Madison D Hogans 1 Will Kretzschmar 1 Payal Agarwal 1 2 Bruce F Smith 1 2 1 Scott Ritchey Research Center College of Veterinary Medicine Auburn University AL 2 Department of Pathobiology College of Veterinary Medicine Auburn University AL ID: 908224
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Elucidation of Canine Adenovirus Type 2 TropismMadison D Hogans1, Will Kretzschmar1, Payal Agarwal1, 2, Bruce F Smith1, 21 Scott Ritchey Research Center, College of Veterinary Medicine, Auburn University, AL2Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL
Gene therapy is the transfer of genetic material into targeted cells and can be used to treat various diseases. The natural tropism of a virus can be modified to transduce specific cells or deliver genes to targeted cells. Adenoviral vectors, especially human adenovirus (Ad5) are excellent contenders. However, Ad5 based vectors have limited utility because a variety of cells are refractory to Ad5 transduction. Therefore, identification of alternative tropism for Ad5 could provide both a mechanism to target refractory cells and to evade preexisting immunity. Canine adenovirus type 2 is a vector that has similar characteristics to Ad5. Although the genome of CAV2 is well characterized, its cellular tropism is still unclear.
Human and canine cell lines were infected with Ad5G/L and CAVGFP for 48 hours and analyzed for green fluorescence using flow cytometry. CAVGFP and Ad5G/L are replication incompetent recombinant adenoviral vectors with Green fluorescence protein (GFP) inserted in the genome under the expression of CMV promoter. All cell lines were cultured according to standard procedures
CAVGFP infects cells that are refractory to Ad5G/L such as, canine lymphoma cell lines 17-71 and OSW and canine mast tumor cell line MPT1. CAVGFP can transduce cells at lower MOIs (10 and 100) in comparison to Ad5G/L in both canine and human cell lines
Canine cell lines
Lymphoma1771, OSWMast cell tumorMPT1Mammary tumorCMT28Histiocytic tumorDH82MelanomaCML7, CML10Human cell linesOvarianSKOV3ProstatePC-3
CAVGFPAd5GFPCell Line0 MOI10 MOI100 MOI1000 MOI0 MOI10 MOI100 MOI1000 MOIOSW0.402.4516.8948.010.200.300.504.9017-710.7116.8163.2475.150.400.400.400.60MPT10.807.3968.0889.150.700.501.601.80DH820.436.1742.2289.720.300.506.2055.00CMT280.364.9431.3583.210.800.9010.1062.90CML70.362.1412.4159.270.401.1011.1074.80CML100.3213.8563.1898.650.200.808.7070.60SKOV30.204.9122.6435.250.400.685.3137.37PC-30.683.9922.5851.210.7716.0570.0392.22
Our objective is to identify the sequence that determines the tropism of CAV2 in order to modify the tropism of AD5 so that it can infect more cell types
The authors would like to acknowledge Allison Church Bird for flow cytometry services and lab members Rebecca Nance, Daniel Patton, and Madeline Matheson for their collegial help and support. Finally, the authors would like to acknowledge the Auburn University Intramural Grants Program (IGP) for funding this project.
Introduction
Objective
Conclusions
Acknowledgements
Results
Methods