Specific Hammerhead Ribozymes Reduce Synthesis of Cation

Gene Therapy (2003) 10, 1567–1574 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Speciﬁc hammerhead ribozymes reduce synthesis of cation-independent mannose 6-phosphate receptor mRNA and protein

A Yaghootfam and V Gieselmann Institut fu¨r Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universita¨t, Bonn, Germany

Storage diseases because of lysosomal enzyme deficiencies integrated into the VaI adenovirus RNA to increase stability may be treated by the transplantation of cells that secrete the and in which they are transcribed from an RNA polymerase enzyme which is deficient in patients. One can expect that III promoter. These plasmids were transiently transfected increasing the amount of secreted enzymes will improve the into BHK cells to investigate in vivo activity. Two ribozymes therapy efficacy. Secretion of lysosomal enzymes can be reduce efficiently the levels of murine M6PR300 mRNA in enhanced by reducing the mannose 6-phosphate receptor transient transfection experiments to 42–45%. This corre- involved in the lysosomal sorting of newly synthesized lates with the reduction of M6PR300 biosynthesis, which is lysosomal enzymes. For this purpose, we have constructed reduced also to 37% of normal. We can also demonstrate hammerhead ribozymes targeting the mRNA of the large that the reduction in M6PR300 is sufficient to increase a murine mannose 6-phosphate receptor (M6PR300). In vitro lysosomal enzyme secretion. ribozymes cleave M6PR300 RNA fragments efficiently with Gene Therapy (2003) 10, 1567–1574. doi:10.1038/ cleavage rates of 69–93% after 3 h of incubation. Ribozymes sj.gt.3302032 were cloned into an expression vector in which they are

Keywords: mannose 6-phosphate receptor; gene therapy; hammerhead ribozymes; lysosomal storage diseases

