Brain Engraftment of Autologous Macrophages Transduced with a Lentiviral flap Vector: an Approach to Complement Brain Dysfunctions
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Gene Therapy (2002) 9, 46–52 2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Brain engraftment of autologous macrophages transduced with a lentiviral flap vector: an approach to complement brain dysfunctions E Mordelet1, K Kissa2, C-F Calvo3, M Lebastard4, G Milon4, S van der Werf1, C Vidal1 and P Charneau5 1Unite de Genetique Moleculaire des Virus Respiratoires, Institut Pasteur, Paris, France; 2Unite d’Embryologie Moleculaire, Institut Pasteur, Paris, France; 3INSERM U114, College de France, Paris, France; 4Unite d’Immunophysiologie et de Parasitisme Intracellulaire, Institut Pasteur, Paris, France; and 5Groupe de Virologie Moleculaire et Vectorologie, Institut Pasteur, Paris, France Transplantation of ex vivo gene-corrected autologous cells expression remained stable for 1 month without cytopathic represents an attractive therapeutic approach for brain dis- effect. In vivo, transplants of transduced macrophages into eases. Among the cells of the central nervous system, brain the striatum of adult rats exhibited long-term expression of macrophages are promising candidates due to their role in GFP up to 3 months. Transduced macrophages were tissue homeostasis and their implication in several neuro- observed around the brain injection site and exhibited the logical diseases. Up to now, gene transfer into macrophages brain macrophage/microglia phenotype. There was no sig- has proven difficult by most currently available gene delivery nificant sign of astrogliosis around the graft. These results methods. We describe herein, an efficient transduction of rat confirm the potential of lentiviral vectors for efficient and bone marrow-derived and brain macrophages with an HIV- stable ex vivo transduction of macrophages. Moreover, 1-derived vector containing a central DNA flap and encoding transduced autologous macrophages appear as a valuable the GFP reporter gene (TRIP-⌬U3-GFP). In primary cultures vehicle for long-term and localized gene expression into of macrophages our results show that more than 90% of the the brain. cells were transduced by the TRIP vector and that GFP Gene Therapy (2002) 9, 46–52. DOI: 10.1038/sj/gt/3301591 Keywords: macrophages; lentiviral vector; brain; ex vivo gene therapy; rat Introduction phages into the brain.9,11–14 In order to target the thera- peutic action of genetically modified macrophages on The transfer of therapeutic genes into CNS-homing cells brain tissues, we have used an alternative approach by is a promising approach for brain therapy. Numerous cell injecting transduced autologous macrophages directly types such as neural progenitors, astrocytes, fibroblasts into the brain. and immortalized cells have been genetically modified Transduction of cells must combine efficient gene 1–6 for their further transplantation into the rat brain. For delivery and long-term gene expression without example, in a rat model of Parkinson’s disease, the intra- inflammatory process. Herpes simplex and adenoviral striatal graft of dopaminergic embryonic neurons vectors provide efficient cell transduction, but their use expressing the human Cu/Zn superoxide dismutase may be restricted in vivo due to their inherent immuno- gene, was found to improve cell survival and to correct genicity.15,16 Recently, Costantini et al17,18 have shown that 7,8 motor symptoms partially. In the same model, the graft a herpes simplex virus/adeno-associated virus amplicon of astrocytes expressing tyrosine hydroxylase signifi- vector injected directly into the brain allowed stable 1 cantly improved motor deficits. expression of the transgene without inflammatory pro- Other CNS diseases like peroxisomal or abnormal lyso- cess. Other studies have used retroviral vectors derived somal storage are known to involve macrophage from Moloney murine leukemia virus19,20 or lenti- 9,10 defects. In the murine Gaucher disease, it has been viruses.21,22 The latter vectors are valuable tools due to reported that glucocerebrosidase-transduced mono- their ability to transduce non-dividing cells, such as nuclear phagocytes injected intravenously migrated into neurons and macrophages.21–24 9,11 the brain. However, only partial improvement of the We have reported that the insertion of the DNA flap neurological deficits were observed, due to the limited sequence into a previously described HIV-1 derived vec- penetration of systemically delivered transduced macro- tor22 strongly stimulates in vitro transduction.25,26 The high efficiency of gene transfer is provided by the triple- stranded DNA flap sequence which increases the nuclear Correspondence: E Mordelet, Groupe de Virologie Moleculaire et Vectorologie, Institut Pasteur, 25–28 rue du Dr Roux, 75724 Paris Cedex import rate of the TRIP vector DNA into the nucleus of 15, France target cells. Received 5 March 2001; accepted 1 October 2001 In the present study, we show that a self-inactivating Brain grafts of transduced macrophages E Mordelet et al 47 TRIP vector, expressing the GFP reporter gene, trans- (2.5 × 107 