Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis

详细信息    查看全文

• 作者：D.D. Hemphill^&dagger ; ; ^{Daniel.Hemphill@colostate.edu" class="auth_mail" title="E-mail the corresponding author} ; C.W. McIlwraith^&dagger ; ; ^{Wayne.Mcilwraith@colostate.edu" class="auth_mail" title="E-mail the corresponding author} ; R.A. Slayden^&Dagger ; ; ^{Richard.Slayden@colostate.edu" class="auth_mail" title="E-mail the corresponding author} ; R.J. Samulski^§ ; ; ^{RJS@med.unc.edu" class="auth_mail" title="E-mail the corresponding author} ; L.R. Goodrich^&dagger ; ; ^{Laurie.Goodrich@colostate.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：AAV ; IGF-I ; Gene therapy ; Joint ; Equine ; NextGen sequencing
• 刊名：Osteoarthritis and Cartilage
• 出版年：2016
• 出版时间：May 2016
• 年：2016
• 卷：24
• 期：5
• 页码：902-911
• 全文大小：692 K

文摘

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.

Design

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.