Introduction However, the secreted enzymes can be recaptured, since the M6PR300 receptor is not only found in the Golgi Lysosomal enzymes are synthesized at the rough apparatus but is present also on the plasma membrane endoplasmic reticulum, where they cotranslationally and mediates the endocytosis of lysosomal enzymes. acquire N-linked oligosaccharide side chains. In the Thus, this receptor also functions in the transport from Golgi apparatus, soluble lysosomal enzymes are specifi- the plasma membrane to the lysosome.1 cally recognized by a phosphotransferase, which initiates Lysosomal enzyme deficiency can cause various a two- step process, which results in the phosphorylation diseases, termed lysosomal storage disorders. Owing to of position 6 of mannose residues of the N-linked the particular sorting pathway of soluble lysosomal oligosaccharide side chains. Via these mannose 6- enzymes, these diseases are suitable candidates for phosphate (M6P) residues lysosomal enzymes can bind enzyme replacement and gene therapy. Enzymes bearing to mannose 6-phosphate receptors (M6PR), which M6P residues can be injected, endocytosed by the mediate the further vesicular transport from the Golgi enzyme-deficient cells of the patient and correct the apparatus to the lysosome.1 In mammalian cells, two metabolic defect.4 receptors of different molecular weights exist. Both Alternatively, cells expressing the missing enzyme can M6PRs are transmembrane glycoproteins and recycle be transplanted. The secreted fraction of this enzyme will between the trans-Golgi network, the plasma membrane be available for endocytosis by the M6PR pathway. It can and the endosomal compartment. The cation-dependent be expected that this therapy will be more efficient, the (CD-) M6PR has an apparent molecular weight of 46 000 more enzyme the transplanted cell secretes. This can be (M6PR46) and the cation-independent (CI-) M6PR of achieved by two means: (i) by increasing the synthesis of 300 000 (M6PR300).1,2 Both receptors bind different but the enzyme via genetic modification and (ii) by increas- overlapping subsets of lysosomal enzymes.3 ing the secretion via downregulation of the M6PRs, The binding of newly synthesized lysosomal enzymes which would enhance the secreted fraction of newly to the receptors in the Golgi compartment is never synthesized M6P-bearing lysosomal enzymes.5 complete. A considerable fraction of these enzymes Thus, tools that would allow reduction of M6PRs escapes receptor binding and is secreted from the cells. synthesis could be of general value in gene therapy of lysosomal storage diseases. One means to achieve this is the expression of ribozymes to cleave the M6PR mRNA, Correspondence: Dr A Yaghootfam, Institut fu¨r Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universita¨t, Nuallee 11, 53115 Bonn, which should lead to the reduction of receptor synthesis. Germany Ribozymes are small catalytic RNA molecules, which Received 28 June 2002; accepted 3 February 2003 are attractive therapeutic agents. The best characterized Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1568 examples of ribozyme-catalyzed cleavage reactions are sites and Rz E, Rz F, Rz G to target the CUC sites, found in plant viroids, virusoids and linear satellite respectively (Figure 2b). The catalytic domain of the RNAs.6 The small, self-cleaving RNAs have conserved ribozymes was identical, the specificity for the potential catalytic core sequences, and need a metal-hydroxide target sites is provided by two short antisense regions, that is involved in the cleavage reaction.7 The cleavage flanking the target sites at the 50- and 30-end, respectively. reaction, which is very specific, generates 20,30-cyclic These regions are 7–8 nt long in the case of Rz A, B, C, D, phosphate and 50-OH products. These self-cleaving F, G and 8–15 nt in the case of Rz E. RNAs can be subdivided into groups depending on the The ribozyme coding cassettes were cloned into vector sequence and secondary structure formed around the pGval (kindly provided by A Lieber).13 Here they are cleavage site. The most studied types are the hammer- embedded in the vaRNA I gene of human adenovirus head and hairpin ribozymes. The hammerhead structure type 2, which can form a stable secondary structure and consists of three base-paired stems (helices I, II and III) is highly