Gene Transfer to the Spinal Cord Neural Scar with Lentiviral Vectors: Predominant Transgene Expression in Astrocytes but Not in Meningeal Cells W. T. J. Hendriks, R. Eggers, J. Verhaagen, and G. J. Boer * Laboratory for Neurodegeneration, Netherlands Institute for Neuroscience, an Institute of the Royal Academy of Arts and Sciences, Amsterdam, the Netherlands Viral vector–mediated overexpression of neurotrophins in cells constituting the neural scar may represent a powerful approach to rendering scar tissue of a central nervous system (CNS) lesion permissive for neuronal regrowth. In this study a lentiviral vector encoding green fluorescent protein (LV-GFP) was injected in and around the neural scar 2 weeks after a dorsal column lesion in the rat spinal cord in order to analyze trans- duction characteristics of the neural scar after 4, 7, and 14 days. GFP expression was found at all points after injection and increased from 4 to 7 days, with no appa- rent difference observed between 7 and 14 days. The core of the lesion was virtually devoid of GFP signal despite direct vector injections in this area. The coloc- alization of GFP with specific cell markers (GFAP, vimentin, Raldh2, NeuN, OX-42, ED-1, and NG-2) indi- cated that the predominant cells transduced in the rim of the lesion were astrocytes, with neurons, microglia, oligodendrocyte precursors, and macrophages trans- duced to a lesser extent. None of the Raldh2-positive meningeal cells, present in the core of the scar, expressed GFP. In vitro meningeal cells were readily transduced, indicating that in vivo the formation of an extracellular matrix might prevent LV particles from transducing cells in the core of the scar. Because astrocytes are important cellular constituents of the glial scar after CNS injury, transduction of astrocytes with LV vectors encoding neurotrophic factors like BDNF or NT-3 may be used to enhance regeneration of severed axonal tracts through or along boundaries of a CNS lesion. V V C 2007 Wiley-Liss, Inc. Key words: lentiviral vector; spinal cord injury; neural scar; astrocytes; meningeal fibroblasts; motoneurons It is generally accepted that the neural scar is a physical and molecular barrier that prevents regeneration of injured neurons in the central nervous system (CNS; reviewed by Schwab and Bartholdi, 1996; Fitch and Silver, 1997). After spinal cord injury, a neural scar is formed in the first weeks after the initial trauma. Reac- tive astrocytes, oligodendrocyte precursor cells, micro- glial cells, and meningeal cells form an extracellular ma- trix (ECM) that contains inhibitory molecules including chondroitin sulfate proteoglycans (CSPGs; reviewed by Morgenstern et al., 2002) and tenascins (reviewed by Joester and Faissner, 2001). Meningeal fibroblasts and re- active astrocytes in the neural scar express chemorepel- lent proteins including secreted semaphorins (reviewed by de Wit and Verhaagen, 2003), ephrins (Goldshmit et al., 2004), and slits (Nguyen-Ba-Charvet et al., 2004). Oligodendrocytes mainly in the white matter express the axon growth–inhibitory molecules Nogo (reviewed by Huber and Schwab, 2000), myelin-associated glycopro- tein (MAG; reviewed by Filbin, 2003), and oligodendro- cyte myelin glycoprotein (OMgp; Wang et al., 2002). In addition to expressing inhibitory molecules, the cellular components of the neural scar also provide a relatively low level of neurotrophic support for injured axons (Liebl et al., 2001; Widenfalk et al., 2001). Modification of the neural scar to create a growth- permissive environment to overcome inhibition of axon regrowth has resulted in some degree of axonal regener- ation after spinal cord injury. Enzymatic removal of the sulfated sugar chains from CSPGs (Bradbury et al., 2002), delayed formation of the ECM by blocking colla- gen synthesis (Stichel et al., 1999; Hermanns et al., 2001), neutralization of the myelin-associated inhibitory protein Nogo (Bregman et al., 1995; Schnell and Schwab, 1993), genetic mutation of the receptors for particular growth-inhibitory ligands EphA4 (Goldshmit et al., 2004) and NgR (Lee et al., 2004), or knockout of the gene encoding the inhibitory ligand Nogo A/B (Kim et al., 2003; Simonen et al., 2003) all result in enhanced regeneration of axotomized nerve fibers. *Correspondence to: Dr. G. J. Boer, Laboratory for Neuroregeneration, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam Z.O., the Netherlands. E-mail: g.boer@nin.knaw.nl Received 28 February 2006; Revised 27 January 2007 and 13 April 2006; Accepted 14 May 2007 Published online 1 August 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21432 Journal of Neuroscience Research 85:3041–3052 (2007) ' 2007 Wiley-Liss, Inc.