Gene Transfer and Expression in Oligodendrocytes Under the Control of Myelin Basic Protein Transcriptional Control Region Mediated by Adeno-Associated Virus

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Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus

H Chen1, DM McCarty2, AT Bruce2, K Suzuki1,3 and K Suzuki1,4 1Neuroscience Center, 2Gene Therapy Center, 4Departments of Pathology and Laboratory Medicine and 3Neurology and Psychiatry, University of North Carolina, Chapel Hill, NC, USA

In this study, a rAAV vector carrying a reporter gene, Infusion of approximately 6 × 10 9 particles (2 × 10 5 infec- ‘humanized’ green fluorescent protein (GFP), linked to the tious units) of rAAV-MBP-GFP into mouse brains resulted transcriptional control region from the myelin basic protein in the GFP expression specifically in white matter. The (MBP) gene (a myelin-forming cell-specific gene) was con- GFP protein was detected 15 days later by immunostain- structed. Transduction of oligodendrocytes was carried out ing, specifically in the oligodendrocytes. No astrocytes both in vitro and in vivo. The GFP expression was detected were transduced. Our studies suggested that cell types for at least 3 weeks in both transduced oligodendrocyte cell other than neurons in the central nervous system can also line (MOCH-1 cells) and primary cultures of rat oligodend- be transduced by rAAV using a cell-type-specific transcrip- rocytes. Preferential GFP expression in oligodendrocytes tional control region or promoter. The MBP transcriptional was observed in the primary cultures. In contrast, transduc- control region might be suitable for gene therapy and other tion with rAAV carrying the CMV promoter produced neurobiology studies requiring direct targeting to the myeli- stronger GFP fluorescence in various cell types, with the nating cells. majority of GFP-expressing cells being the astrocytes.

Keywords: gene therapy; central nervous system; green ﬂuorescent protein (GFP); AAV; MBP promoter