resistant against nuclease degradation. (Figure 1). The catalytic domain of the ribozyme includes The plasmid contains the T7 promoter for in vitro highly conserved nucleotides and is essential for transcription and a pol III promoter for in vivo expression ribozyme activity. Two stretches of oligonucleotides, of ribozymes.13 which give the hammerhead construct its specificity, flank the central core of the ribozyme. The target site of Ribozymes cleave murine M6PR300 RNA in vitro the ribozyme can be any NUH sequence (where H¼A, C We examined the potential of ribozymes to cleave or U, and N is any nucleotide).8 Hammerhead ribozymes murine M6PR300 RNA in vitro. The size of mouse require a divalent metal ion, such as Mg2+, to mediate M6PR300 mRNA (8.8 kb) excludes that the entire mRNA catalytic cleavage.9 In this work, we have constructed is used as a ribozyme substrate in vitro. Thus, fragments specific ribozymes directed at the murine M6PR300 of the M6PR300 cDNA encompassing the potential mRNA with the aim to develop therapeutic tools for ribozyme cleavage sites were subcloned into the Blue- improved secretion of lysosomal enzymes from trans- script II SK +/À plasmid (Stratagene). These were planted platform cells. linearized with HindIII restriction enzyme and transcribed in vitro. The RNA fragments, which were designated as sA, sB, sC, and sD, were labelled with Results [g-32P] ATP at the 50-end (see Materials and methods). Four different ribozymes (Rz A, Rz B, Rz C, and Rz D) Construction of specific hammerhead ribozymes targeting GUC sites within fragments sA, sB, sC, sD were In order to identify potential ribozyme cleavage sites of incubated in vitro with the respective M6PR300 RNA murine M6PR300 mRNA, predictions of the secondary fragments without prior heat denaturation. A typical in structure of M6PR300 RNA fragments were generated vitro cleavage reaction contained 0.66 pmol 50-labelled 10,11 by the program MFOLD (Figure 2a). A comparison substrate RNA, 1.32 pmol ribozyme RNA and 10 mM

of cleavage rates of individual NUH triplets revealed MgCl2 (see Material and methods). After incubation for that the reaction rates decrease in the following order: 1 h at 421C Rz B, C, and D produced cleavage products of GUC4CUC4AUU4GUU.12 Accordingly, we chose the expected size (Figure 3). Even after extended time of only GUC and CUC cleavage sites, which were located incubation (3 h), the Rz A did not generate any cleavage in putative loop structures of the murine M6PR300 fragments (Figures 3 and 4). An experiment, in which the mRNA, since single-stranded loop structures are more cleavage reactions were done for varying length of time likely to be accessible by ribozymes. This led to the revealed extensive cleavage after 3 h of incubation of identification of four potentially single-stranded GUC M6PR300 RNA fragments sD (93.5% cleaved), of sC and three CUC sites. We constructed four ribozymes (80.5%) and 69% of sB (Figure 4). designated as Rz A, Rz B, Rz C, Rz D to target the GUC Effects of ribozymes on M6PR300 synthesis Since the ribozymes are specific for the murine receptor, we initially transfected NIH/3T3 and other murine cell lines (Swiss 3T3, murine embryonic fibroblasts) with the ribozymes to investigate the reduction of the endogeneous M6PR300 mRNA and protein. However, although we applied various transfection reagents and protocols, we were not able to transfect more than 20% of this murine cells. If the ribozymes reduce the endogeneous M6PR300 mRNA by about 50% this would result in an overall reduction of mRNA by not more than 10%. This small difference cannot be reliably detected in Northern blot analysis. Cotransfection, however, of murine M6PR300 cDNA and ribozyme expressing plasmids into BHK cells over- comes this problem. First, the endogeneous hamster M6PR300 mRNA is not detected in the Northern blot Figure 1 The secondary structure model of the trans-acting hammerhead ribozyme (Rz B). The arrow indicates the predicted site of cleavage at under stringent washing conditions, thus there is no nucleotide 2130. All constructed ribozymes in this study contained the endogeneous M6PR300 mRNA background. Second, same catalytic core (II). Stems I and III contain the recognition site for in our transfection protocol we can transfect 60–70% of each ribozyme. BHK cells. Therefore, we cotransfected the cDNA

