Gene Therapy (1997) 4, 736–743  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Intracellular application of hairpin against hepatitis B

PJ Welch, R Tritz, S Yei, J Barber and M Yu Immusol, Inc, 3050 Science Park Road, San Diego, CA 92121, USA

HBV, a partially double-stranded DNA virus, replicates modifications were performed on these two Rzs, with the through a pregenomic RNA (pgRNA) intermediate, which goal of increasing catalytic efficiency both and in provides a therapeutic opportunity for a novel antiviral cells. To determine the Rz activities in liver cells, the therapy based on ribozyme RNA cleavage. Three hairpin cDNAs for each of the anti-HBV Rzs (and their catalytically (Rzs) were designed which have the potential disabled negative controls) were cloned into retroviral vec- to disrupt HBV replication by targeting the pgRNA as well tors. Unmodified ribozymes co-expressed with HBV in as specific mRNAs encoding the HBV surface antigen human liver Huh7 cells reduced the level of viral particle (HBsAg), the and the X . The ability of production by up to 66% based on the endogenous poly- each ribozyme to cleave approximately 0.3 kb HBV merase assay, while the structurally modified ribozymes subgenomic RNA fragments was tested in vitro. Two of the inhibited HBV production up to 83%. These encouraging three Rzs tested (BR1 and BR3) were capable of cleaving results indicate the feasibility of ribozyme-mediated gene their respective RNA substrates, while their catalytically therapy for the treatment of HBV . disabled mutated counterpart Rzs were not. Structural

Keywords: -independent ; endogenous polymerase assay; ribozyme kinetics; RNA cleavage; antiviral

Introduction problems associated with the direct injection of antisense DNA. Stable expression of the anti-HBV ribozymes in Hepatitis B is a major worldwide health problem. liver cells could theoretically lead to lifelong intracellular Although a is currently available for hepatitis B immunity against HBV. virus (HBV), it is estimated that no less than two billion HBV undergoes replication through reverse transcrip- people are infected globally and 300 million are chron- tion of a pregenomic RNA intermediate5,6 and is thus 1,2 ically infected carriers. Approximately 10% of infected somewhat analogous to HIV and other . This adults will develop a chronic, -long often offers an excellent rationale for the use of ribozymes as 1,2 with devastating consequences. About 2 million deaths antiviral agents for HBV based on the successful preclini- occur each year as a direct result of chronic HBV infection cal studies of ribozymes against HIV infections (for due to either cirrhosis or primary liver cancer. Presently review see Refs 7 and 8). An anti-HBV ribozyme can the only partially effective treatment for hepatitis B is potentially be multifunctional, targeting both the pregen- ␣ interferon- , which is effective in only less than half of omic RNA as well as the viral mRNAs (see Figure 1a). all patients. In addition, by targeting the ribozymes to small highly One recently investigated approach to the treatment of conserved regions of the viral , the possibility of HBV infection has been the utilization of antisense DNA generating escape mutants can be greatly reduced. 3,4 directed against HBV. Unfortunately, In this report, we have designed several ‘hairpin’ the simple antisense approach is subject to several limi- ribozymes against different conserved regions of the tations when used in vivo, including toxicity, degradation HBV genome and have demonstrated their ability to of the oligos, lack of tissue specificity and transient avail- cleave HBV RNA in vitro and to reduce the level of HBV ability. An alternative to antisense technologies is the use expression and viral production in . Hairpin of ribozymes directed against HBV RNA. Ribozymes ribozymes are small catalytic RNA molecules derived may be a more effective tool since they possess both anti- from the negative strand RNA of the tobacco sense