AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo

Intervertebral disc degeneration is the main contributor to low back pain,now the leading cause of disability worldwide.Gene transfer,either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration,is a fascinating and promising tool to manipulate the complex physiology of the disc.Viral vectors based on the adeno-associated virus(AAV)have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism,transduction efficiency,and relative toxicity have not yet been performed in the disc in vivo.Herein,we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes,injection volumes,titers,promoters,and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus(NP)cells in vivo.We find that AAV6 using a CAG promoter to drive transgene expression,delivered into the NP of murine caudal discs at a titer of 1011 GC/mL,provides excellent transduction efficiency/kinetics and low toxicity in vivo.We also show,for the first time,that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides.Finally,to our knowledge,we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc,which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery.Taken together,these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes.However,its role in articular cartilage remains obscure.Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes,the distribution of Twist1 proteins in normal versus OA human articular cartilage,and its role in modulating OA development in mice.High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features.The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro.In normal human articular cartilage,Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones.However,our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage.Moreover,Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples.To assess the role of Twist1 in OA pathophysiology,we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus.At 12 weeks post-surgery,micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice.Collectively,the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.

Constructing the toolbox:Patient-specific genetic factors of altered fracture healing

The multifaceted sequence of events that follow fracture repair can be further complicated when considering risk factors for impaired union,present in a large and growing percentage of the population.Risk factors such as diabetes,substance abuse,and poor nutrition affect both the young and old,and have been shown to dramatically impair the body’s natural healing processes.To this end,biotherapeutic interventions such as ultrasound,electrical simulation,growth factor treatment(BMP-2,BMP-7,PDGF-BB,FGF-2)have been evaluated in preclinical models and in some cases are used widely for patients with established non-union or risk/indication or impaired healing(i.e.ultrasound,BMP-2,etc.).Despite the promise of these interventions,they have been shown to be reliant on patient compliance and can produce adverse side effects such as heterotopic ossification.Gene and cell therapy approaches have attempted to apply controlled regimens of these factors and have produced promising results.However,there are safety and efficacy concerns that may limit the translation of these approaches.In addition,none of the above mentioned approaches consider genetic variation between individual patients.Several clinical and preclinical studies have demonstrated a genetic component to fracture repair and that SNPs and genetic background variation play major roles in the determination of healing outcomes.Despite this,there is a need for preclinical data to dissect the mechanism underlying the influence of specific gene loci on the processes of fracture healing,which will be paramount in the future of patient-centered interventions for fracture repair.