Gene Therapy Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1569

Figure 2 Selection and location of ribozymes within the mouse M6PR300 mRNA. (a) The ﬁgure shows one example of a secondary structure prediction of a fragment of murine M6PR300 RNA generated by the program MFOLD.10,11 Owing to its large size, secondary structure predictions of total M6PR300 mRNA are not possible. The selected potentially single-stranded GUC target site of Rz B is indicated. (b) A schematic drawing of the entire murine M6PR300 cDNA is shown. The 50- and 30-ends are indicated, the coding sequence is depicted by a box, ATG and TAA indicate the initiation and termination codon, respectively. The position of Rz A to Rz G and the cleavage sites for each ribozyme are shown by bold letters and numbers, respectively. expressing the murine M6PR300 mRNA with the caused a reduction of murine M6PR300 mRNA in ribozymes into BHK cells. transfected cells. To investigate the potential of the ribozymes to reduce Rz A, Rz C, Rz E, Rz F, and Rz G did not decrease M6PR300 mRNA in vivo, 50 ng pBEH plasmid containing M6PR300 mRNA level in transfected cells. Both the the complete murine M6PR300 cDNA downstream of the ribozymes, which reduce M6PR300 mRNA in eucaryotic SV 40 early promoter, and 2 mg pGval vectors containing cells to 42 and 45% of control (Figure 5c), respectively, different ribozyme cassettes downstream of the Pol III target sites within the translated region of the murine promoter were cotransfected into BHK-21 cells. At 48 h M6PR300 mRNA. after transfection, cells were lysed, and total RNA was isolated. A Northern blot of total RNA was hybridized Ribozymes affect the biosynthesis of M6PR300 with a mouse M6PR300 cDNA probe (Figure 5a). We further investigated whether the ribozymes reducing Transcripts of ribozyme cassettes were detected with a mouse M6PR300 mRNA in vivo also cause a reduction radioactively labelled oligonucleotide hybridizing to the of M6RP300 synthesis. BHK-21 cells were transiently catalytic domain of the hammerhead ribozymes (Figure transfected with plasmids encoding the mouse M6PR300, 5b). Only two of seven ribozymes (Rz B and Rz D) and the hammerhead ribozyme cassettes. At 48 h after

Gene Therapy Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1570

Figure 3 In vitro cleavage of M6PR300 RNA fragments. M6PR300 RNA fragments sA, sB, sC, and sD containing the cleavage sites of Rz A, B, C, and D, respectively, were transcribed in vitro and labelled with [g-32P]ATP at the 50-end. Labelled substrate RNA (0.66 pmol) was incubated on ice (incubation À) or for 1 h at 421C (incubation +) without (À) or with (+) the addition of 1.32 pmol of Rz A, Rz B, Rz C, and Rz D. Reactions were

started by the addition of 10 mM MgCl2 and terminated by ethanol precipitation. Reactions were subjected to denaturing gelelectrophoresis and autoradiography. sA, sB, sC, and sD depict the uncleaved M6PR300 RNA fragments. nt gives the length of the substrate fragments, respectively.