and RNA cleavage activities. The enzymatic nature ringspot virus (see Figure 1b for the structures). Two of ribozymes may facilitate effectiveness even at low lev- hairpin ribozymes, BR1 and BR3, targeting the viral els of expression. In a setting, ribozyme envelope (S), polymerase (P) and X (Figure 1a) genes can be delivered via various delivery can efficiently cleave HBV RNA in vitro and are shown systems and expressed constitutively within the target to reduce the production of hepatitis B viral particles in cells, thereby overcoming the toxicity and degradation cultured human liver cells, when produced intracellu- larly by ribozyme and expression. Some regions of a can be modified for its opti- Correspondence: J Barber mized cleavage activity for each individual ribozyme.9–13 Received 13 September 1996; accepted 10 March 1997 The length of loop 3, for example, is one of Hairpin ribozymes as gene therapy for hepatitis B PJ Welch et al 737

Figure 1 Schematic representation of HBV and ribozyme expression vectors, and wild-type and tetraloop hairpin ribozyme structures. (a) HBV: ‘Over- length’ HBV as cloned into pGem7 (see Materials and methods). The pregenomic RNA (pgRNA) start site and common polyA signal are indicated. All RNA transcripts are driven by authentic HBV promoters. Cleavage sites for hairpin ribozymes BR1, BR2 and BR3 are indicated by downward arrows. HBV-encoded proteins X, C (core), S (envelope) and P (polymerase) are shown as boxes below. Ribozyme: Retroviral vector, pLNT-Rz, used for ribozyme expression in mammalian cells. Ribozymes are cloned under the control of the human tRNAval promoter (see Materials and methods). LTR: Moloney long terminal repeat. (b) The hairpin ribozyme consists of a 50 to 54 nucleotide RNA molecule (shaded, in uppercase letters) which binds and cleaves an RNA substrate (lowercase letters). The catalytic RNA folds into a two-dimensional structure that resembles a hairpin, consisting of two helical domains (helix 3 and 4) and 3 loops (loop 2, 3 and 4). Two additional helices, helix 1 and 2, form between the ribozyme and its substrate. Recognition of the substrate by the ribozyme is via Watson–Crick base pairing (where N or n = any nucleotide; b = C, G or U; and B = the nucleotide complementary to b). The length of helix 2 is fixed at 4 basepairs and the length of helix 1 typically varies from 6 to 10 basepairs, with 8 being experimentally optimal for most ribozymes. The substrate must contain a GUC in loop 5 for maximal activity, and cleavage occurs immediately 5′ of the G as indicated by an arrow. The catalytic, but not substrate binding, activity of the ribozyme can be disabled by mutating the AAA in loop 2 to CGU, as shown.9,16 The tetraloop hairpin ribozyme differs from the hairpin ribozyme by replacement of loop 3 and helix 4 with the RNA-stabilizing tetraloop configuration, originally observed in the 16S rRNA.11 In addition to increasing RNA stability, tetraloop additions have been shown to alter the catalytic properties of hairpin ribozymes.13 those regions (Figure 1b).12,13 Modification in the loop 3 ribozyme target site was chosen due to high sequence region of BR1 and BR3 resulted in improved RNA cleav- conservation between different HBV strains. To deter- age in vitro and antiviral properties in tissue culture. mine if these ribozymes were capable of cleaving their Together, our data demonstrate the value of hairpin respective targets, fragments of HBV RNA were used as ribozymes as potential therapeutic agents for treatment substrates for in vitro cleavage reactions (Figure 2, see of HBV infection. Materials and methods for reaction conditions). BR1 and BR3 ribozymes cleaved 35 and 15%, respectively, of their Results substrate in 60 min, without any prior treatment or denaturation of the substrate HBV RNAs (Figure 2, Specific cleavage of HBV RNA in vitro by hairpin lanes 2 and 6). This level of in vitro cleavage is significant ribozymes compared to previous reports.14,15 As controls, the