al., 2012; Ahmed et al., 2013). The amount of 1× PBS needed will vary depending on the application. For example, 5 to 10 μl per injection would be the maximum volume for intracranial injection, but other applications might require 100 μl.
13. Keep the virus-loaded SCs on ice until use. It is recommended to use the virus-loaded SCs as soon as possible, but they can be stored on ice for up to an hour.
COMMENTARY Background Information Initially isolated from human adenoids as infectious particles in the mid-20th century, adenoviruses may now be engineered for use as delivery vehicles for gene therapy and for their oncolytic activity against cancer (Nandi and Lesniak, 2009). More specifically, adenovirus-mediated cancer therapy has sparked particular interest in treating glioma (Nandi and Lesniak, 2009). This is due to the fact that these viruses function particularly well as delivery vehicles, offering specific tropism to the engineered site so desired (Zheng et al., 2007; Tyler et al., 2009; Kim et al., 2013). However, despite many advantages of adenoviral vectors, there have been major challenges for their use, such as pre-existing anti-adenoviral immunity within the population, and minimal penetration and spreading of viruses in the tumor mass (Hendrickx et al., 2014). The use of a stem cell carrier can overcome these challenges and enhance the oncolytic activity of adenovirus. Since the discovery of stem cells (SCs), their biological classification, function, and potential for therapeutic use has been investigated in a litany of preclinical and translational studies. The self-renewal and regenerative capabilities of SCs make them attractive agents for treatment of neurodegenerative disorders. However, it is their tropism to brain tumors that motivated the study of their potential as therapeutic delivery vehicles (Aboody et al., 2008). This remarkably intrinsic tumor tropism of neural stem cells allows them to specifically deliver a therapeutic agent, such as an oncolytic adenovirus, to a tumor, drastically reducing the off-target side effects all too
commonly experienced. Using SCs to deliver oncolytic viruses can enhance viral distribution throughout the tumor bed and simultaneously shield the viruses from the immune system (Ahmed et al., 2010). Furthermore, SCs can deliver oncolytic viruses across the blood– brain barrier to a tumor, making multiple, lessinvasive intravenous doses of therapy possible (Aboody et al., 2000). The groundbreaking use of SCs as a carrier for therapeutic agents has already progressed to early clinical trials, and hopefully, clinical trials exploring stem cell-mediated delivery of oncolytic adenoviruses will commence in the near future. Preclinical studies have demonstrated that SCs can successfully be loaded with conditionally replicative viruses, deliver them to a tumor, and provide a therapeutic benefit in animal models (Ahmed et al., 2011; Ahmed et al., 2012; Thaci et al., 2012; Tobias et al., 2013). The innate limitations and obstacles facing oncolytic virotherapy can be overcome by delivering them with stem cell carriers like neural SCs and mesenchymal SCs, and the future is bright for this promising combination of molecular agents against currently devastating malignancies.
Critical Parameters and Troubleshooting Several steps are critical for the efficient loading of adenoviruses on SCs. The initial recovery of cells from the frozen stock takes about 3 days. At this point, SCs grow slowly. However, SCs grow exponentially after 2 to 3 passages. Therefore, during the initial passages, extra careful observation is absolutely required.
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SCs can be cultured for 25 to 30 passages, after which SCs start to grow extremely slow. Cells should not be cultured or used for any experiments after 30 passages. Moreover, it is highly recommended to make frozen stocks at the earliest time point. In addition, SCs are highly sensitive to pH change. When SCs are confluent and left to themselves for too long, the pH of the media acidifies, causing SCs to die. Observe the SCs on daily, and change the media if any media acidification is observed. Optimization of virus titer to maximize viral progeny production and minimize cytopathic effects on SCs is the most important step for a stem cell-mediated adenovirus delivery approach. Depending on how well it is optimized, it will completely change the therapeutic effect from the SC-mediated adenovirus delivery approach. As mentioned above, within 72 hr the viral progeny production should reach the maximum without killing the SCs. At least 80% to 90% of virus loaded SCs should be healthy but detached from the bottom of the well, which should generate approximately 104 virus particles per cell. For optimized virus titer, it is recommended to dilute an original virus stock to 100-fold in 1× PBS and use it for infection. Keep in mind that the dissociation factor of adenovirus particles is low and the sample must be thoroughly mixed to achieve homogeneity of virus particles in the solution and minimize variation between experiments.
Anticipated Results It takes approximately 48 to 72 hr for adenovirus to produce the maximum number of virus progeny (104 virus particles) in a host cell and to be released from the host cell. When healthy and minimally passaged, SCs will be loaded with optimized virus particles/cell and these SCs will migrate to areas around the tumor during this progeny production period and then release the virus progeny. When 106 SCs are loaded with adenoviruses at an optimal MOI (between 10 and 100 infectious units), they are expected to produce and release approximately 108 to 1010 virus particles at their destination.