transfection, cells were labelled for 16 h with [35S]methionine. After pulse labelling cells were lysed, M6PR300 was immunoprecipitated from the lysates and analyzed by autoradiography (Figure 6a). The amount of synthesized M6PR300 was quantified using a phosphoimager BAS 1000 (Fuji). Results of four different experiments are shown in Figure 6b. These experiments show that synthesis of murine M6PR300 can be reduced to 37% of control levels by Rz B and 62% by Rz D, respectively Figure 4 Time dependence of ribozyme cleavage reactions. Ribozymes were incubated under conditions described in Figure 3 with M6PR300 RNA (Figure 6b). fragments. Reactions were terminated after 15, 30 , 60, 120, and 180 min. Cleavage reactions were subjected to denaturing gelelectrophoresis and Effects of ribozymes on secreation of arylsulfatase A autoradiography. The relative amount of the cleavage product was To demonstrate an effect of ribozymes in vivo, we choose quantified with a Fuji phosphoimager BAS 1000 (Fuji). Top panel the MEF-mpr46 cell line. These cells are deficient in the summarizes the data for all ribozymes tested. An example of the reaction of small 46 kDa M6PR46 and only express the large Rz C is shown at the bottom. nt gives the length of sC and cleavage product, respectively. M6PR300.14 Since the transfection efficiency of these cells is only about 10%, we used an assay in which ribozymes and the cDNA of human lysosomal arylsulfa- determined in the immunoprecipitates of cells and tase A encoding plasmids were cotransfected into MEF- media. Cells expressing the irrelevant control ribozyme mpr46 cells. Since transfected cells receive both the (Rz Co) secrete about 15% of human ASA, whereas this ribozyme and the cDNA of a lysosomal enzyme, a amount is increased to 25% in cells transfected with Rz B ribozyme-induced reduction of the amount of endoge- (Figure 7). Rz D is less effective, which is in accordance neous M6PR300 in the transfected cells should enhance with M6PR300 biosynthesis experiments, in which this the secretion of this lysosomal enzyme. As a lysosomal ribozyme was less effective in reducing the amount of enzyme, we used human ASA since a monoclonal receptor. antibody specific for the human enzyme allows to measure selectively the human enzyme in the massive background of endogenous murine sulfatases that are Discussion secreted by all transfected as well as untransfected cells.15 Thus, human ASA was immunoprecipitated with Lysosomal storage diseases are good candidates for the monoclonal antibody from cells and media of cells enzyme replacement and gene therapy approaches. This which were cotransfected with Rz B, Rz D and an is because of M6P residues, which play a central role in irrelevant ribozyme (Rz Co). Enzyme activity was efficient endocytosis and delivery of lysosomal enzyme

Gene Therapy Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1571

Figure 5 Ribozyme induced in vivo reduction of mouse M6PR300 mRNA. BHK-21 cells were transiently transfected with a plasmid containing the murine M6PR300 cDNA and the pGval plasmid carrying the respective ribozyme cassettes (Rz B and Rz D). At 48 h after transfection total RNA was isolated from the transfected cells and subjected to Northern blot analysis. (a) Total RNA isolated from transfected cells was separated in a 1% denaturing agarose gel (see Materials and methods). As a positive control, RNA isolated from Figure 6 Immunoprecipitation of murine M6PR300 protein. (a) At 2 days nontransfected murine NIH/3T3 cells was included, mock-transfected cells after transfection of BHK-21 with plasmids encoding the mouse M6PR300 were used as an negative control. The blot was hybridized to an M6PR300 (pBEH-M6PR300), and the hammerhead ribozyme cassettes (Rz B, Rz D, cDNA and to a GAPDH probe. M6PR300 and GAPDH signals were and Rz Co), cells were labelled for 16 h with [35S]methionine. After pulse quantified with a Fuji phosphoimager and the M6PR300 signal was labelling cells were lysed, M6PR300 was immunoprecipitated from the normalized for the GAPDH signal. (b) Northern blot analysis of lysates, subjected to SDS-PAGE and quantified with the phosphoimager ribozymes. Total RNA isolated from transfected cells was separated in a BAS 1000. As a positive control, M6PR300 from metabolically labelled 2% denaturing agarose gel. The blot was hybridized with an oligonucleo- nontransfected murine NIH/3T3 cells was immunoprecipitated. Rz Co is a tide complementary to the catalytic domain of ribozymes. 5 S indicates the control ribozyme targeting an irrelevant mRNA. (b) Columns show the position of ribosomal 5 S RNA. (c) Columns show the mean of three mean of four independent experiments. Bars indicate minimum and independent transfections and Northern blot analysis. Bars indicate maximum values for these experiments, respectively. minimum and maximum values for these experiments, respectively. from the extracellular space to the lysosomes. Genetically of fragments of the M6PR300 RNA to identify single- modified cells, which are transplanted for therapeutic stranded GUC- or CUC-containing sites, which are reasons, secreting the enzyme that the respective patient considered to be preferential targets for ribozyme is missing, can be expected to be more efficient, the more cleavage.12 enzyme they secrete. The amount of secreted enzyme can Four GUC-targeting ribozymes were tested for their either be enhanced by overexpression or by interference capability to cleave M6PR300 RNA fragments in vitro, with the intracellular sorting events of lysosomal three of which proved to be very efficient, whereas one enzymes in the transplanted cell. Both approaches are a showed no cleavage activity. Nevertheless, all ribozymes means to improve the bioavailability of all lysosomal were tested in cultured cells since it was recently shown enzymes sorted in an M6P-dependent way. Here we that in vitro activity and in vivo activity of ribozymes does have constructed seven hammerhead ribozymes, which not correlate.18 The latter is most likely because of the target the large murine M6PR300 mRNA. This receptor fact that in vivo the substrate binding rather than the functions in the sorting of lysosomal enzymes from the cleavage reaction is the rate-limiting step. The experi- Golgi apparatus to the lysosomes. Deficiency of the ments we performed in cultured cells clearly revealed receptor in fibroblasts of mice has been shown to cause that the two ribozymes had the capacity to reduce mouse enhanced secretion of newly synthesized lysosomal M6PR300 mRNA levels in cultured cells to about 40% enzymes.16,17 We have used RNA structure predictions of control. In accordance with these data, the two

