Detectable reporter gene expression following transduction of adenovirus and adeno‐associated virus serotype 2 vectors within full‐thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

Abstract

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adenoassociated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and fullthickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. ß

Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis

“…An additional three guinea pigs received TV in one knee or an identical vector dose of NTC in the other knee. Administered vector doses were consistent with other peer-reviewed manuscripts 25,26 . Two months post-injection, animals were humanely euthanized; cartilage was collected from the patella and patellar surface of the femur for RNA extraction to detect IL-1β transcript expression, as described below.…”

Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model

“…Recombinant E-1 deleted human serotype 5 adenovirus preparations, single-stranded human serotype 2 AAV2, and scAAV2 containing GFP under the control of the cytomegalovirus (CMV) promoter were generated and purified by cesium chloride density gradient method [12, 15]. Particle count of Ad vectors was determined by optical density at 260 nm, and particle titers of rAAV2 and scAAV2 vectors were determined by DNase-resistant particle (DRP) values using real-time PCR assay as described previously [16].…”

“…In contrast, recombinant AAV2 (rAAV2) has gained much attention as a safer vector for gene delivery to joints, because it can induce sustained transgene expression in the absence of significant inflammation [6, 9–11]. In addition, rAAV2-mediated joint therapy may be advantageous by its greater penetration into articular cartilage, likely due to smaller particle size [12]. Moreover, AAV2 vectors have been modified to package self-complementary DNA, capable of bypassing the rate-limiting step of second-strand synthesis in order to accelerate and improve the transduction efficiency [12, 13].…”

“…In addition, rAAV2-mediated joint therapy may be advantageous by its greater penetration into articular cartilage, likely due to smaller particle size [12]. Moreover, AAV2 vectors have been modified to package self-complementary DNA, capable of bypassing the rate-limiting step of second-strand synthesis in order to accelerate and improve the transduction efficiency [12, 13]. Such self-complementary AAV2 (scAAV2) vectors have been shown to effectively transduce chondrocytes and synovial cells [14] and cartilage explants [12] in vitro, and induce superior transgene expression in cartilage and synovium tissues after injection of 5 × 10 11 particles in vivo in rodent models [3].…”

“…Moreover, AAV2 vectors have been modified to package self-complementary DNA, capable of bypassing the rate-limiting step of second-strand synthesis in order to accelerate and improve the transduction efficiency [12, 13]. Such self-complementary AAV2 (scAAV2) vectors have been shown to effectively transduce chondrocytes and synovial cells [14] and cartilage explants [12] in vitro, and induce superior transgene expression in cartilage and synovium tissues after injection of 5 × 10 11 particles in vivo in rodent models [3]. Taken together, intra-articular administration of scAAV2 vectors has potential for future clinical application, but the inflammation and immune reactions, cell tropism, and application to larger joints as in people have not been directly compared among rAAV2, scAAV2, or Ad vectors in vivo.…”