Introduction disease has been reported to be associated with AAV. Co- infection by helper viruses (eg adenovirus or herpes sim- Gene transfer into the central nervous system (CNS) as a plex virus) is required for productive AAV infections. possible route for gene therapy of neurological diseases, Without helper viruses its genome integrates into host either by in vivo or ex vivo methods, has gained attention chromosome DNA, preferentially into the human chro- 1–4 in recent years (see reviews ). Among various gene mosome 19.8 Recombinant AAV vector is generated by delivery systems, viral vector-mediated gene transfer is removing the rep and cap genes and placing a ‘transgene’ considered to be the most efficient approach. Herpes sim- between the ITRs. plex virus and adenovirus are both capable of infecting The oligodendrocyte is the myelin-forming cell in the post-mitotic cells such as neurons and glial cells and have CNS. It plays a major role in function and maintenance been used for gene transfer into the CNS. However, their of the myelin sheath.9 Metabolic perturbation of oligo- applications in gene therapy have been associated with dendrocytes resulting from genetic defects, toxins, infec- problems of immunogenicity and cytopathogenicity. tion, etc causes defective myelination and/or demyelin- The adeno-associated virus (AAV) vector has gained ation. The specific targeting of gene transfer and increasing attention recently. Its nonpathogenicity and a expression into the oligodendrocytes may facilitate wide host range make it a good candidate as a gene deliv- understanding of the pathogenesis of demyelination as 5,6 ery system in the CNS. Adeno-associated virus is a well as a possible gene therapy for demyelinating dis- small nonenveloped parvovirus. The wild-type AAV-2 eases. Genes transferred by rAAV carrying the cytomega- genome contains a single-strand DNA of 4680 nucleo- lovirus (CMV) immediate–early promoter have been 7 tides. It consists of one structural gene (cap) and one found to be expressed preferentially in neurons.10 In this nonstructural gene (rep) responsible for the virus encap- study, we explored the possibility of gene expression in sidation and replication, respectively, and two inverted oligodendrocytes mediated by rAAV, using the green repeat sequences (ITRs) of 145 bases which are minimal fluorescent protein (GFP)11,12 as a reporter gene under the essential cis-acting sequences required for virus repli- control of the myelin basic protein (MBP) transcriptional cation, host integration, packaging, etc. So far, no human control region.13 Results Correspondence: K Suzuki, Department of Pathology and Laboratory Medicine, CB# 7525, Brinkhous-Bullitt Building, University of North Construction of rAAV-MBP-GFP Carolina, Chapel Hill, NC 27599-7525, USA To investigate cell-specific expression in oligodendro- Received 28 February 1997; accepted 7 August 1997 cytes mediated by rAAV, the myelin basic protein (MBP) AAV-mediated oligodendrocyte-specific expression H Chen et al 51 transcriptional control region,13 which has been found fluorescence. Approximately 5, 10 and 30% of cells previously to direct MBP gene expression specifically in expressed GFP at 2 days after infection at a multiplicity oligodendrocytes by transgenic mouse studies, was of infection (MOI) of 2.5, 5 and 7.5, respectively. The GFP chosen to control the expression of the GFP gene selected fluorescence was located in the cytoplasm and nucleus, as a reporter. The rAAV vector carrying the MBP tran- as well as the processes of MOCH-1 cells (Figure 2) and scriptional control region and GFP gene was constructed was detectable for at least 3 weeks. by replacing the CMV promoter in plasmid pTR-UF2 To evaluate transduction on primary oligodendrocyte with 1.9 kb of the MBP transcriptional control region13 cultures, oligodendrocyte progenitor cells were purified (Figure 1). To maintain a viral genome size with from primary rat glial cells and allowed to differentiate maximum packaging efficiency, the neoR cassette in the to mature oligodendrocytes in the supplemented pTR-UF2 plasmid was retained, thus generating a 4.76 kb DMEM/F12 medium. The oligodendrocytes were ident- AAV cassette which is close to the size of wild-type AAV. ified by morphology and immunostaining with anti-O1 antibody. Between 30 and 50% of cells, after 4 days in Recombinant AAV viruses the supplemented DMEM/F12 medium, were immuno- Recombinant AAV-MBP-GFP and rAAV-CMV-GFP virus stained by anti-O1 antibody (data not shown). The num- particles were generated by transfection of 293 cells with ber of mature oligodendrocytes with myelin sheath rAAV plasmid and a helper plasmid providing the rep (immunostained with antibodies against MBP) increased and cap genes in trans- and co-infection by helper adeno- gradually and reached maximum level (50–60%) at virus. Southern analysis of Hirt14 extracted viral DNA approximately day 10. The remaining cells mostly after DpnI digestion confirmed the successful rescue and appeared to be type 1 astrocytes with some type 2 astro- replication of the rAAV cassette by the presence of full cytes. length DpnI resistant viral DNA (data not shown). After Primary cultures of oligodendrocytes were infected isopycnic gradient centrifugation, purified rAAV stocks with rAAV-MBP-GFP viruses between 5 and 11 days of up to 1012 virus particles per milliliter based on dot blot after initial plating. The GFP fluorescence was observed analysis were produced. The infectious titer measured by at 2 to 3 days after infection at a MOI of 10 based on the replication center assay on 293 cells (reflecting the the infectious units. The number of GFP-expressing cells number of virus particles containing intact ITRs and reached a maximal level (approximately 35%) at 5–8 days being able to enter the nucleus) was approximately after infection and remained relatively stable for another 10 000-fold less than the particle units. The infectious tit- 2 weeks. The fluorescence was observed in both the cell ers of the virus stocks used in vitro were 3 × 107 infection bodies and the fine processes, but was weak (Figure 3). units (IU) /ml for rAAV-CMV-GFP and 8 × 107 IU/ml for To determine the GFP-expressing cell types, cells were rAAV-MBP-GFP and residual adenovirus estimated to be fixed and double-immunostained with anti-GFP antibody 0.5 and 0.1% of rAAV IU, respectively. The replication and anti-O1 antibody. Immunostaining with anti-GFP center assay indicated that there was some wild-type antibody resulted in stronger and more stable fluor- AAV contamination (at the level of 10% rAAV IU for escence. Double-immunostaining confirmed that the rAAV-CMV-GFP stocks and less than 1% for rAAV-MBP- GFP-expressing cells transduced with rAAV-MBP-GFP GFP stocks). The in vitro data presented in this report were almost exclusively oligodendrocytes, as demon- were based on the studies with purified rAAV prep- strated by typical oligodendrocyte morphology and anti- arations without further heat inactivation. For in vivo O1 immunostaining (Figure 4). There were some GFP- studies, virus stocks containing approximately 8 × 107 positive/O1-negative cells which displayed small cell IU/ml to 2 × 108 IU/ml were subjected to heat inacti- bodies bearing a few coarse processes, probably rep- vation (30 min at 56°C). After heat inactivation no detect- resenting type 2 astrocytes.16 No polygonal cells (typical able adenovirus (less than 1 × 103 p.f.u./ml) was found type 1 astrocytes) were found expressing GFP. In con- by plaque assay. The level of wild-type AAV was less trast, parallel studies with rAAV-CMV-GFP resulted in than 1% of rAAV IU in these stocks. stronger GFP-expressing in cells with different morpho- logies (Figure 5): stellate cells with a few coarse radial Transduction of oligodendrocyte cell line and primary processes and with negative anti-O1 immunostaining; cultures and polygonal cells with a large cell body and negative MOCH-1 is a transformed oligodendrocyte cell line.15 anti-O1 immunostaining. Although some cells with mul- Under 1% FBS conditions, the cells display an oligodend- tiple processes and with anti-O1 immunostaining also rocyte-like morphology and express MBP and other mye- expressed the GFP, the anti-O1-negative types comprised lin-specific proteins. Transduction of MOCH-1 cells by approximately 90% of cells with GFP immuno- rAAV-MBP-GFP produced GFP protein as early as 24 h fluorescence. after infection as demonstrated by the natural GFP green To examine whether expression of GFP with rAAV-

