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Influences of Age on scAAV Transduction of Chondrocytes and Synoviocytes

Take Home Message

Age does not appear to significantly influence the efficacy of gene transduction using scAAV in synoviocytes and chondrocytes in horses. Because the adult population may be more prone to receiving gene therapy than neonatal of young animals this is valuable information.

Introduction

Self-complementary adeno-associated viral vectors (scAAV) have shown promising results for gene delivery to cells of joint tissues. To test these vectors in vitro, it is most common to utilize cell cultures from skeletally immature animals due to the increased metabolic activity and recovery rate from cryopreservation of these cells compared to those from older animals.[1, 2] While initial investigation of transduction in neonatal tissue is relevant, it is important to determine if transduction of scAAV vectors is similar in adolescent and adult tissue since this is the population that would most frequently require gene therapeutic measures. This study was done by Nicki Phillips, Drs. Goodrich and McIlwraith of CSU and Dr. Jude Samulski of the Gene Therapy Center, University of North Carolina, and examined the transduction efficiency of scAAV in joint tissues from animals that represent a range of clinically applicable ages. Viral transduction efficiency was measured along with protein production from chondrocytes and synoviocytes of equine neonates (0-3 mth), weanlings (4-8 mth) and adults (2-5 yr). We hypothesized that scAAV transduction efficiency in chondrocytes and synoviocytes would decrease with age.

Methods

Cell Culture: Cartilage and synovial samples were taken from normal cadaveric stifle joints of neonates, weanlings, and adult equines within 4 hours of death. Age groups consisted of three to five individuals per group. Isolated chondrocyte cultures were plated immediately for use at passage 0 (P0), while synoviocytes were expanded to P2 to ensure a pure population of cells prior to experimental use.
 
ScAAV Transduction: Chondrocytes and synoviocytes were plated to 50% confluency 48 hours prior to transduction with scAAV constructs expressing GFP and IL1-ra at a concentration of 6000 viral particles per cell (vpc). Transductions were carried out for three hours and evaluated at four and eight days post transduction. GFP Evaluation: GFP (green fluorescent protein) expression was determined with a spectrophotometer and samples were normalized against the negative controls to account for auto fluorescence. Equine IL1-ra ELISA Evaluation: Culture media samples were collected at four and eight days post-transduction. Non-transduced samples and those transduced with IL1-ra were evaluated using a commercially available Equine IL1-ra ELISA kit (R&D Systems). Cultures expressing IL1-ra were normalized to their negative controls. Cell Scoring: Cultures were scored on a scale 0-5 based on the number of cells exhibiting abnormal morphology and complete cell death. A score of 0 had no abnormalities, 1 = 1-20% abnormality, 2 = 20-40%, 3 = 40-60%, 4 = 61-80% and 5 = >80%.
 
A one way ANOVA was performed to detect difference between age groups. A p value of <0.05 was considered significant.

Results

There were minimal differences observed in the transduction efficiency of synoviocytes between age groups at day 4 (data not shown) and day 8 post-transduction (Fig. 1). Fluoroscopic analysis revealed a gradual increase in fluorescence of chondrocytes with age and this trend was further emphasized by fluorescent microscopy (Fig. 2, C and D). However; this increase was not seen with IL1-ra protein production (Fig. 2). Transduction of synoviocytes with scAAVGFP did not change with age group.
 
Similarly, there were minimal differences in the associated protein production of synoviocytes between age groups at day 4 (data not shown) and day 8. Protein production in weanling chondrocytes were markedly reduced compared to those of neonates and adults (with similar production) (Fig. 3).
 
Cell morphology scores for adult synoviocytes were considerably higher (worse) at day eight, with minimal changes observed between chondrocytes and synoviocytes for neonates and weanlings (data not shown).

Discussion

Our results suggest that age does not significantly decrease scAAV transduction and associated protein production. Surprisingly, transduction with scAAVGFP actually increased in chondrocytes in adults although this was not demonstrated in IL1-ra production. This data is important when considering gene therapeutic protocols in adult animals since this population is more likely to receive gene therapeutic measures to treat joint disease. To our knowledge, age differences of scAAV transduction has not been investigated. This information will be important to clinicians choosing viral dosages for in vivo gene therapy trials.

Acknowledgements

Funded by NIH grant K08AR054903-01A2.

References

  1. Nixon A.J., Lust G., and Singer V.M. Isolation, propagation and preservation of equine articular chondrocytes, Am. J. Vet. Res, 1992; 53:2364-70.
  2.  
  3. Goodrich L.R., Choi V.W., DudaCarbone B.C., McIlwraith C.W., Samulski R.J. (2009). Serotype-specific transduction of equine joint tissue by self-complementary AAV vectors, Human Gene Therapy, 20:1697-702.
Figure 1. D8 GFP expression in chondrocyte and synoviocte cultures taken from neonates, weanlings, and adults.
 
Figure 2A.
Figure 2. Fluorescent microscopy of chondrocytes (A) and synoviocytes (B) 8 days post-transduction with IRAP (top) and GFP (bottom) scAAV.
 
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