dis- Three hairpin ribozymes were designed to cleave HBV abled forms of BR1 and BR3 (termed dBR1 and dBR3) RNA, each targeting both pgRNA and mRNA (see Figure which inactivate the RNA cleavage activity without 1 and Materials and methods). Ribozyme BR1 targets affecting its substrate binding9,16 were generated by intro- HBV RNA at nucleotide position 1698 (pgRNA start site ducing a three nucleotide mutation in loop 2 of the hair- = 1), which is located within the HBV surface antigen pin ribozyme (Figure 1b). Neither dBR1 nor dBR3 dem- (HBsAg) and polymerase (P) coding regions (see Figure onstrated any RNA cleavage activity (Figure 2, lanes 4 1a). Ribozymes BR2 and BR3 target the HBV regulatory and 8), indicating that the cleavage by BR1 and BR3 protein X and polymerase coding regions at requires ribozyme enzymatic activity. Ribozyme BR2, 2816 and 2933, respectively (see Figure 1a). Each however, showed no detectable in vitro cleavage activity Hairpin ribozymes as gene therapy for hepatitis B PJ Welch et al 738

Figure 2 In vitro cleavage of HBV RNA by BR1 and BR3 ribozymes. Ribozymes BR1 and BR3, as well as their disabled counterparts dBR1 and dBR3 were tested. Ribozymes and radiolabeled HBV substrates were synthesized by in vitro transcription (see Materials and methods). HBV substrates are as follows: BR1 substrate = HBV nucleotides 1629–1919 (pgRNA start site is nucleotide 1), generated from EcoNI-linearized pGem-HBV-X/B; BR3 substrate = HBV nucleotides 2782–3023, generated from EcoNI-linearized pGem-HBV-B/E. Cleavage reactions contain 40 nm ribozyme and 200 nm substrate (ribozyme:substrate = 1:5) and were incu- bated for 0 or 60 min at 37°C. Cleavage products were resolved on 5% polyacrylamide/7 m urea gels. Uncleaved substrate and the 5′ and 3′ cleav- age products are indicated. Refer to Figure 1 for the locations of the tar- geted genes (S, X and P).

after 60 min (data not shown). Since both the BR2 Figure 3 In vitro kinetic studies for (a) BR1 and tetraloop BR1; and (b) ribozyme and its substrate target site were verified by BR3 and tetraloop BR3. Shown are typical in vitro cleavage reaction time- m DNA sequencing, the lack of cleavage activity could be course experiments (see Materials and methods), including 5.2 n ribozyme and 72 nm substrate. The reactions were assembled on ice and due to the presence of RNA secondary structure within then incubated at 37°C and aliquots were removed every 30 min from 0 the region targeted by BR2, as is suggested by computer- to 120 min, as indicated. During assembly of the reactions at 4°C, a small assisted RNA folding analysis. Indeed, substrate second- amount of cleavage activity is occasionally observed prior to addition of ary structure has been previously shown to prevent stop buffer, as seen for BR1 tetraloop at 0 min. Cleavage products were ribozyme binding and cleavage in studies with hepatitis quantified by phosphorimager analyses (Molecular Dynamics) and data C.17 Conversely, the observed BR1 and BR3 efficient in from three experiments are plotted. Km and kcat were determined in two vitro cleavage activities on these long substrates indicate separate experiments as described in Materials and methods. that the target sites for these two ribozymes are probably located in relatively open regions in the RNA, making tetraloop enhanced cleavage activity (Figure 3). To deter- these two sites worthy of further evaluation. mine if the tetraloop affected Km, kcat or both, kinetic stud- ies were performed by holding the ribozyme concen- Kinetic studies of BR1, BR3 and their tetraloop variants tration constant while varying the amount of substrate, Modifications can be made to several regions including and the cleavage data were plotted in an Eadie–Hofstee loop 3, helix 4 and helix 1 (Figure 1b) of a specific hairpin plot from which the value of Km and kcat can be determ- ribozyme that may affect the Km, the rate of (kcat) ined (Figure 3). For BR1 and BR3, the replacement of loop and the catalytic efficiency (kcat/Km). One modification 3 with this tetraloop increased the catalytic efficiency that has previously been shown to increase the enzymatic (kcat/Km) three- or 10-fold, respectively, over that of the activity of a ribozyme directed against HIV, is to replace unmodified ribozyme. In both cases, addition of a tetra- the loop 3 region of the hairpin ribozyme with an RNA- loop mainly enhanced kcat, while having only a modest 13 stabilizing ‘tetraloop’ motif. For this reason, tetraloops to insignificant impact on the Km. were used to replace loop 3 of BR1 and BR3 and the resulting in vitro activities were compared with the proto- Effects of anti-HBV ribozymes in liver culture type BR1 and BR3 (Figure 3). Cleavage time-course The effectiveness of anti-HBV ribozymes as therapeutic experiments (see Materials and methods) with each agents in vivo will depend on their ability to function in ribozyme indicated that in both cases the addition of a a complex cellular environment. To that end, ribozyme Hairpin ribozymes as gene therapy for hepatitis B PJ Welch et al 739 genes encoding BR1 and BR3, their tetraloop variants (BR1-TL and BR3-TL) and their disabled counterparts (dBR1 and dBR3) were cloned into a retroviral vector pLNT, as one of the possible gene therapy delivery vehicles, under the control of the human tRNAval pol III promoter17,18 (see Figure 1a). This retroviral vector was chosen due to its successful application in delivering ribozymes against HIV.13 As the first step of testing the principle of ribozyme gene therapy for the treatment of HBV infection, we employed transient DNA co-transfec- tion to facilitate the rapid test of intracellular ribozyme efficacy. Following individual into the human hepatocellular carcinoma cell line Huh7, each ribozyme was expressed at similar levels as determined by RNase protection (Figure 4), considering that the dif- ferent sizes of each detected ribozyme RNA were due to the specific design of the probe (Materials and methods and Figure 4b). To date, use of infectious HBV serum productively to infect human liver cells in culture has proven problem- atic. However, it has been demonstrated that of greater than full-length HBV into liver cell lines will result in HBV protein expression, replication and production of infectious HBV virions.19–21 These ‘overlength’ HBV DNA constructs mimic the circularity of the HBV genome by duplicating the HBV sequences containing the pgRNA promoter and polyA signal on both ends of a linear genome (see Figure 1a). Quantifi- cation of HBV virus particles produced in tissue culture is best achieved using the endogenous polymerase assay (EPA).20,21 This assay relies on the fact that the packaged HBV genome is only partially double stranded and that the HBV-specific polymerase is also packaged in each virion. Addition of radiolabeled nucleotide triphosphates to purified HBV particles allows the endogenous poly- merase to ‘fill-in’ the partially single-stranded region, thus allowing detection of HBV replication and virus pro- duction (see Materials and methods). To evaluate the ability of each anti-HBV ribozyme to inhibit HBV replication and production, each pLNT-Rz was co-transfected with the ‘overlength’ HBV into Huh7 cells and virus production was monitored by EPA. In a typical co-transfection experiment, HBV particles sub- jected to EPA produce DNA bands representing the fully repaired relaxed circular (RC) form as well as faster Figure 4 Ribozyme expression in vivo verified by RNase protection. (a) migrating linear forms (Figure 5a). When overlength Huh7 cells were transfected with each of the ribozymes indicated. Twenty- five micrograms of total cellular RNA was hybridized to 128 nucleotide HBV was co-transfected with BR1 or BR3, HBV pro- long radiolabeled probes, of equal specific activity, antisense to either BR1 duction was reduced by 66 and 50%, respectively (Figure or BR3, as indicated (see Materials and methods). Following RNase diges- 5). Co-transfection with dBR1, however, had