Figure 1 Schematic map of pTR-MBP-GFP construct. ITR, 145 bp of palindromic terminal repeated sequence of AAV; MBP, 1.9 kb of myelin basic protein transcriptional control region; S, 180 bp splice signal from SV40 late viral protein gene; GFP, 715 bp of humanized green ﬂuorescence protein; and PA, 200 bp of SV40 polyadenylation signal. The neoR cassette includes an enhancer region from the polyoma virus mutant pYF441, the TK promoter of HSV, the neomycin resistance gene, and the bovine growth hormone polyadenylation signal. The small arrow represents the transcription start site. The viral vector sequences were cloned into pBS M13+ plasmid (Stratagene, La Jolla, CA, USA). AAV-mediated oligodendrocyte-speciﬁc expression H Chen et al 52 a b

c d

e f

Figure 2 Green fluorescent protein expression in the MOCH-1 cells transduced with rAAV. (a) A MOCH-1 cell displaying a native GFP fluorescence at 3 days after infection with rAAV-MBP-GFP. (b) The same field as (a) under phase contrast. Photographs were taken under an Olympus IMT2 inverted light microscope with a FITC filter. (c–f) MOCH-1 cells were infected with increasing amounts (MOI = 0, MOI = 2.5, MOI = 5 and MOI = 7.5 in c, d, e and f, respectively) of rAAV-MBP-GFP and examined under Nikon microphoto FXA light microscope 2 days after infection.

MBP-GFP transduction was restricted to oligodendro- due to contaminating adenovirus or a wild-type AAV, cytes, the 293 cells and primary cultures of astrocytes we assayed the promoter-reporter constructs by transfec- were infected by rAAV-MBP-GFP viruses at an MOI of tion of 293 cells, HeLa cells, and primary cultures of 10. The GFP expression was observed in 293 cells and a astrocytes and oligodendrocytes with pTR-UF2 plasmid few type-2-like astrocytes in the primary culture of astro- (the CMV-GFP construct) and pTR-MBP-GFP plasmid. cytes. We do not know why the MBP promoter in the Forty-eight hours after transfection with the CMV-GFP rAAV infection showed relatively high expression in 293 construct, the GFP was expressed in more than 60% of cells. However, it remains a possibility that ampliﬁcation 293 or HeLa cells. Approximately 35% of cells in the pri- of rAAV by contaminating adenovirus and a wild-type mary culture of astrocytes expressed the GFP driven by AAV could have contributed to the high transduction in the CMV promoter, suggesting that the CMV promoter this cell type. was active in this cell type in vitro. Less than 5% of cells in the primary cultures of oligodendrocytes expressed Transfection of cell cultures with plasmid constructs GFP after transfection with the CMV-GFP construct and In order to show that the relative activity of each pro- appeared to be all large polygonal astrocytes which moter was an intrinsic property of the construct and not typically constituted a small percentage and increased AAV-mediated oligodendrocyte-speciﬁc expression H Chen et al 53 a a

b b

c c

Figure 3 Green fluorescent protein expression under the control of the MBP transcriptional control region in primary cultures of oligodendrocytes transduced with rAAV. Primary cultures of oligodendrocytes (in defined DMEM-F12 medium for 10 days) were infected with rAAV-MBP- GFP at an MOI of 10. Cells were fixed at 12 days after infection and stained with anti-O1 monoclonal antibody. Native GFP fluorescence and O1 immunofluorescence were observed under a FITC filter (a) and a Figure 4 Double-immunostaining of rAAV-MBP-GFP transduced oligo- TRITC filter (b), respectively. Two oligodendrocytes expressed the GFP dendrocytes with anti-GFP and anti-O1 antibodies. Primary cultures of fluorescence in both the cell bodies and the multiple fine processes. (c) The oligodendrocytes (in defined DMEM-F12 medium for 10 days) were same field under phase contrast. infected with rAAV-MBP-GFP at an MOI of 10. Cells were fixed at 3 weeks after infection and double-immunostained with anti-GFP and anti- O1 antibodies as described in the text. The GFP and O1 immunofluorescence was observed under a FITC filter (a) and a TRITC filter (b), respectively. Several oligodendrocytes including the processes were stained with anti-GFP antibody (a) and anti-O1 antibody (b). (c) The phase contrast view of the same field. AAV-mediated oligodendrocyte-specific expression H Chen et al 54 a b c

