Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction

Schorge S (2013) Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction. PLoS ONE 8(1): e53269. doi:10.1371/journal.pone.0053269 Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction Klaus Wanisch 0 Stjepana Kovac 0 Stephanie Schorge 0 William Phillips, University of Sydney, Australia 0 1 Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London , London , United Kingdom , 2 Department of Neurology, University of Muenster , Muenster , Germany Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required. - In order to understand how different genes can modify specific neuronal functions, it is necessary to manipulate gene expression in neurons. However, reliably introducing genetic material into neurons has been problematic (for review see [1]). Recently lentiviral vectors have emerged as a powerful tool capable of delivering DNA to neurons in vitro and in vivo. While the transduction efficiency with viral vectors in general is largely dependent on titre, further limitations of the ability to transduce cells are imposed, in vivo mainly through the restricted diffusion of lentivector particles around the injection site within the extracellular matrix of the brain [2], and in vitro, especially for lower multiplicities of infection (MOIs), through an as yet poorly understood restriction. In many studies this is circumvented by transducing at early time points or simultaneously with seeding cells [3,4,5,6,7,8,9,10]. In general transduction efficiency is thought to decrease with increasing age of the neuronal culture (days in vitro; DIV) but few groups have systematically investigated this. In cases where virally transduced genes are intended to take effect in mature neurons only, or where transduced genes may disrupt development and differentiation of plated neurons, the requirement to transduce neurons in vitro at early stages in order to achieve high transduction efficiency presents a major drawback. One possibility is that the age-related decrease in transduction efficiency is linked to the maturation of neuronal cells, with changes of the outer cellular surface during this phase restricting viral entry into cells. All types of cells are surrounded by extracellular matrix (ECM), and one of the main constituents of the ECM is hyaluronic acid (HA), a long polysaccharide molecule which is composed of N-acetyl-glucosamine and Dglucuronic acid [11,12]. HA is anchored to extracellular receptor CD44 and CD168 [13] and serves as a scaffold to keep proteins and molecules that support cellular viability in close proximity to the cell surface (for review see [14]). The importance of HA in the brain has been recognised since the 1970s [15,16]. It entirely covers neurons, including cell bodies, dendrites and axons [17], and in conjunction with other molecules such as chondroitin sulphate proteoglycan and various proteins like tenascins, reelin, laminin, HA is central to building up a net like structure surrounding neurons, known as perineural nets (PNNs). In the brain, HA is thought to maintain the physicochemical properties of the ECM [18,19], but there is increasing evidence that HA also alters functional properties of neurons [20,21,22]: The characteristic distribution of HA and changes during cerebral development are indicative of functional properties (n.b. there is no HA in the adult cerebellum); neurite growth is altered by HA, and neurites tend to avoid HA containing collagen substrates. HA can also have an impact on membrane potential, as indicated by the depolarization observed when HA was added to cultured neurons [23]. The mechanisms of these interactions are still a matter of speculation, but could be related to altered distribution of extracellular ions or signalling via CD44 receptors. Recently HA has been shown to influence neurotransmission and signalling, and also to contribute to synaptic plasticity by regulating usedependent Ca2+ currents via Cav1.2 channels [24], thus manipulation of HA might have consequences for neuronal viability. Hyaluronidase is an enzyme which cleaves HA, and could be used to degrade the PNNs and increase access to the surface of neurons. We report that treating cells with hyaluronidase improves transduction efficiency with lentiviral vectors in vitro and in vivo. This was done after evaluating potential toxic effects in vitro and in vivo on neuronal survival. Materials and Methods All experimental procedures of this study involving animals were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and following ethical approval from UCL Institute of Neurology. Chemicals if not specified are from Sigma (St. Louis, Missouri, USA). Production of Lentiviral Vectors and Titration Second and third generation lentiviral vectors have been produced as described previously [25,26]. Both express different GFP variants driven by different promoters, with pGIPZ (second generation; Open Biosystems, ThermoScientific, Waltham, Massachusetts, USA) expressing turboGFP (tGFP) under the control of CMV promoter, and pCDH1-MCS1-EF1-copGFP (third generation; System Biosciences, Mountain View, California, USA) expressing copGFP under the EF1a promoter. Packaging was done with plasmids pCMVDR8.91 for second generation vector [27], or pMDLg/pRRE and pRSV-Rev [26] for third generation vectors, together with the vesicular stomatitis virus protein G envelope plasmid pMD2.G expressing VSV-G surface protein for both (packaging and envelope plasmids were kindly provided by D. Trono, Geneva, Switzerla (...truncated)