no inhibi- tion, reactions were electrophoresed on an 8% polyacrylamide/7 m urea tory effect on HBV replication (lane 7). The lack of an gel and quantified by phosphorimager analysis. Due to the three nucleotide difference between the active and the disabled form of each ribozyme, the effect by dBR1 strongly suggests that the effects of BR1 + are due to intracellular RNA cleavage and not simply an expected protected fragments are 64 nt for BR1 and BR3 and 32 nt 29 nt for dBR1 and dBR3 (see b for schematic representation of protected ‘antisense’ effect, since dBR1 maintains a perfect comp- fragments). lementary target binding antisense sequence, but is cata- lytically inactive. Furthermore, co-transfection with increasing molar amounts of BR1, from 1:5 to 1:20 infected livers, especially with the potent tetraloop vari- resulted in an increasing anti-HBV effect, indicating a ants of each ribozyme. dose–response to the presence of more ribozyme in the cell (data not shown). Consistent with the in vitro obser- Discussion vations, addition of the tetraloop to either BR1 or BR3 enhanced each of their activities in liver cells. When the As the first step towards the treatment of HBV infection tetraloop was added to BR1 and BR3, HBV production by ribozyme gene therapy, two hairpin ribozymes, BR1 was reduced 83 and 82%, respectively (Figure 5). and BR3, were generated and shown to be capable of Together, these data indicate that both BR1 and BR3 HBV cleaving HBV RNA in vitro. The in vitro catalytic activities ribozymes could potentially inhibit HBV replication in of these two ribozymes were further enhanced by as Hairpin ribozymes as gene therapy for hepatitis B PJ Welch et al 740 have also described hammerhead ribozymes capable of cleaving HBV RNA in vitro.14,15 To the best of our know- ledge, however, this is the first demonstration of ribozyme-mediated reduction of HBV production in a tissue culture system. Previous work with hairpin ribozymes against HIV have revealed that adding a tetraloop to the loop 3 region can enhance the catalytic efficiency (kcat/Km)ofa ribozyme.13 It is noteworthy that the addition of a tetra- loop to either BR1 or BR3 increases the catalytic efficiency by increasing kcat, without significantly altering Km. Based on our studies with HIV, the in vitro catalytic efficiency of each ribozyme usually serves as a good indicator of its catalytic efficiency in cells (unpublished data). Indeed, addition of tetraloops to both BR1 and BR3 enhanced their anti-HBV activities in liver cells. While the increase in their intracellular activities was significant, it did not fully represent the increase seen in vitro. Obviously other currently unknown factors influence ribozyme activity in vivo. Nevertheless, the tetraloop modifications for both resulted in a more potent ribozyme. Other anti-HBV strategies that have been reported pre- viously include treatment with various interferons,25,26 analogs25,27–30 and antisense molecules.3,4,28,31 Drug treatment for HBV infection, such as interferon or nucleoside analogs, can result in 50 to 90% inhibition of in and have shown efficacy in the clinic.25–30,32 However, these treatments yield only temporary efficacy, require continued administration and can have debilitating side-effects. Antisense treatment also shows efficacy in cell culture, and inhibition of HBV replication ranging between 50 and 90% has been demon- strated, depending on the target sequence.4,28,31 However, as a potential therapy, it is also subject to several limi- Figure 5 Expression of anti-HBV ribozymes reduces Hepatitis B virus tations not shared by ribozyme gene therapy, including production in human liver-derived cell culture. Huh7 cells were co- the requirement for delivery of stoichiometric amounts, transfected with overlength HBV (see Figure 1a) and pLNT-Rz for each in vivo toxicity and transient availability after adminis- ribozyme (unmodified ribozyme or tetraloop ribozyme). The DNA molar tration. We have shown here that our