d e f

g h

Figure 5 Green fluorescent protein expression under the control of the CMV promoter in primary cultures of oligodendrocytes mediated by rAAV. Primary cultures of oligodendrocytes (in defined DMEM-F12 medium for 10 days) were infected by rAAV-CMV-GFP at an MOI of 10. (a–f), Cells were fixed 3 weeks after infection, double-immunostained with anti-GFP polyclonal antibody and anti-O1 monoclonal antibody and observed with a FITC filter for GFP expression (a and d), a TRITC filter for O1 expression (c and f) and phase contrast (b and e). Green fluorescent protein immunofluorescence was observed in a polygonal cell (a) and a stellate cell with a few coarse processes (d). Neither cell was immunostained with anti-O1 antibodies (c and f). (g) Two polygonal cells on the edge of cell cultures displayed native GFP fluorescence at 12 days after infection. (h) The phase contrast view of g.

gradually in the primary culture of oligodendrocytes. promoter was silent or exhibited very low activity in this Transfection with pTR-MBP-GFP plasmid resulted in the cell type. We did not detect any GFP fluorescence in the GFP expression in a few cells in both 293 cells and HeLa primary culture of oligodendrocytes, which was probably cells, indicating there was some ‘leaking expression’ in due to the low transfection efficiency in this cell type. this cell type by the MBP promoter. Inconsistent with the Nevertheless, the data of astrocytes suggested that the transduction studies in vitro, no GFP fluorescence was pattern of expression in the transduction studies observed in the primary cultures of astrocytes transfected described above was attributed mainly to the intrinsic with the pTR-MBP-GFP plasmid, suggesting that this properties of the promoters. AAV-mediated oligodendrocyte-specific expression H Chen et al 55 In vivo infection with rAAV-MBP-GFP To determine whether the preferential oligodendrocytes a expression observed in vitro was retained in vivo, one microliter (containing approximately 8 × 104 to 2 × 105 infection units) of rAAV-MBP-GFP virus which had been subjected to heat inactivation was injected into the left cerebral hemisphere (approximately 0.6 mm lateral to the midline and 0.8 mm caudal to the bregma and 1.5 mm deep). Fifteen days later, the mice were perfused and post-fixed for 6 h. Vibratome sections of 40–50 ␮m were immunostained with anti-GFP antibody alone or double- immunostained with anti-GFP antibody and anti-GFAP antibody, a specific antibody against astrocytes. The GFP immunofluorescence was observed in the white matter, mainly in the corpus callosum (Figure 6a). Approxi- mately 20–50 GFP-expressing cells were observed per section. The fluorescent cell bodies generally appeared to be oval shaped, approximately 15–25 ␮m in size, and b arranged horizontally along the white matter tract. Fine processes stained with GFP immunofluorescence were also observed (Figure 6c). The immunostained processes appeared to be either longitudinal processes parallel to the long axis of nerve fibers or fine radial processes. Double-immunostaining demonstrated that there was no colocalization of GFP and GFAP immunofluorescence (Figure 6b). No colocalization of immunostaining was observed either between anti-GFP antibody and F4/80 antibody, a specific antibody against mouse microglia (data not shown). Cells, immunostained with anti-GFP antibody, were located in the regions stained heavily with anti-MBP antibody. The fine processes, immunostained with anti-GFP antibody, were not correlated with the neuronal processes immunostained with antibody against neurofilaments 200 (data not shown). The GFP c immunofluorescent cells were detected approximately up to 1.5 mm in a rostral–caudal direction and 2 mm in a medial–lateral direction. In some cases, the GFP immunofluorescent cells dispersed approximately 4 mm along the corpus callosum. No GFP immunofluorescence was observed in all brain samples of mice injected with Indian ink only.

