Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis

Abstract

Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200–800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.

scAAVIL-1ra dosing trial in a large animal model and validation of long-term expression with repeat administration for osteoarthritis therapy

“…24 Contralateral joints in horses receiving the middle or the low dose scAAV2IL-1ra had barely detectable levels of IL-1ra.…”

“…22–26 Further, specific genetic alterations of the therapeutic transgene such as codon optimization as well as testing various promoters in joint tissues, revealed that the scAAVoptimizedequineIL-1ra vector produced significant and therapeutic protein production within the middle carpal and metacarpophalangeal joints of horses. 24 Horses have now been used for decades as animal models to mimic osteoarthritic conditions of human joint disease and are considered a highly relevant animal model due to the size of their joints, the thickness of their cartilage and the character of their subchondral bone. 27,28 Thus, we further pursued a scAAVIL-1ra dosing protocol to determine proper dosing of vector genomes to be injected into joints that have similar size and synovial volumes to the human knee.…”

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

“…A deno-associated virus (AAV) is frequently utilized as a gene delivery vector for clinical application; thus, several approaches have been undertaken to increase efficacy, including transgene optimization (1)(2)(3), capsid alteration (reviewed in reference 4), and drug treatments to enhance transduction (5)(6)(7)(8)(9)(10)(11)(12)(13). Proteasome inhibitors (PIs) were first described to enhance recombinant AAV (rAAV) polarized airway cell transduction (6), and since then PIs, including N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) (6,(14)(15)(16)(17)(18)(19), MG132 (5,6,8,11,14,17,(20)(21)(22)(23)(24), bortezomib (11,25), and celastrol (26), have been observed to enhance transduction in many cell types both in vitro and in vivo.…”

Viruses: Friends and Foes

“…Since that study, rAAV vectors have demonstrated a great efficiency at transducing a variety of joint/articular cells, both in vitro and in vivo, including chondrocytes [31][32][33]. In the hopes of treating osteoarthritis, not only has the transduction of chondrocytes been investigated but also other important cells, including osteocytes, meniscal fibrochondrocytes, tendon/ligament cells, muscle cells, cells of the synovial lining and progenitor cells that may differentiate to form joint tissues [6].…”