tetraloop ratio for HBV:ribozyme was 1:10. Three days after transfection, cells were ribozymes can inhibit HBV replication up to 83% in cell lysed and HBV particles were immunoprecipitated for the endogenous polymerase assay (see Materials and methods). (a) Shown is a typical culture, which is similar to the therapeutic levels endogenous polymerase assay with radiolabeled HBV DNA resolved by observed for other HBV inhibitors. Ribozyme gene ther- agarose , with relaxed circular (R.C.) and linear forms apy, however, has the added advantage that delivery and indicated. (b) Quantification was performed by phosphorimager analysis stable expression of one or more effective ribozyme genes (Molecular Dynamics) and data averaged from two to four independent could obviate the need for repeat administration and may experiments were plotted. Percentages listed below graph bars indicate offer lifelong protection. percentage reduction of HBV production by each ribozyme, relative to HBV transfections without ribozyme (first bar on graph). Our ongoing studies also include investigation of other hairpin ribozyme sites within the HBV pgRNA, as well as co-expression of multiple ribozymes (such as BR1 and much as 10-fold by replacing the loop 3 region of the BR3 together) or ribozymes in combination with other ribozymes with a tetraloop motif. Expression of ribozyme anti-HBV strategies. Naturally, selection of new ribozyme BR1 or BR3, but not their disabled counterparts, in Huh7 sites will focus on highly conserved regions of the HBV cells resulted in up to 66% decrease in HBV particle pro- genome and the simultaneous in vivo expression of sev- duction and their tetraloop variants demonstrated even eral different anti-HBV ribozymes will not only allow higher antiviral activity, reducing HBV production by up cleavage of all currently known strains of HBV but will to 83%. These findings support the conclusion that these also prevent the generation of resistant mutants. ribozymes cleave HBV RNA under physiological con- One of the major challenges associated with gene ther- ditions within the complex cellular environment. These apy is the gene delivery system. Currently, murine-based encouraging in vitro and intracellular results support the amphotropic retroviral vectors are widely used in gene feasibility of ribozyme-mediated gene therapy for human therapy, as they provide efficient of a wide hepatitis B. spectrum of cell types and result in stable integration and The genetic application of ribozymes as antiviral expression of genes delivered to the host cell. Such retro- agents is not new, and their utility is quickly becoming viral vectors were also used in this study as an initial test widely appreciated. Indeed, both hairpin and hammer- of principle for gene therapy for HBV infections. For head ribozymes have been used successfully against future studies, both adenovirus and adeno-associated HIV-18,22 and hepatitis C virus.17,23,24 Previous reports virus (AAV) vectors will be tested as vectors for Hairpin ribozymes as gene therapy for hepatitis B PJ Welch et al 741 ribozyme gene delivery. Unlike retroviral vectors, AAV ture Collection (ATCC, Rockville, MD, USA). can stably transduce cells that have not been stimulated for HBV substrate production were constructed by clon- to divide. This property, and the observation that injected ing two HBV fragments into pGem7Z as follows. pGem- AAV is hepatotropic,33–35 make AAV vectors an attractive HBV-X/B was created by ligating the XbaI/BamHI frag- gene delivery system for the treatment of HBV infection. ment of HBV (nucleotides 1629–2782, pgRNA start site = Adenoviral vectors also have several advantages. Firstly, 1) into pGem7Z digested with XbaI and BamHI. pGem- and most importantly, when infused into the blood- HBV-B/E was created by ligating the BamHI/EcoRI frag- stream, adenoviral vectors are also hepatotropic.36 Sec- ment of HBV (nucleotides 2782–1380, pgRNA start site = ondly, they can be produced in very large amounts