Discussion Adeno-associated virus (AAV) vectors have been demonstrated as transducing a variety of cell types both in vivo and in vitro. Gene transfer experiments in the CNS are, however, relatively new. The majority of recent studies have focused on vectors carrying the CMV promoter.5,6,10 Figure 6 Green fluorescent protein expression in mouse brain tissues. The CMV immediate–early promoter is one of the strong- One microliter of rAAV-MBP-GFP virus was injected into the left hemisphere of the mouse brain. Tissue sections were double-immunostained est constitutively active virus-derived transcription with anti-GFP and anti-GFAP antibodies 15 days after injection. (a) GFP- elements and has been used for high–level expression of expressing cells were labeled with FITC-conjugated antibody and detected a multitude of genes in many different cell types. In stud- only in the corpus callosum under a FITC filter. The arrowhead represents ies of virus–mediated gene transfer in CNS in vivo, how- the needle injection site. C, cortex; CC, corpus callosum; LV, lateral ven- ever, gene expression under the control of the CMV pro- tricle; LS, lateral septal; solid arrow, fissure. Bar = 200 ␮m. (b) A higher moter has often resulted in expression of the foreign gene magnification view of the box region in (a) under a triple filter (FITC, 6,10,17 TRITC and UV). GFP-expressing cells were labeled with FITC-conjugated primarily in neurons and has decreased over time. antibody and GFAP-expressing cells were labeled with TRITC-conjugated The use of endogenous transcriptional control sequences antibody. The number of green fluorescence cells appearing in the photo- may provide a distinct advantage in terms of stability of graph was lower than actually observed due to thick sections. No overlap- gene expression and cell-type specificity over viral tran- ping immunostaining between GFP and GFAP was observed. Bar = 30 18 ␮m. (c) A different section examined under a FITC filter. Some fine pro- scriptional elements. Recently, Peel et al demonstrated = the efficient transduction of green fluorescent protein in cesses (arrows) were also labeled with GFP immunofluorescence. Bar 30 ␮m. neurons using neuron cell-type-specific promoter. How- ever, whether rAAV can transfer genes into cells other than neurons in the brain remains to be tested. AAV-mediated oligodendrocyte-specific expression H Chen et al 56 Myelin basic protein is the second most abundant mye- by rAAV is efficient in a variety of cell types including lin protein both in the CNS and in the peripheral nervous oligodendrocytes. However, the transduction efficiency system (PNS). The activity of the endogenous MBP tran- observed in specific cell types was greatly influenced by scriptional control region is limited to oligodendrocytes the transcriptional control regions used to transcribe the in the CNS and Schwann cells in the PNS. Previous stud- reporter gene. Our results indicate that previous obser- ies have suggested that a 1.9 kb fragment of the MBP vations of neuron-dominated expression of rAAV vectors transcriptional control region was sufficient for appropri- under the control of the CMV promoter6,10 may be ate spatial and temporal specificity in transgenic mice.13 attributable to the relatively low level of activity of this We therefore used this fragment to test the possibility of promoter in glial cells, including oligodendrocytes in oligodendrocyte-specific expression mediated by rAAV. vivo, rather than a host range restriction of rAAV vectors. Due to the large size of the fragment of the control region Cells other than neurons in the CNS can be efficiently and the limited capacity of rAAV, the relatively small transduced using the cell-type-specific transcriptional ‘humanized’ green fluorescent protein12 was chosen to be control region. This vector system may be useful for stud- the reporter gene. The in vitro studies demonstrated that ies of gene therapy of demyelinating diseases as well as rAAV carrying the MBP promoter transduced oligodend- for neurobiological studies of myelin-forming cells. rocytes preferentially over astrocytes, while the inverse was observed with rAAV using the CMV promoter to transcribe the identical reporter. Although the presence Materials and methods of adenovirus and wild-type AAV in some of our rAAV vector stocks used in in vitro studies might increase the Cell cultures ° transduction rates, parallel transfection studies suggested All cell cultures were incubated at 37 C in a 5% CO2 that it did not affect the cell-type-specific expression dis- atmosphere. An adenovirus transformed human embry- played by the promoters, especially in the primary glial onic kidney cell line (293 cells) was maintained in Dulbec- cultures. Transfection of astrocytes