Time Considerations Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
Initial cell recovery time from a frozen stock can vary depending on the condition of the cell stock and personal skills, but subculturing of SCs should be done within 72 hr of plating to keep them healthy. When the SCs do not grow well at the initial recovery or during
the subculture period, aspirate and discard the old media, and replace with fresh cell-growth medium. All of the procedures, from the preparation of SCs and adenovirus infection (loading) to delivery, should be done in 3 to 4 hr to maximize the therapeutic efficacy of SC-mediated adenovirus delivery.
Acknowledgements This work was supported by NIH/NINDS grant U01NS069997 to MSL.
Literature Cited Aboody, K.S., Najbauer, J., and Danks, M.K. 2008. Stem and progenitor cell-mediated tumor selective gene therapy. Gene. Ther. 15:739-752. Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., and Snyder, E.Y. 2000. Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U.S.A. 97:12846-12851. Ahmed, A.U., Alexiades, N.G., and Lesniak, M.S. 2010. The use of neural stem cells in cancer gene therapy: Predicting the path to the clinic. Curr. Opin. Mol. Ther. 12:546-552. Ahmed, A.U., Ulasov, I.V., Mercer, R.W., and Lesniak, M.S. 2012. Maintaining and loading neural stem cells for delivery of oncolytic adenovirus to brain tumors. Methods Mol. Biol. 797:97109. Ahmed, A.U., Thaci, B., Alexiades, N.G., Han, Y., Qian, S., Liu, F., Balyasnikova, I.V., Ulasov, I.Y., Aboody, K.S., and Lesniak, M.S. 2011. Neural stem cell-based cell carriers enhance therapeutic efficacy of an oncolytic adenovirus in an orthotopic mouse model of human glioblastoma. Mol. Ther. 19:1714-1726. Ahmed, A.U., Thaci, B., Tobias, A.L., Auffinger, B., Zhang, L., Cheng, Y., Kim, C.K., Yunis, C., Han, Y., Alexiades, N.G., Fan, X., Aboody, K.S., and Lesniak, M.S. 2013. A preclinical evaluation of neural stem cell-based cell carrier for targeted antiglioma oncolytic virotherapy. J. Natl. Cancer Inst. 105:968-977. Bolontrade, M.F., Sganga, L., Piaggio, E., Viale, D.L., Sorrentino, M.A., Robinson, A., Sevlever, G., Garcia, M.G., Mazzolini, G., and Podhajcer, O.L. 2012. A specific subpopulation of mesenchymal stromal cell carriers overrides melanoma resistance to an oncolytic adenovirus. Stem Cells Dev. 21:2689-2702. Davydova, J. and Yamamoto, M. 2013. Oncolytic adenoviruses: Design, generation, and experimental procedures. Curr. Protoc. Hum. Genet. 78:12.14:12.14.1-12.14.21. He, T.-C. 2004. Adenoviral vectors. Curr. Protoc. Hum. Genet. 40:12.4:12.4.1-12.4.25. Hendrickx, R., Stichling, N., Koelen, J., Kuryk, L., Lipiec, A., and Greber, U.F. 2014. Innate immunity to adenovirus. Hum. Gene Ther. 25:265284.
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Kim, J.W., Glasgow, J.N., Nakayama, M., Ak, F., Ugai, H., and Curiel, D.T. 2013. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer. PLoS One 8:e55533. Nandi, S. and Lesniak, M.S. 2009. Adenoviral virotherapy for malignant brain tumors. Expert. Opin. Biol. Ther. 9:737-747. Phelan, M. C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Hum. Genet. 51:A.3G.1-A.3G.18. Shabram, P., Vellekamp, G., and Scandella, C. 2002. Purification of Adenovirus, Elsevier. Thaci, B., Ahmed, A.U., Ulasov, I.V., Tobias, A.L., Han, Y., Aboody, K.S., and Lesniak, M.S. 2012. Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: Targeted delivery of the therapeutic payload in an orthotopic brain tumor model. Cancer Gene Ther. 19:431-442. Tobias, A.L., Thaci, B., Auffinger, B., Rincon, E., Balyasnikova, I.V., Kim, C.K., Han, Y., Zhang,
L., Aboody, K.S., Ahmed, A.U., and Lesniak, M.S. 2013. The timing of neural stem cellbased virotherapy is critical for optimal therapeutic efficacy when applied with radiation and chemotherapy for the treatment of glioblastoma. Stem Cells Transl. Med. 2:655-666. Tyler, M.A., Ulasov, I.V., Sonabend, A.M., Nandi, S., Han, Y., Marler, S., Roth, J., and Lesniak, M.S. 2009. Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo. Gene Ther. 16:262-278. Yong, R.L., Shinojima, N., Fueyo, J., Gumin, J., Vecil, G.G., Marini, F.C., Bogler, O., Andreeff, M., and Lang, F.F. 2009. Human bone marrowderived mesenchymal stem cells for intravascular delivery of oncolytic adenovirus Delta24RGD to human gliomas. Cancer Res. 69:89328940. Zheng, S., Ulasov, I.V., Han, Y., Tyler, M.A., Zhu, Z.B., and Lesniak, M.S. 2007. Fiber-knob modifications enhance adenoviral tropism and gene transfer in malignant glioma. J. Gene Med. 9:151-160.
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