com- 1) into pGem7Z digested with BamHI and EcoRI. Both pared with retroviral and AAV vectors (with titers plasmids were linearized with EcoRI prior to in vitro tran- approaching 1 × 1011/ml, as compared with 1 × 107/ml scription. HBV substrates were then synthesized in the for retroviral and AAV vectors).37 Therefore, adenovirus presence of alpha-32P-UTP, DNase treated and gel pur- and AAV are currently being investigated as potential ified, according to the manufacturer (Promega). The in gene delivery systems for our hairpin ribozyme gene vitro cleavage reactions contained 40 nm ribozyme and m m m therapy for the treatment of HBV infection. 200 n substrate in 12 m MgCl2/2 m spermidine/40 mm Tris-HCl, pH 7.5, were incubated at 37°C for 60 min and were terminated by the addition of 7 m urea gel load- Materials and methods ing buffer. Products of the cleavage reactions were resolved by electrophoresis on 5% polyacrylamide/7 m Ribozyme construction urea gels and analyzed by autoradiography. For each ribozyme constructed, a specific was synthesized (Retrogen, San Diego, CA, USA) that Ribozyme kinetic studies shared 16 bases of complementary sequence (shown in bold below) to a ‘universal’ hairpin ribozyme oligonucle- Preparation of ribozymes and substrates: Ribozyme and otide (wild-type, CC; tetraloop, CC-TL; disabled, dCC) substrate synthesis was achieved by a new method of (see sequences below). To create a clonable, double- plasmid-independent in vitro transcription. Oligonucleo- stranded DNA containing the complete ribozyme tides were synthesized (Retrogen) that contained the T7 sequence, the specific ribozyme oligonucleotide is RNA polymerase promoter and the ribozyme or sub- hybridized to the universal oligonucleotide and the strate sequences (see below). The universal T7 promoter single-stranded regions are converted to double-stranded oligo contains 63 nucleotides of random sequence DNA using Klenow DNA polymerase (Promega, Madi- (‘template arm’) and 17 nucleotides that make up the T7 son, WI, USA). promoter. The ribozyme oligo contains the 17 nucleotides BR1 GCGGATCCAGGTTGGGAGAAGCGAACCA- complementary to the T7 promoter followed by a CCC GAGAAACACACG (for optimal transcription) and the 50 bases encoding the BR2 GCGGATCCTCAGCGCCAGAAGGACACCA- ribozyme. Similarly, the substrate oligos contain the T7 GAGAAACACACG promoter, a CGC and the 33 or 34 nucleotides encoding BR3 GCGGATCCAAGGCACAAGAAGGGAACCA- the substrate. To synthesize each ribozyme or substrate, GAGAAACACACG the two oligonucleotides were annealed and converted to CC GGGACGCGTACCAGGTAATATACCACAA- double-stranded DNA using Klenow polymerase. In vitro transcription was carried using 4 ␮g of dsDNA template CGTGTGTTTCTCTGGT ° CC-TL GGGACGCGTACCAGGTAATATACCACGGA- at 37 C for 2 h using T7 RNA polymerase (Promega), according to the manufacturer’s instructions. RNAs were CCGAAGTCCGTGTGTTTCTCTGGT 32 dCC GGGACGCGTACCAGGTAATATACCACAAC- labeled by incorporation of alpha P-CTP during the GTGTGACGCTCTGGT transcription reaction and purified by denaturing gel electrophoresis. For purposes, the specific ribozyme oligonuc- leotides contain a BamHI restriction site In vitro cleavage reactions: Time-course reactions were (underlined), and the universal oligonucleotides contain prepared in a final volume of 25 ␮l with a ribozyme con- an MluI site (underlined). The three centration of 5.2 nm and substrate concentration of 72 nm. nucleotide change that inactivates the ribozyme cleavage The reactions were assembled on ice and 5 ␮l was activity, without affecting its ability to bind its target removed for the 0 min time-point and mixed with 5 ␮l sequence7,15 (termed a ‘disabled’ ribozyme) is double loading buffer and stored at −85°C. The remainder of the underlined. Following conversion to double-stranded reaction was incubated at 37°C for up to 2 h in 40 mm m m DNA, the ribozyme were digested with BamHI Tris pH 7.5, 10 m MgCl2,2m spermidine, with time- and MluI and cloned into the in vitro transcription vector