with pTR-UF2 that co’s modified Eagle’s medium (DMEM-H) supplemented carried the CMV promoter showed high GFP expression. with 10% fetal bovine serum (FBS), penicillin (100 IU/ml) ␮ In contrast, no GFP expression was observed in the astro- and streptomycin (100 g/ml) (P/S). An oligodendrocyte 14 cytes transfected with the construct carrying the MBP cell line (MOCH-1 cells) (provided by Dr B Popko, Neu- promoter. The lack of MBP-driven GFP expression in roscience Center, University of North Carolina at Chapel astrocytes was further supported by the in vivo studies. Hill, NC, USA) was cultured in DMEM-H medium sup- Infusion of rAAV-MBP-GFP virus that was heat treated plemented with 1% FBS and P/S. Primary rat glial cul- to inactivate adenovirus, without a significant amount of tures were maintained in Earle’s basal medium (BME) wild-type AAV, into mouse brains resulted in the supplemented with glutamine, 0.6% glucose, 10% FBS expression of GFP specifically in the white matter. These and P/S. Purified oligodendrocytes were maintained in GFP-expressing cells generally contained oval shaped DMEM/F12 medium supplemented with 0.5% FBS, bov- somata of 15–25 ␮m and were arranged in parallel with ine serum albumin (1.0 mg/ml), biotin (10 ng/ml), pro- ␮ ␮ each other and with the long axis of the nerve fibers. The gesterone (20 nm), insulin (5 g/ml), putrescine (100 m), processes that were immunostained with GFP appeared sodium bicarbonate (3.6 g/l), selenium (5 ng/ml), trans- ␮ to be either radial processes near the cell somata or longi- ferrin (50 g/ml), triiodothyronine (30 nm) and P/S. tudinal processes which passed parallel along the long axis of the nerve fibers, resembling oligodendrocytes.19,20 Preparation of primary cultures of oligodendrocytes Although various types of antibodies specific for differ- Primary rat glial cultures were prepared from cerebral ent stages of oligodendrocytes were explored, their utility cortices of 1- to 3-day-old normal rat pups as described for the morphology studies of oligodendrocytes in the by McCarthy and de Vellis.22 After 7–10 days in vitro, oli- mature brains was very limited.21 We could not observe godendrocyte progenitor cells were separated from the the cell body staining with antibodies specifically against confluent astrocyte bed by the shaking method of McCar- mature oligodendrocytes such as anti-MBP and anti- thy and de Vellis.23 Cells were suspended in sup- CNPase antibodies due to the heavily myelin immunos- plemented BME medium and seeded on to coverslips taining in adult mice by these antibodies. Nevertheless, precoated with 100 ␮g/ml of poly-d-lysine (Sigma double-immunostaining with anti-GFP and anti-GFAP Chemical, St Louis, MO, USA) at a density of 4– antibodies clearly showed that there was no overlapping 5 × 104/cm2. On the next day, cells were fed with sup- immunostaining between anti-GFP and anti-GFAP anti- plemented DMEM/F12 medium and then were fed every bodies in all tissue samples observed, which was consist- 2–3 days thereafter. The astrocytes were maintained in ent with the in vitro transfection and transduction results. the supplemented BME medium. Furthermore, the possibility of transduction of microglial cells by rAAV-MBP-GFP was ruled out since there was Plasmid constructions no overlapping immunostaining between anti-GFP and The construct of pTR-MBP-GFP plasmid is depicted in F4/80 antibodies. Cells located in the white matter are Figure 1. Briefly, a 1.9-kb fragment of MBP transcrip- mainly oligodendrocytes, astrocytes and a small number tional control region13 from the MBP/IFN-␥ construct of of microglia and endothelial cells. Based on the results of Corbin et al24 was cleaved by SacI and XbaI digestions. double-immunostaining of anti-GFP antibody with anti- Plasmid pTR-UF2 (Provided by Dr S Zolotukhin, Gene GFAP antibody and anti-GFP antibody with F4/80 anti- Therapy Center, University of Florida, USA) was treated body and the morphology of the GFP immunofluorescent by KpnI and XbaI digestions to remove the CMV tran- cells, it is reasonable to suggest that the majority of GFP- scriptional control region. Both the SacI site in the expressing cells, if not all, were oligodendrocytes. MBP/IFN-␥ construct and the KpnI site in the pTR-UF2 In conclusion, we believe that gene transfer mediated plasmid were converted to blunt-ends before ligation. AAV-mediated oligodendrocyte-specific expression H Chen et al 57 The excised fragment of 1.9-kb MBP transcriptional con- dishes at a density of 60–75%. Primary cultures of oligo- trol region was gel purified by QiAquick gel extraction dendrocytes were prepared as described before. Three kit (Qiagene, Chatsworth, CA, USA) and subcloned into days after plating, the cultures of oligodendrocytes were the pTR-UF2 