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Gene Therapy Approach to Treat Osteoarthritis Using AAVIRAP

Take Home Message

Gene therapy vectors were used to deliver IL-1ra to joint tissues and appeared to significantly increase protein production in equine joint tissues. When scAAVIL-1ra was injected into metacarpal phalangeal and middle carpal joints of horses, IL-1ra was produced for 23 and 183 days, respectively. No evidence of intra-articular toxicity was noted throughout the study and horses remained sound for the duration of the testing periods.

Introduction

Osteoarthritis (OA) is a chronic and incurable disease that affects over 40 million Americans per year with estimated costs of over $100 billion yearly. Gene therapy offers a radically different approach to the treatment of osteoarthritis. Interleukin receptor antagonist protein (IRAP) is antiarthritic due to its ability to effectively block or antagonize interleukin-1 (IL-1) which is a cytokine present in OA joint fluid and considered pivotal in the induction of osteoarthritis. An effective AAVIRAP vector would result in long-term abrogation of IL-1 and its devastating effects in the joint. This collaborative study was done by Nikki Phillips with Drs. Goodrich and McIlwraith of CSU, along with Drs. Samulski and Foti of the University of North Carolina, with the objective of constructing an AAVIRAP vector that would inhibit inflammation within the joint. Our hypothesis was that this vector would produce high levels of functional IRAP protein in the joint for up to six months.

Materials and Methods

An equine optimized IL-1ra gene was cloned into a mammalian expression vector, and transfected into AD293 cells. The vector was then cloned into an adeno-associated viral vector containing a CMV promoter. The scAAVIRAP vectors were tested for functionality against IL-1 stimulation in synoviocytes, and evaluated with a PGE2 ELISA. Two equines were evaluated for a pilot study in which joints were dosed with scAAVIRAP, scAAVGFP or saline. Synovial fluid was collected prior to dosing, two weeks after dosing, every week thereafter for 14 weeks, and every other week until termination of the study. IRAP levels were evaluated with an equine ELISA kit. White blood cell counts and total protein values were also measured.

Results

The equine optimized IL-1ra gene produced IRAP in transfected 293 cells, with levels greater than 15ng/ml two days post-transfection. The scAAVIRAP vector produced similar levels in transduced synoviocytes (12 ng/ml), and was able to reduce the response of synoviocytes to IL-1 stimulation by 90%. Levels of IRAP protein were consistently high in the scAAVIRAP dosed joints of the pilot study animals with a peak occurring around week 8 (Fig. 1). Levels were between 400 and 1200 ng/ml and stayed above 400 ng for at least four months following injection. Study animals are ongoing and will continue to be monitored for at least two additional months. White blood cell count and total protein values rose only slightly in the scAAVIRAP or scAAVGFP injected joints and values stayed below that which is consistent with inflammation. Elevations of IRAP in saline injected joints were not detected.

Discussion

Our results suggest that the scAAVIRAP vector will produce extremely high levels of IRAP protein in the cells of joints and cause minimal to no toxicity in these tissues. The functional assay reveals that the IRAP protein produced effectively ameliorates the inflammatory cascade as evidenced by extreme reduction in PGE2. The viral vectors (scAAVGFP or scAAVIRAP) injected into the joints were well tolerated and didn't result in pain or joint swelling. This is the beginning of a gene therapy protocol that will be tested in horses with induced osteoarthritis to reveal if OA can be reduced or halted. Our objectives of this study were met and our hypothesis that scAAVIRAP would produce high levels of functional IRAP in the joint was proven. Further studies will define whether this gene therapy approach can result in the effective treatment and prevention of OA.

Acknowledgement

Funded by the National Institute of Health (NIH) National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), and the Grayson-Jockey Club Research Foundation.

References

  1. 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.
  2.  
  3. Evans C.H., Gouze J.N., Gouze E., Robbins P.D., Ghivizzani S.C. Osteoarthritis gene therapy. Gene Ther 2004;11:379–89.
Figure 1A. Clinical design for the dosing of Horse 2. The blue circles correspond to the carpal joints, and the green circles correspond to the metacarpal phalangeal joints. ScAAV constructs were given at specific concentrations per joint, whereas 5 ml of saline was administered into the joint receiving that treatment.
 
Figure 1B. IL1ra expression and WBC counts (103 /ul) in the synovial fluid of the carpus and metacarpal phalangeal joints of Horse 2. Each joint is graphed separately over time. IL1ra levels were assessed with an equine IL1ra ELISA kit. Note: the WBC numbers on the top right graph are higher due to a higher WBC count in that joint. WBC counts were not determined at D23.
 
Figure 1C. Clinical design for the dosing of Horse 1. The blue circles correspond to the carpal joints, and the green circles correspond to the metacarpal phalangeal joints. ScAAV constructs were given at specific concentrations per joint, whereas 5 ml of saline was administered into the joint receiving that treatment.
 
Figure 1D.IL1ra expression and WBC counts (103 /ul) in the synovial fluid of the carpus and metacarpal phalangeal joints of Horse 1. Each joint is graphed separately over time. IL1ra levels were assessed with an equine IL1ra ELISA kit.
 
 
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