Multiple Recombinant Adeno-Associated Viral Vector Serotypes Display PersistentIn VivoGene Expression in Vector-Transduced Rat Stifle Joints

Abstract

Our aim was to investigate serotype-specific cell and tissue-transduction tropisms, transgene expression levels and longevity, and immunogenicity of candidate rAAV serotypes in rat osteochondral cells, tissues, and stifle joints. In vitro, we used six rAAV serotypes and two promoters to transduce synoviocytes and chondrocytes. Serotypes rAAV2/5 and 2/2 yielded the highest transduction efficiency 4 days after transduction. No differences were detected between cytomegalovirus and chicken b-actin promoters. In vivo, intra-articular injection was used to introduce four rAAV serotypes into 4-month-old rats in the left stifle joint. Eleven months later, serotype 2/5 vector, diluted with saline or surfactant, was injected into the right stifle joint of the same rats. Rats were analyzed up to 12 months after initial injection. Bioluminescence was detected at 7 days and all serotypes tested displayed bioluminescence above controls after 1 year in the left stifle. Gene expression was detected in the right stifle joints of all rats with the exception of rats previously injected with serotype 2/5. We observed no difference irrespective of whether the luciferin was injected subcutaneously or intraperitoneally. However, surfactantdiluted vectors led to increased gene expression compared with saline-diluted vectors. Cell-and tissue-specific transduction was observed in rat stifles injected with an nLacZ-containing rAAV. Transduction was greatest in stromal tissues and mesenchymal cell types. Exposure to a specific serotype did not inhibit subsequent transduction with a different serotype at a second vector injection. Including surfactant as a vector diluent increased gene expression within the stifle joint and should be considered for in vivo gene therapy applications.

Delivery and evaluation of recombinant adeno‐associated viral vectors in the equine distal extremity for the treatment of laminitis

“…In situ β‐gal staining of the intact hoof slice provided a valuable context in which to observe the distal migration of transduced cells/tissues/proteins from the coronary band toward the toe, in the direction of hoof growth only 1 week after vector injection. Previous in vivo results showed that gene expression can increase significantly between Day 7 and Day 28 . Conversely, multiple rAAV vector studies identify a drop in transgene expression over time due to pre‐existing immune responses to vector capsid proteins and acquired responses against vector and transgene products, as well as high cell turnover .…”

“…Incubation times from 7 to 21 days were unlikely to expose potential long‐term gene silencing in these experiments. However, in previous experiments in our lab, gene expression with a CB promoter continued for a minimum of 1 year .…”

“…Previously, the inclusion of surfactant as a component of the vector diluent increased in vivo gene expression in rodent stifle joints, as measured with in vivo bioluminescence imaging . In the current experiments in the horse, when vector was diluted with surfactant‐containing saline for delivery to the equine foot, the level of transduction increased dramatically.…”

“…In these previous experiments, serotype 2/1 demonstrated more promising levels of transduction . In contrast to these in vitro results, previous in vivo gene therapy results with serotype 2/9 demonstrated superior performance .…”

Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system

“…2 Direct administration of rAAV is a simple and cost-effective gene transfer approach but it requires the availability of a considerable cell population in a damaged tissue susceptible to transduction for expression of the transgene being delivered at appropriate therapeutic levels. Also, direct rAAV gene transfer by intra-articular injection as commonly performed in musculoskeletal translational research may lead to the dissemination of the vectors to non-target tissues early on (liver, kidney, lymph nodes) [17][18][19][20] while vector DNA might be rapidly cleared, becoming detectable only at the site of injection at extended periods of time 17,[19][20][21][22][23][24][25][26][27][28][29][30][31] and leading to prolonged transgene local expression (at least a year, the longest time points examined). 31,32 Another issue is a possible contralateral effect of the gene treatment in nonmodified locations (joints) upon circulation of the therapeutic product and/or by trafficking of vector-modified cells, even though this observation has been mostly reported when using adenoviral and retro-/lentiviral vectors [33][34][35][36][37][38] and only in rare cases with rAAV.…”