Gene Therapy Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1572 affecting the survival of such cells cannot be expected. It was reported recently that in M6PR300-deficient cells lysobisphosphatidic acid accumulates in the late endosomal/lysosomal compartment.22 A similar phenomen- on has also been described for cells from patients with various forms of glycolipidosis.23 Since these cells can also be cultured in vitro and so far no toxic effects of lysobisphosphatidic acid have been described, it seems unlikely that this compound reduces the fitness of the modified cell. It should also be taken into account that ribozymes will not allow to generate a complete reduction of the murine M6PR300, such that putative adverse effects in ribozyme-modified cells can be expected to be milder than in completely deficient cells. In summary, the ribozyme-induced reduction of M6PR biosynthesis may prove applicable to enhance the bioavai- Figure 7 Human arylsulfatase A activity in cells and media of MEF- mpr46 cells. Mouse embryonic fibroblasts were transiently transfected with lablity of lysosomal enzymes in the treatment of lysosomal a plasmid encoding the hASA cDNA and plasmids containing Rz B, Rz D storage diseases, in particular for sulfatase deficiencies. and Rz Co. At 48 h after transfection, cells were harvested, hASA was immunoprecipitated with a monoclonal antibody from cell and the media and the enzyme activity in the immunoprecipitates was measured. Materials and methods Columns give the average of four independent experiments. Bars indicate standard deviation. Rz Co is a control ribozyme targeting an irrelevant Ribozyme and substrate construction mRNA. Differences between Rz Co and Rz B (Po0.0015), Rz Co and Rz Mouse wild-type M6PR300 cDNA (kindly provided by P D(Po0.014) are statistically (Student’s t-test) significant. Lobel) was subcloned into the expression vector pBEH (Artelt), resulting in the plasmid pBEH-M6PR300.24–26 ribozymes reduce the biosynthesis of the murine M6PR For the construction of plasmids that encode ribozyme protein to 37 and 62% of normal in the BHK cell (Figures cassettes, overlapping sense and antisense oligonucleo- 5 and 6). tides encompassing the catalytic domain were designed. 0 Thus, these results clearly demonstrate that reduction NsiI and XhoI restriction sites were included at the 3 - 0 of M6PR biosynthesis by a ribozyme-mediated approach and 5 -end, respectively. All of the ribozyme cassettes is possible. This approach is particularly suitable for the have a size of 44 bp, except of the Rz E, which has a size treatment of diseases because of the deficiency of of 52 bp (Figure 8). sulfatases, like mucopolysaccharidosis VI, Morquio dis- After annealing, the double-stranded oligonucleotides 0 0 ease, metachromatic leukodystrophy and others. Proper were digested with XhoI(5-end) and NsiI(3-end) and function of sulfatases depends on a sulfatase-specific cloned into the SalI and PstI sites of pGval plasmid post-translational modification.19 This modification, (kindly provided by A Lieber and M Strauss). This vector which takes place in the endoplasmatic reticulum, has a T7 and Pol III promoters allowing for both in vitro converts a cysteine residue in the active center into a and in vivo transcription.13 formylglycine residue.19 In the case of overexpression, one sulfatase competes with the modification of others, In vitro transcription reactions and 50-labelling of RNA indicating that the post-translational modification is the Transcription reactions of linearized ribozyme plasmids step limiting the amount of active sulfatases that can (with HindIII) were carried out with T7 RNA polymerase be expressed by a cell.20 In these cases, secretion by as described.27 transplanted cells needs to be enhanced by an approach other than massive overexpression, namely interference with intracellular sorting of lysosomal enzymes in the transplanted cell. Using arylsulfatase A as an indicator enzyme we could indeed demonstrate that ribozyme- induced reduction of M6PR300 synthesis enhances the secretion of this enzyme by a factor of about 1.7. The reduction of M6PR300 will not only enhance the secretion of an overexpressed enzyme, but also the secretion of all lysosomal enzymes synthesized by the transplanted cell. So far there have been no reports that overexpression or the enhanced secretion of lysosomal enzymes have any adverse side effects. In particular for arylsulfatase A, this has been excluded.21 Thus, the combination of genetic modification and ribozyme- induced receptor reduction seems to be a suitable approach for lysosomal storage diseases. Figure 8 Sequences of the ribozyme cassettes. For construction of plasmids It can also be expected that the downregulation of the that encode ribozyme cassettes, overlapping sense and antisense oligonu- M6PR300 does not affect the life cycle of the modified cleotides encompassing the catalytic domain were designed. The bold cell. Since M6PR300-deficient cells can be grown in tissue nucleotides indicate the antisense regions of ribozymes (Rz A to Rz G), culture without special requirements, severe side effects which flank the target sites at the 50- and 30-end, respectively.