cells/ml) were injected into the brain. The GFP- duced macrophages derived from bone marrow (BMDM) positive BMDM or bM cells were injected into the right or embryonic brain (bM) in an efficient and stable man- striatum, whereas control untransduced cells (Figure 3a ner in vitro. In vivo, transplants of transduced macro- and c (BMDM); e and g (bM)) or PBS were injected into phages into the striatum of adult rats showed long-term the contralateral striatum. Animals were killed at 5, 15, expression of GFP up to 3 months without any significant 30 and 90 days after transplantation and brain sections sign of astrogliosis. Thus, such transduced macrophages were analyzed by confocal microscopy as described in appear as particularly attractive vehicles for long-term Materials and methods. gene expression into the brain. For each transplanted cell type, the GFP fluorescence was detected around the injection site at day 5 (Figure 3b and f), and at day 15 and 30 (data not shown). At day Results 90, GFP-positive cells surrounding the injection site were still present, thus indicating sustained GFP expression Highly efficient gene transfer into primary cultures of and long-term cell survival, together with no or limited brain (bM) or bone marrow-derived macrophages cell migration (Figure 3d and h). It is noteworthy that at (BMDM) day 90 as compared with day 5 most of the transplanted Non-dividing terminally differentiated bM and BMDM bM cells exhibited an intense ramified phenotype as were transduced with a TRIP vector deleted in the U3 shown on Figure 3h. The BMDM adopted the same region of the LTR and expressing the GFP reporter gene phenotype, but the ramifications were less extended than 27 under the control of the CMV internal promoter. TRIP for the bM (Figure 3d). vector particles were pseudotyped using the VSV-G envelope protein.28 Three, 10 and 15 days after transduc- Immunohistochemical analysis of transplanted cells tion, FACS analysis was performed to assess gene trans- The phenotype of transplanted BMDM or bM cells was fer by detection of GFP fluorescence. We observed that characterized using ED1 and Mac-1 antibodies. At day 5, more than 90% of both BMDM (Figure 1a) or bM (Figure we observed that bM expressing GFP were both ED1 and 1b) were transduced using a concentration of TRIP vector Mac-1 positive (Figure 4a and b). Additional ED1 and particles corresponding to as little as 30 ng/ml of p24. Mac-1-positive cells were also observed which might cor- Pseudotransduction of GFP was assayed by treating cells, respond to resident macrophages or monocytes that have before the transduction, with 1 m nevirapine infiltrated the tissue as a consequence of a local inflam- (Boehringer Ingelheim, Ridgefield, CT, USA), an HIV-1 matory process. reverse transcriptase inhibitor. The weak signal observed At day 90, all the GFP-expressing cells were strongly 3 days after transduction in the presence of nevirapine Mac-1-positive (Figure 3d), but the ED1 staining became undetectable 15 days after transduction, whereas decreased dramatically (Figure 3c). This observation sug- a strong GFP signal was still detected in the absence of gests that the resting ramified phenotype adopted by the nevirapine up to 1 month after transduction (data not bM cells at day 90 is the result of the integration of the shown). The sustained GFP expression in vitro and the cells in the brain microenvironment. Moreover, no longer cell viability for up to 1 month after transduction was local inflammation process was detected indicating the indicative of the absence of lentiviral vector toxicity in presence of local and transient monocytes/macrophages the primary BMDM or bM cultures. infiltration due to the inflammation caused by the injection needle. Characterization of the population of transduced cells For BMDM cells, similar results were obtained both at The purity of primary cultures of BMDM and bM cells day 5 and at day 90 after transplantation (data not was assessed using macrophage monoclonal antibodies. shown). ED1 recognizes a single chain glycoprotein expressed predominantly on the lysosomal membrane of macro- Assessment of the astrocytic gliosis phages and Mac-1 labels, a cell surface protein present on In numerous brain transplantation studies in animals, resident or activated macrophages. As shown in Figure 2, local astrocytic gliosis is a well-known phenomenon whole cells were stained with ED1 and Mac-1 antibodies, which needs to be monitored for gene therapy appli- indicating that they belonged to the mononuclear phago- cations. We therefore examined each transplanted brain cyte lineage. Almost all cells expressed GFP when section for astrocytes reactivity as an assessment of local observed by confocal microscopy, thus confirming the astrocytic gliosis using the GFAP astrocytic marker. highly efficient gene transduction by the TRIP vector. The Enhanced GFAP staining was observed around the injec- BMDM cells exhibited a rounded cell morphology with a tion site 5 days after PBS injection (data not shown), as large cytoplasm (Figure 2a and b), while bM cells showed well as after the graft of untransduced (Figure 5a) or highly branched extensions (Figure 2c and d).