points being taken every 30 min. Five microliters of reac- pGem7Z (Promega) and the sequences verified. tion was removed for each time-point and mixed with loading buffer as above. Reaction products were ana- In vitro cleavage of HBV RNA lyzed by urea denaturing polyacrylamide gel electro- pGem7Z-ribozyme plasmids were linearized with NsiI phoresis (PAGE) and cleavage products were quantified (immediately downstream of the cloned gene) and tran- by phosphorimager analysis (Molecular Dynamics). scribed in vitro by T7 RNA polymerase. The reactions Percentage cleavage was defined as (radioactivity of the were treated with RQ1 DNase, according to the manufac- products)/((radioactivity of the products) + (radio- turer’s recommendation (Promega) and the transcripts activity of remaining substrate)). were gel-purified. The full-length HBV genome, in The Michaelis constant (Km) and kcat were determined plasmid pEco63, was obtained from American Type Cul- for each of the ribozymes by performing multiple turn- Hairpin ribozymes as gene therapy for hepatitis B PJ Welch et al 742 over kinetic experiments. Kinetic reactions were continued for another hour. HBV DNA was purified from assembled on ice in a final volume of 10 ␮l. Ribozyme the intact particles by digestion with Proteinase K (500 concentration was held constant at either 2 or 4 nm and ␮g/ml final) and SDS (1% final) for 2 h at 37°C, followed substrate concentrations ranged from 2 to 200 nm. Reac- by phenol extraction and ethanol precipitation. Purified tions were incubated at 37°C for as little as 15 min for DNA was separated on 0.8% agarose gels, dried and ana- the fastest reactions and up to an hour for the slowest lyzed by autoradiography and phosphorimager analysis reactions. Cleavage reactions were done in the same (Molecular Dynamics). buffer as described above and reactions were terminated by the addition of 10 ␮l of loading buffer. Products were Acknowledgements resolved by urea denaturing PAGE and analyzed as We would like to thank Dr Aleem Siddiqui (University of above. The K and k for the ribozymes were estimated m cat Colorado) for the Huh7 cells as well as valuable scientific from plotting the data in an Eadie–Hofstee plot input. We also thank Drs Mark Leavitt and Patrick Gilles (Deltagraph, Deltapoint Inc, Monterey, CA, USA) with R2 for useful scientific discussions, and Andrew Craven Ͼ 0.90. Catalytic efficiency = k /K = 104 per min/m. cat m and Kevin Buckley for technical assistance and DNA sequencing. Construction of ribozyme and HBV expression vectors For expression of each ribozyme in mammalian cells, the previously described pLNT-Rz retroviral vector was References used,16 and the HBV Rzs were cloned via BamHI and 1 Margolis HS, Alter MJ, Hadler SC. Hepatitis B: evolving epi- MluI downstream of the human tRNAval promoter (see demiology and implications for control. Semin Liver Dis 1991; Figure 1a). The ‘overlength’ HBV plasmid was con- 11: 84–92. structed by ligating two HBV DNA fragments into 2 Yoffe B, Noonan CA. Hepatitis B virus: new and evolving issues. ′ Digest Dis Sci 1992; 37: 1–9. pGEM7Z (Promega). The 5 end of HBV was supplied by 3 Offensperger W-B et al. In vivo inhibition of duck hepatitis B the 1966 bp SphI/EcoRI HBV fragment (nucleotides 2614– = ′ virus replication and by phosphorothioate 1380, pgRNA start site 1), and the 3 portion of HBV modified antisense oligonucleotides. EMBO J 1993; 12: 1257– consisted of the 1988 bp EcoRI/BglII HBV fragment 1262. (nucleotides 1380–166). A three-way, directional ligation 4 Korba BE, Gerin JL. Antisense oligonucleotides are effective was performed wherein the two HBV fragments were lig- inhibitors of hepatitis B virus replication in vitro. Antiviral Res ated into pGem7Z digested with SphI and BamHI (see 1995; 28: 225–242. Figure 1a). The resulting plasmid, pGEM-HBV-over- 5 Ganem D, Varmus HE. The molecular of the hepatitis length, contains the entire pgRNA and polyA signal, as B . 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