Gene Therapy Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1573 In order to prepare [g-32P] 50-end-labelled transcripts Northern blot analysis of M6PR300 mRNA used as substrates for the in vitro cleavage reactions, Total RNA (6 mg) isolated from transfected cells we used a protocol described previously.28 Briefly, to and 40 mg isolated from NIH/3T3 cells were separated transcribe RNA with a free 50-OH group the initiator in a 1% denaturing agarose gel. After the transfer dinucleotide (ApG) was included into the reaction. After of RNA to a Hybond-N+ membrane (Amersham completion of in vitro transcription T4 polynucleotide Pharmacia), the membrane was hybridized with kinase is used to transfer the terminal phosphate group an [a-32P]dCTP labelled cDNA probe encompassing from [g-32P]ATP to a free 50-OH of the RNA. nucleotides 450–1900 of the M6PR300 cDNA. 50-[g-32P]RNAs were prepared by incubating 4 pmol of Labelling and hybridization conditions have been 50-OH transcripts in the presence of 50 mCi [g-32P]ATP, described.32 and 1 U of T4 polynucleotide kinase in 70 mM Tris/HCl A glyceraldehyde 3-phosphate dehydrogenase (pH 7.5), 15 mM MgCl2,7mM DTT at 371C for 30 min. (GAPDH) cDNA probe was used as reference for The labelling of the RNA was terminated by ethanol quantitation for M6PR300 mRNA. Autoradiographs precipitation.29 were performed at different exposure times with hyper Transcription reactions of the M6PR300 cDNA frag- films (Kodak) at À801C. ments were initiated in the presence of 100 mM each NTP (ATP, UTP, CTP and ApG), 2 mg of linearized plasmid template and 20 U T7 RNA polymerase. After incubating Northern Blot analysis of ribozyme mRNA the reaction at 371C for 10 min, 100 mM GTP was added Total RNA (8 mg) isolated from transfected cells were and the reaction proceeded for another 2 h. loaded on a 2% denaturing agarose gel and transferred to a Hybond-N+ membrane. For the detection of ribozyme mRNA, an oligonucleotide (10 pmol) containing the In vitro cleavage reaction 0 Single-turnover cleavage reactions of hammerhead ribo- catalytic domain of the ribozyme cassette (5 -CTGAT GAGTCCGTGAGGACGAAA-30) was labelled with zymes were performed by incubating 0.66 pmol 50- g 32 [32P]M6PR300 RNA fragments, 1.32 pmol of in vitro [ - P]ATP. transcribed ribozymes in 50 mM Tris/HCl (pH 7.5). All reactions were performed at 421C. The mixtures of substrate, ribozyme and buffer were heated to 421C, Metabolic labelling and immunoprecipitation finally 10 mM MgCl was added. The cleavage reactions Transfected cells were incubated on 6-cm dishes in 2 methionine-free DMEM medium (Invitrogens) for 1 h, were terminated by ethanol precipitation. Fragments 35 generated in the reactions were separated on an 8% and then labelled for 16 h with 50 mCi [ S]methionine polyacrylamide/8 M urea gel and visualized by auto- (Amersham Pharmacia) in a medium containing 4% radiography. dialyzed FCS. M6PR300 was immunoprecipitated from extracts of cells as described for M6PR46 with antisera against rat M6PR300.33 Transfections The antiserum crossreacts with mouse M6PR300 and The hamster kidney cell line BHK-21 and the murine was kindly provided by K von Figura. fibroblast cell line NIH/3T3 were maintained in Dulbec- The quantitation of Northern blots and immunopreci- co’s modified Eagle medium (DMEM) with 4.5 g/l pitations were performed using a phosphoimager BAS glucose, 3.7 g/l NaHCO3, 10% (v/v) heat-inactivated 1000 (Fuji). fetal calf serum, 100 U/ml penicillin, 100 mg/ml strep- tomycin and 2 mML-glutamine (Invitrogens)ina humidified atmosphere containing 5% CO2/95% air at Arylsulfatase A activity determination 371C. MEF-mpr46 cells were lysed 48 h after transfection. Plasmids were purified by CsCl gradient centrifuga- Human ASA was immunoprecipitated from the media tion.30 For transient transfections, 2 Â 106 BHK-21 cells and the cell homogenates with a monoclonal antibody were seeded on 6-cm dishes and grown overnight. Cells against hASA.15 were transfected with circular plasmids of pBEH- Human ASA activity in medium and homogenate was M6PR300 (50 ng/dish), pGvalRz (2 mg/dish) and se- measured with 200 ml substrate solution consisting creted alkaline phosphatase (SEAP) (100 ng/dish) using 10 mM p-nitrocatecholsulfate in 170 mM NaCl, 0.5 M the Lipofectamin Plust protocol as provided by the sodium acetate (pH 5.0), 0.3% Triton X-100 and 1 mg/ manufacturer (Invitrogens). ml BSA. Reaction was performed at 371C for 90 min and stopped with 1 M NaOH. Absorption was read at 515 nm. Extraction of total cellular RNA Transfected BHK-21 cells were lysed 48 h post-transfection by the addition of 0.6 ml/dish of lysis buffer containing 200 mM sodium acetate (pH 4.0), 4 M guani- Acknowledgements dium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium N-lauryl-sarcosyl and 0.1 M b-mercap- We thank P Lobel for kindly providing the wild-type toethanol.31 The lysate was extracted once with phenol cDNA of murine M6PR300, A Lieber for pGval plasmid, followed by one extraction with chloroform. Total R Pohlmann for the MEF-mpr46 cell line and K von cellular RNA was obtained after precipitation with Figura for providing rat antisera against M6PR300 ethanol. The RNA pellets were washed once with 70% protein. This work was supported by the Bundesminister- ethanol and resolved in DEPC-treated water. ium fu¨r Bildung und Forschung.

