title: Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.
abstract: OBJECTIVE
  IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I.
  
  
  METHODS
  Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes.
  
  
  RESULTS
  Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis.
  
  
  CONCLUSIONS
  This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response.
authors.affiliation: Orthopaedic Research Center, College of Veterinary Medicine, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Daniel.Hemphill@colostate.edu.
authors.forename: D D
authors.initials: DD
authors.lastname: Hemphill
authors.affiliation: Orthopaedic Research Center, College of Veterinary Medicine, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Wayne.Mcilwraith@colostate.edu.
authors.forename: C W
authors.initials: CW
authors.lastname: McIlwraith
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Richard.Slayden@colostate.edu.
authors.forename: R A
authors.initials: RA
authors.lastname: Slayden
authors.affiliation: University of North Carolina Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: RJS@med.unc.edu.
authors.forename: R J
authors.initials: RJ
authors.lastname: Samulski
authors.affiliation: Orthopaedic Research Center, College of Veterinary Medicine, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Laurie.Goodrich@colostate.edu.
authors.forename: L R
authors.initials: LR
authors.lastname: Goodrich
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D063990:Gene Ontology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006736:Horses / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D007334:Insulin-Like Growth Factor I
keywords: AAV; Equine; Gene therapy; IGF-I; Joint; NextGen sequencing