Transplantation of Human Adult Astrocytes: Efficiency and Safety Requirements for an Autologous Gene Therapy Jean-Luc Ridet, 1 Chamsy Sarkis, 1 Che Serguera, 1 Ve ´ronique Zennou, 2 Pierre Charneau, 2 and Jacques Mallet 1 * 1 CNRS UMR 9923, Laboratoire de Ge ´ne ´tique Mole ´culaire de la Neurotransmission et des Processus Neurode ´ge ´ne ´ratifs, ba ˆt. CERVI, Ho ˆpital Pitie ´-Salpe ´trie `re, Paris, France 2 Laboratoire d’Oncologie Virale, Institut Pasteur, Paris, France Ex vivo gene therapy is emerging as a promising ap- proach for the treatment of neurodegenerative diseases and central nervous system (CNS) trauma. We have shown previously that human adult astrocytes can be expanded in vitro and can express various therapeutic transgenes (Ridet et al. [1999] Hum. Gene Ther. 10:271– 280; Serguera et al. [2001] Mol. Ther. 3:875– 881). Here, we grafted normal and lentivirally-modified human adult astrocytes into the striatum and spinal cord of nude mice to test whether they are suitable candidates for ex vivo CNS gene therapy. Transplanted cells survived for at least 2 months (longest time analyzed) and sustained transgene expression. Importantly, the absence of pro- liferating cell nuclear antigen (PCNA) staining, a hallmark of cell division, ascertains the safety of these cells. Thus, adult human astrocytes are a promising tool for human CNS repair; they may make autologous ex vivo gene transfer feasible, thereby avoiding the problems of im- munological rejection and the side effects of immunosuppressors. © 2003 Wiley-Liss, Inc. Key words: human astrocytes; gene therapy; neurode- generative diseases; spinal cord trauma Gene therapy has great potential for the treatment of neurodegenerative diseases and traumatic injuries. This approach requires optimized gene delivery systems for therapeutic molecules. Several cellular vehicles have been investigated for ex vivo gene therapy of the central ner- vous system (CNS) (Gage, 1998). The development of cellular vectors allowing autologous transplantation would be extremely beneficial, as they would avoid immunolog- ical rejection and side effects of immunosuppressors. In- terestingly, an autologous strategy for CNS repair using carotid body cell aggregates has been tested in a primate model of Parkinson’s disease and led to anatomical and functional improvements (Luquin et al., 1999). Other autologous strategies have involved mainly primary fibro- blasts, but have shown limitations due to safety concerns. The use of primary astrocytes offers specific advan- tages for autologous CNS gene therapy. They can be expanded in vitro, they can be easily modified genetically using various vectors, and they can be transplanted into their natural CNS environment. Their characteristics as efficient cellular “minipumps” and neuronal supportive substrate (Ridet et al., 1997; Ridet and Privat, 1999) make them promising tools to deliver therapeutic molecules into the brain. Many studies have demonstrated that astrocytes from different sources can be transplanted successfully into the CNS after genetic modification (Cunningham et al., 1994; Yoshimoto et al., 1995; Lundberg et al., 1996; Keir et al., 1997; Lin et al., 1997; Ljungberg et al., 1999; Cortez et al., 2000; Ericson et al., 2002). We have established previously experimental culture conditions for 1) obtaining and expanding pure astroglial cultures from human adult cortex, and 2) genetically mod- ifying these cells by viral vectors (Ridet et al., 1999). More recently, we have shown that human adult astrocytes modified with an adenovector expressing the -glucuronidase gene integrate into rodent brain and sus- tain a high level expression for a relatively short period after engraftment (1 month) (Serguera et al., 2001). Here, we report a detailed analysis of the suitability of retrovirally-modified human adult astrocytes for ex vivo Contract grant sponsor: IRME; Contract grant sponsor: AFM; Contract grant sponsor: Aventis; Contract grant sponsor: BIOMED; Contract grant sponsor: Re ´gion Ile-de-France; Contract grant sponsor: Ministe `re de l’Education Nationale; Contract grant sponsor: VML Association; Contract grant sponsor: CNRS; Contract grant sponsor: Universite ´ Pierre et Marie Curie (Paris VI). J-L. Ridet and C. Sarkis contributed equally to this work. Jean-Luc Ridet’s current address is: Nestle ´ Research Center, PO Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26 Switzerland. Che Serguera’s current address is: EMBL, Mouse Biology Program, via Ramarini 32, I-00016 Monterotondo-Scalo (RM), Italy. *Correspondence to: Dr. Jacques Mallet, CNRS UMR 9923, Laboratoire de Ge ´ne ´tique Mole ´culaire de la Neurotransmission et des Processus Neu- rode ´ge ´ne ´ratifs, ba ˆt. CERVI, Ho ˆpital Pitie ´-Salpe ´trie `re, 83 boulevard de l’Ho ˆ pital, F-75013 Paris, France. E-mail: mallet@infobiogen.fr Received 21 October 2002; Revised 21 January 2003; Accepted 14 Feb- ruary 2003 Journal of Neuroscience Research 72:704 –708 (2003) © 2003 Wiley-Liss, Inc.