Gene Therapy Hammerhead ribozymes reduce synthesis of M6PR300 A Yaghootfam and V Gieselmann 1574 References 18 Kato Y et al. Relationship between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular 1 Kornfeld S. Structure and function of the mannose 6-phosphate/ localization in mammalian cells. J Biol Chem 2001; 276: 15378– insulinlike growth factor II receptors. Annu Rev Biochem 1992; 61: 15385. 307–330. 19 Schmidt B, Selmer T, Ingendoh A, von Figura K. A novel amino 2 Hoflack B, Kornfeld S. Purification and characterization of a acid modification in sulfatases that is defective in multiple cation-dependent mannose 6-phosphate receptor from murine sulfatase deficiency. Cell 1995; 82: 271–278. P388D1 macrophages and bovine liver. J Biol Chem 1985; 260: 20 Brooks DA et al. Two site-directed mutations abrogate enzyme 12008–12014. activity but have different effects on the conformation and 3 Sleat DE, Lobel P. Ligand binding specificities of the two cellular content of the N-acetylgalactosamine 4-sulphatase mannose 6-phosphate receptors. J Biol Chem 1997; 272: 731–738. protein. Biochem J 1995; 307: 457–463. 4 Medin JA et al. Correction in trans for Fabry disease: expression, 21 Ohashi T, Matalon R, Barranger JA, Eto Y. Overexpression of secretion, and uptake of a-galactosidase A in patient-derived arylsulfatase A gene in fibroblasts from metachromatic leuko- cells driven by a high-titer recombinant retroviral vector. Proc dystrophy patients does not induce a new phenotype. Gene Natl Acad Sci USA 1996; 93: 7917–7922. Therapy 1995; 2: 363–368. 5 Kasper D, Dittmer F, von Figura K, Pohlmann R. Neither type of 22 Reaves BJ et al. Loss of cation-independent mannose 6- mannose 6-phosphate receptor is sufficient for targeting of phosphate receptor expression promotes the accumulation of lysosomal enzymes along intracellular routes. J Cell Biol 1996; lysobisphosphatidic acid in multilamellar bodies. J Cell Sci 2000; 134: 615–623. 113: 4099–4108. 6 Uhlenbeck OC. A small catalytic oligoribonucleotide. Nature 23 Puri V et al. Cholestrol modulates membrane traffic along the 1987; 328: 596–600. endocytic pathway in sphingolipid-storage diseases. Nat Cell 7 Scott WG, Klug A. Ribozymes: structure and mechanism in RNA Biol 1999; 1: 386–388. catalysis. Trends Biochem Sci 1996; 21: 220–224. 24 Lobel P et al. Cloning of the bovine 215-kDa cation-independent 8 Haseloff J, Gerlach WL. Simple RNA enzymes with new and mannose 6-phosphate receptor. Proc Natl Acad Sci USA 1987; 84: highly specific endoribonuclease activities. Nature 1988; 334: 2233–2237. 585–591. 25 Lobel P, Dahms NM, Kornfeld S. Cloning and sequence analysis 9 Forster AC, Symons RH. Self-cleavage of plus and minus RNAs of the cation-independent mannose 6-phosphate receptor. J Biol of a virusoid and a structural model for the active sites. Cell 1987; Chem 1988; 263: 2563–2570. 49: 211–220. 26 Artelt P et al. Vectors for efficient expression in mammalian 10 Zuker M. On finding all suboptimal foldings of an RNA fibroblastoid, myeloid and lymphoid cells via transfection or molecule. Science 1989; 244: 48–52. infection. Gene 1988; 68: 213–219. 11 Zuker M. Computer prediction of RNA structure. Methods 27 Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC. Enzymol 1989; 180: 262–288. Oligoribonucleotide synthesis using T7 RNA polymerase 12 Ludwig J, Blaschke M, Sproat BS. Extending the cleavage rules and synthetic DNA templates. Nucleic Acids Res 1987;15: for the hammerhead ribozyme mutating adenosine15.1 to 8783–8798. inosine15.1 changes the cleavage site specificity from 28 Gaur RK, Krupp G. Preparation of templates of ribozymes and N16.2U16.1H17 to N16.2C16.1H17. Nucleic Acids Res 1998; 26: substrates. Methods Mol Biol 1997; 74: 69–78. 2279–2285. 29 Krupp G. Enzymatic synthesis and characterization of un- 13 Lieber A, Strauss M. Selection of efficient cleavage sites in target modified ribozymes and substrates. Methods Mol Biol 1997; 74: RNAs by using a ribozyme expression library. Mol Cell Biol 1995; 79–89. 15: 540–551. 30 Heilig JS, Elbing KL, Brent R. Large-scale preparation of plasmid 14 Pohlmann R, Wendland M, Boeker C, von Figura K. The two DNA. In: Ausubel FM et al (eds). Current Protocols in Molecular mannose 6-phosphate receptors transport distinct complements Biology. John Wiley & Sons: New York, 1997, pp 1.7.1–1.7.16. of lysosomal proteins. J Biol Chem 1995; 270: 27311–27318. 31 Chomczynski P, Sacchi N. Single-step method of RNA isolation 15 Sommerlade HJ, Hille-Rehfeld A, von Figura K, Gieselmann V. by acid guanidinium thiocyanate–phenol–chloroform extraction. Four monoclonal antibodies inhibit the recognition of arylsul- Anal Biochem 1987; 162: 156–159. phatase A by the lysosomal enzyme phosphotransferase. 32 Brown T, Mackey K. Analysis of RNA by Northern and slot blot Biochem J 1994; 297: 123–130. hybridization. In: Ausubel FM et al (eds). Current Protocols in 16 Wang ZQ, Fung MR, Barlow DP, Wagner EF. Regulation of Molecular Biology. John Wiley & Sons: New York, 1997, pp 4.9.1– embryonic growth and lysosomal targeting by the imprinted 4.9.16. Igf2/Mpr gene. Nature 1994; 1: 464–467. 33 Punnonen EL, Wilke T, von Figura K, Hille-Rehfeld A. The 17 Ludwig T et al. Differential sorting of lysosomal enzymes in oligomeric state of 46-kDa mannose 6-phosphate receptor does mannose 6-phosphate receptor-deficient fibroblasts. EMBO J not change upon intracellular recycling and binding of ligands. 1994; 13: 3430–3437. Eur J Biochem 1996; 237: 809–818.

Gene Therapy