Abstract
PurposeInflammatory and oxidative stress upregulates matrix metalloproteinase (MMP) activity, leading to intervertebral disc degeneration (IDD). Gene therapy using human tissue inhibitor of metalloproteinase 1 (hTIMP1) has effectively treated IDD in animal models. However, persistent unregulated transgene expression may have negative side effects. We developed a recombinant adeno-associated viral (AAV) gene vector, AAV-NFκB-hTIMP1, that only expresses the hTIMP1 transgene under conditions of stress.MethodsRabbit disc cells were transfected or transduced with AAV-CMV-hTIMP1, which constitutively expresses hTIMP1, or AAV-NFκB-hTIMP1. Disc cells were selectively treated with IL-1β. NFκB activation was verified by nuclear translocation. hTIMP1 mRNA and protein expression were measured by RT-PCR and ELISA, respectively. MMP activity was measured by following cleavage of a fluorogenic substrate.ResultsIL-1β stimulation activated NFκB demonstrating that IL-1β was a surrogate for inflammatory stress. Stimulating AAV-NFκB-hTIMP1 cells with IL-1β increased hTIMP1 expression compared to unstimulated cells. AAV-CMV-hTIMP1 cells demonstrated high levels of hTIMP1 expression regardless of IL-1β stimulation. hTIMP1 expression was comparable between IL-1β stimulated AAV-NFκB-hTIMP1 cells and AAV-CMV-hTIMP1 cells. MMP activity was decreased in AAV-NFκB-hTIMP1 cells compared to baseline levels or cells exposed to IL-1β.ConclusionAAV-NFκB-hTIMP1 is a novel inducible transgene delivery system. NFκB regulatory elements ensure that hTIMP1 expression occurs only with inflammation, which is central to IDD development. Unlike previous inducible systems, the AAV-NFκB-hTIMP1 construct is dependent on endogenous factors, which minimizes potential side effects caused by constitutive transgene overexpression. It also prevents the unnecessary production of transgene products in cells that do not require therapy.
Highlights
An estimated 84% of adults experience low back pain during their lifetime [1]
To confirm that IL-1β was an appropriate surrogate for inflammatory stress, nucleus pulposus (NP) cells were exposed to IL-1β and nuclear translocation of NFκB from the cytoplasm was assessed
These findings demonstrated that the NFκB signalling pathway can be robustly activated by the pro-inflammatory cytokine IL-1β in disc cells, which justifies the use of IL-1β to investigate the expression of the human tissue inhibitor of metalloproteinase 1 (hTIMP1) transgene from associated viral (AAV)-NFκB-hTIMP1
Summary
An estimated 84% of adults experience low back pain during their lifetime [1]. Of the many causes for low back pain, intervertebral disc degeneration (IDD) is a common contributor [2]. Loss of the ECM proteoglycan leads to dehydration and fibrosis of the nucleus pulposus (NP), which commonly results in formation of fissures in the annulus fibrosis (AF) [4]. These disc fissures are responsible for the abnormal biomechanical changes seen in IDD, but are associated with neoinnervation, which has been implicated in pain [5]. HTIMP1 inhibits the activities of most of the known matrix metalloproteinases (MMPs), the catabolic enzymes involved in degrading the ECM and facilitates collagen and aggrecan expression in the disc [8,9,10,11] Our lab introduced human tissue inhibitor of metalloproteinase 1 (hTIMP1) and bone morphogenetic protein (BMP) into the NP of rabbits and demonstrated delayed disc degeneration in a rabbit annulotomy model of IDD [7]. hTIMP1 has demonstrated promise as a therapeutic factor to ameliorate IDD. hTIMP1 inhibits the activities of most of the known matrix metalloproteinases (MMPs), the catabolic enzymes involved in degrading the ECM and facilitates collagen and aggrecan expression in the disc [8,9,10,11]
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