plasmid. Plasmids pTR-MBP-GFP and pTR- used for the transfection studies. The transfection was UF2 were used to generate rAAV-MBP-GFP and rAAV- carried out using SuperFect transfection kit (Qiagene) MBP-GFP viral vectors, respectively. according to the manufacturer’s instructions. Briefly, between 2 and 4 ␮g of pTR-UF2 and pTR-MBP-GFP DNA Generation of rAAV vectors were mixed with the SuperFect reagents. After 10 min Recombinant AAV vectors were produced in 293 cells by the mixture was applied to the cells. Two hours later, calcium phosphate-mediated cotransfection of the vector cells were washed with medium and then fed with the plasmid with a complementing plasmid containing the appropriate medium. At 48 and 72 h after transfection AAV genes, rep and cap.25 The cells were infected with cells were observed for GFP fluorescence under an adenovirus type 5 dl309 helper virus at a MOI of 5 at 16 inverted microscope with a FITC filter. h after transfection and harvested at 48 h after infection when full cytopathic effects were observed. The cells Animals and microinjection were harvested and lysed by three cycles of freeze–thaw. C57BL/6 mice, female or male, 20 to 25 days old were Virus was precipitated by (NH ) SO . The precipitate was 4 2 4 used in this study. Mice were bred within the animal col- redissolved in 4.5 g/cm3 CsCl followed by centrifugation ony. All animal surgical and care procedures were at 288 000 g for 36 h in a SW41 rotor to band the recombi- approved by the Institutional Animal Care and Use Com- nant virus at its buoyant density. The gradient was frac- mittee at the university. The microinjection procedures tionated and each fraction assayed for rAAV by dot blot were based on the technique described by McCown et hybridization26 or marker gene expression. Fractions con- al.10 In brief, the mice were anesthetized with 2.5 % of taining virus were pooled, diluted with 4.5 g/cm3 CsCl, avertin (0.014 ml per gram of body weight). The skin over and subjected to a second round of centrifugation at the skull was incised. The injection sites were determined 400 000 g in an NVT 65 rotor for 4 h. The gradients were based on the coordinates of sagittal suture and bregma. fractionated and assayed as above. The fraction contain- A small hole was drilled in the skull with a 26-gauge ing the highest amount of virus particle was dialyzed needle. The injector was lowered to a depth of 1.5 mm. against 20 mm Hepes containing 15% glycerol at 4°C. Ali- Recombinant AAV-MBP-GFP virus of 1 ␮l and a trace of quots were subjected to heat treatment at 56°C for 30 min Indian ink were co-injected into the brains over 5 min. to inactivate residual adenovirus. The level of residue The injector was left in place for 3 min. The incision was adenovirus was determined by plaque assay. The virus then sutured. The operated mice were either returned to titer was determined by the dot blot assay27 or replication the mother cage after they regained consciousness or to center assay28 which was also used to determine the wild- separate cages. For controls, the same volume of dialysis type AAV level. buffer with a trace of the ink was substituted for the rAAV. DNA hybridization All DNA probes were labeled by the random priming method using the ready-to-go kit (Pharmacia Biotech, Immunohistochemistry and immunocytochemistry Piscataway, NJ, USA). Blots were hybridized overnight Fifteen days after injection, the animals were anesthetized in a SSPE buffer solution containing 50% formaldehyde, and perfused with 4% paraformaldehyde for 10 min. Six 10% dextran sulfate (Sigma Chemical), 1% powder milk hours after fixation, the brains were removed and trans- and 0.1% SDS at 42°C. All later steps were performed ferred to PBS solution containing 20% sucrose and stored following the standard method.26 at 4°C overnight. Tissue sections were cut 40–50 ␮m thick with a vibratome and mounted on probe-on slides (Fisher Infection of cell cultures Scientific, Pittsburgh, PA, USA). Following 5 min immer- MOCH-I cells were seeded at 2 × 104/cm2 on a 12-mm sion into PBS containing 50 mm ammonium chloride to coverslip or directly in 24-well plates. Next day, cells quench free aldehyde group, the tissue sections were were infected by rAAV at a MOI of 10. Oligodendrocyte- blocked for 1.5 h in PBS containing 10% normal goat progenitor cells were maintained in the supplemented serum, 0.2% of Triton X100. After blocking, sections were DMEM/F12 medium and differentiated into mature olig- exposed to primary antibodies, either single-immuno- odendrocytes. At 5–11 days after initial seeding, these stained with polyclonal antibody against GFP (Clontech, primary cultures of oligodendrocytes were infected by Palo Alto, CA, USA) (1:150, diluted in PBS containing 5% aliquots of