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scAAV Transduction Efficiencies in Joint Tissue Monolayer and Explant Cultures and the Effects of Synovial Fluid Neutralization

Take Home Message

Gene transduction of chondrocyte and synovial monolayer is significantly different than cartilage and synovial explants and this study highlights the necessity to test gene therapeutic tissue in situ. Furthermore, neutralizing antibodies exist in both the synovial fluid and serum which can reduce efficient gene transduction. It is important to determine which serotypes have antibodies that exist in the horse.

Introduction

Within the joint there are two major tissues currently targeted by gene therapy vectors to treat osteoarthritis. Synoviocytes are within the synovial membrane which has fimbria that extend into the synovial fluid. Chondrocytes are surrounded by matrix in the cartilage layer. Although transduction efficiency has been well quantified in chondrocyte and synoviocyte monolayers, transduction of explants of cartilage and synovium has not been thoroughly investigated. Although efficient transduction of synoviocytes and chondrocytes is important to ex vivo gene therapy, it is important to determine if self-complimentary adeno-associated viral (scAAV) vectors can transduce cells in situ. scAAV is currently the vector of choice being investigated to carry therapeutic genes to both the synovium and cartilage.1 There are many serotypes of AAV each with specific and variable tissue tropisms. Furthermore, neutralizing antibodies have been identified in the synovial fluid of humans which results in decreased scAAV transduction of tissue.2 The purpose of this study done by Daniel Hemphill together with Drs. McIlwraith and Goodrich of the ORC and Dr. Jude Samulski of the University of North Carolina was to compare scAAV transduction in monolayer to explant tissue of synovium and cartilage and to investigate whether the presence of synovial fluid inhibits efficient transduction in joint tissue. Our goal was to indentify which serotype of AAV leads to efficient transduction in both synovium and cartilage in the horse model. We hypothesized that monolayer transduction efficiency is similar to explant transduction efficiency and that neutralizing antibodies exist in synovial fluid that will decrease transduction efficiency.

Methods

Synovium from the femoropatellar joint capsule and cartilage from the patella were aseptically harvested post-mortem from three horses 2-5 years of age. Each tissue was cut into pieces of similar size for explants with the remainder digested overnight in collagenase type II solution. The isolated chondrocytes and synoviocytes were expanded one passage and then plated in 48 well plates at 50,000 cells/cm^2 for synoviocytes and 100,000 cells/cm^2 for chondrocytes. After two days all explants and monolayers were transduced with scAAV-GFP at 8000 virus particles per cell for four hours at 37C. Fluorescent microscopy pictures were taken every three days and flow cytometry was performed 35 days after the transduction. Multivariable ANOVA was performed on the data and results with p < 0.05 chosen as statistically significant.
Figure 1. Explant experiment transduction efficiencies for serotypes averaged across all tissue types. On left showing the percentage of cells expressing GFP at significant levels and on right average intensity of fluorescence.
 
Synovial fluid was aseptically collected post-mortem from five horses 2-5 years of age. Synoviocytes and chondrocytes were plated in 48 well plates at the densities previously mentioned. Three days after plating, 4000 virus particles per cell were incubated with a 1:2 or 1:200 synovial fluid dilution for one hour at 37C. Immediately following, the cells were transduced with the virus and synovial fluid mixture for four hours at 37C. Plates were read using a fluorometric plate reader every two days. One way ANOVA was performed and p < 0.05 chosen as statistically significant.

Results

In vitro, explants and monolayers were successfully transduced and there was no difference in transduction efficiency between monolayer and explants when analyzing the percentage of cells transduced. The mean fluorescence intensity of the vector gene expression revealed that monolayer cells appeared more fluorescent than explants cells. When percentage of cells transduced and mean expression intensity were combined, horses did not show statistical difference, but virus serotypes did show statistical differences with S2 > (S3 , S5 , S2.5 , S6); only these serotypes were significantly different from the control. Furthermore, cartilage showed statistically higher transduction efficiency than synovium.
Virus incubation with synovial fluid decreased GFP production in synoviocytes for several serotypes of virus, although averaging over all serotypes showed no difference. While specifically testing individual serotypes: 2, 2.5, 5, and 6 revealed significant differences following synovial fluid incubation. There was no statistical difference between the five horses tested.

Discussion

The results of this study suggest that cell culture monolayer is a good indicator for actual tissue tropism of scAAV serotypes in the joint as tissue explants (both cartilage and synovium) are efficiently transduced with various serotypes. These results suggest that when scAAV vector is placed into the joint environment, vector can transduce chondrocytes and synoviocytes efficiently in situ. Furthermore serotype 2 appears to be the best option for total transduction of joint tissue. However, synovial fluid has a negative effect on transduction efficiency with AAV serotypes 2, 2.5, and 6. This suggests that, in addition to identifying the optimal serotype for transducing joint tissues, some attention must be paid to identifying a serotype that will not be inhibited by synovial fluid; or the removal of synovial fluid through joint lavage should be considered in a treatment. Despite monolayers and explants having similar transduction efficiencies, the intensity of gene expression differed between the two and this deserves further investigation. The clinical significance of this study is that specific serotypes have been identified that will efficiently transduce synoviocytes and chondrocytes in situ and while neutralizing antibodies appear to exist, significant transduction can still occur.
Figure 2. Transduction efficiency as mean cell GFP intensity from chondrocyte monolayer and cartilage explants.
 

References

  1. Evans, C.H. Curr Rheumatol 2004 Rep 6: 31–40.
  2.  
  3. Boissier M.C., et al. Hum Gene Ther. 2007;18:525-535.
​S2 ​No SF > 1:200 > 1:2
​S2.5 ​No SF > (1:200, 1:2)
​S3 ​(No SF , 1:200, 1:2)
​S5 ​1:200 > (No SF, 1:2)
​S6 ​(No SF, 1:200) > 1:2
 
Table 1. Synovial fluid dilution experiment – Comparison of GFP expression in synoviocytes. "No SF" indicates the control in which virus was incubated with only media and no synovial fluid.
 
 
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