rAAV virus at a MOI of 10. After 1.5–4 h the normal goat serum/1% BSA) overnight at 4°C or double- cells were washed twice and maintained in the sup- immunostained with various combinations, including plemented DMEM/F12 medium. At various time-points, anti-GFP antibody with anti-GFAP monoclonal antibody living cells were examined directly under an Olympus (1:450, Sigma Chemical); anti-GFP antibody with F4/80 IMT2 inverted light microscope (Olympus, Lake Success, rat antibody (1:100, Serotec, Washington DC, USA). After NY, USA) with a FITC filter (450–500 nm excitation, 515 extensive washing in PBS containing 0.2% Triton X100, nm long path emission). To identify cell types, cells were tissue sections were exposed to FITC-conjugated goat fixed with 4% paraformaldehyde, then subjected to the anti-rabbit IgG (1:50, Sigma Chemical) only or with rho- following immunocytochemical studies. damine (TRITC)-conjugated goat anti-mouse IgG or TRITC-conjugated goat anti-rat IgG (1:100, Cappel, Dur- Transfection of cell cultures ham, NC, USA). No cross-reactions in the double- One day before transfection, 293 cells, HeLa cells and immunostained sections were found by the single astrocytes, purified as above, were plated in six-well immunostaining of anti-GFP antibody with anti-mouse AAV-mediated oligodendrocyte-specific expression H Chen et al 58 or anti-rat goat IgG and anti-GFAP antibody or F4/80 9 Pfeiffer SE, Warrington AE, Bansal R. The oligodendrocyte and antibody with anti-rabbit goat IgG. its many cellular processes. Trends Cell Biol 1993; 3: 191–197. MOCH-1 cells and oligodendrocytes in the primary 10 McCown TJ et al. Differential and persistent expression patterns cultures were identified by anti-O1 monoclonal antibody of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res 1996; 712: 99–107. (1:10, Boehringer Mannheim, Indianapolis, IN, USA). 11 Chalfie M et al. Green fluorescent protein as a marker for gene Astrocytes were identified with polyclonal antibody expression. Science 1994; 263: 802–805. against GFAP (1:150; Dako, Carpinteria, CA, USA). 12 Zolotukhin S et al. A ‘humanized’ green fluorescent protein Double-immunostaining with anti-GFP and anti-GFAP cDNA adapted for high-level expression in mammalian cells. antibodies or anti-GFP and anti-O1 antibodies was car- J Virol 1996; 70: 4646–4654. ried out sequentially. 13 Gow A, Friedrich VL, Lazzarini RA. Myelin basic protein gene After immunostaining, slides were coverslipped with contains separate enhancers for oligodendrocyte and Schwann anti-photobleaching mounting medium (0. 25% DABCO, cell expression. J Cell Biol 1992; 119: 605–616. 2% n-propyl gallate in polyvinyl alcohol/glycerol) and 14 McMaster GK, Beard P, Engers HD, Hirt B. Characterization of examined under either a Nikon microphoto FXA micro- an immunosuppressive parvovirus related to the minute virus of mice. J Virol 1981; 38: 317–326. scope (Nikon, Garden City, NJ, USA) equipped with an 15 Hayes C et al. Expression of the neu oncogene under the tran- FITC filter, TRITC filter, and a FITC-TRITC-UV triple fil- scriptional control of the myelin basic protein gene in transgenic ter or confocal microscope equipped with a Zeiss micro- mice: generation of transformed glial cells. J Neurosci Res 1992; scope (Carl Zeiss, Thornwood, NY, USA). 31: 175–187. 16 Raff MC, Miller RH. A glia progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture Acknowledgements medium. Nature 1983; 303: 390–396. 17 Doll RF et al. Comparison of promoter strengths on gene deliv- We wish to thank Dr Sergei Zolotukhin for the pTR-UF2 ery into mammalian brain cells using AAV vectors. Gene Therapy construct and Dr Joshua Corbin for the MBP-IFN-r plas- 1996; 3: 437–447. mid. We thank Dr Thomas McCown for his technical help 18 Peel AL et al. Efficient transduction of green fluorescent protein of microinjection and his generous donation of micro- in spinal cord neurons using adeno-associated virus vectors con- injectors. We thank Dr Robert Bagnell and Ms Victoria taining cell type-specific promoters. Gene Therapy 1997; 4: 16–24. Madden for microscope assistance, Ms Janice Weaver for 19 Szuchet S. The morphology and ultrastructure of oligodendro- supplying us with rat pups, Ms Carolyn Lloyd for her cytes and their functional implications. In: Kettenmann H, Ran- editing work, Ms Shin-ja Kim for perfusion and animal som BU (eds). Neuroglia. Oxford University: New York, 1995, pp 23–44. care. Also, we thank Dr Brain Popko for his critical 20 Butt AM et al. Three-dimensional morphology of astrocytes and review and supplying us with the MOCH-1 cells. This oligodendrocytes in the intact mouse optic nerve. J Neurocytol work was supported by research grants, NS24453, 1994; 23: 469–485. 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