1 School of Life Sciences and Technology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, PR China
2 School of Pharmacy, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, PR China
3 Shanghai GenePharma Co. Ltd, 501 Newton Road, Shanghai, PR China
Correspondence
Yuhong Xu
yhxu{at}sjtu.edu.cn
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ABSTRACT |
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INTRODUCTION |
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It has been suggested that the preferred way of controlling the virus is to interfere with the viral gene replication and expression processes. Studies employing antisense and ribozyme technologies have demonstrated some promise, but the poor in vivo stability of these molecules, limited access to infected cells and secondary-structure interference of the DNA or RNA sequences all limited their clinical efficacy (Aoki et al., 2003; Goodarzi et al., 1990
; von Weizsacker et al., 1992
).
Most recently, RNA interference (RNAi) technology has emerged not only as an extremely powerful tool for functional genomic studies, but also as a potentially useful method for developing specific gene-silencing therapeutics, especially for the treatment of viral diseases (Marathe et al., 2000; Yang et al., 2000
). Specifically designed RNAi molecules can target viral mRNAs and initiate their degradation. Several pioneering studies have demonstrated great possibilities for treating serious viral diseases, including those caused by human immunodeficiency virus, HBV and hepatitis C virus (Adelman et al., 2001
; Gitlin et al., 2002
; Jacque et al., 2002
; Lee et al., 2002
; Wilson et al., 2003
). For HBV, RNAi molecules have been shown to have impressive inhibitory effects against viral gene transcription and expression (Giladi et al., 2003
; Hamasaki et al., 2003
; McCaffrey et al., 2003
; Ying et al., 2003
). The inhibitory effects were similar against both replication-competent and -incompetent HBV (Shlomai & Shaul, 2003
). Therefore, this strategy could be used for treatment at various stages of disease progression. One recent study further demonstrated viral clearance from the liver of transgenic mice (Uprichard et al., 2005
), which provides high hopes for the use of RNAi molecules as HBV therapeutics.
However, to advance such a strategy towards clinical use, there are still many aspects of the molecule's pharmacological properties that need to be well-defined. Several construct structures that have RNAi activity have been proposed, including double-stranded RNA (dsRNA) synthesized by chemical methods or T7 RNA polymerase, and small hairpin RNA transcripts from plasmid vectors containing the U6/H1 promoter (Brummelkamp et al., 2002). Furthermore, some structural modifications have been made to the dsRNA constructs to improve molecular stability and efficacy (Chiu & Rana, 2003
; Czauderna et al., 2003
; Morrissey et al., 2005
). All of these different structures may affect in vivo RNAi efficacy, as well as their pharmacokinetic/pharmacodynamic properties and the delivery requirements. These parameters are essential for the development of therapeutic applications based on RNAi mechanisms.
In this paper, we present a detailed comparison of the in vitro and in vivo activities and the pharmacodynamic behaviours of several different RNAi molecular constructs. Although they all had specific inhibitory effects in vitro, their stability in vitro and in vivo and their dose responsiveness and effective duration were distinctively different. These parameters all have to be taken into account when designing possible therapeutic regimes for chronic HBV treatment.
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METHODS |
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RNAi constructs.
Three RNAi sequences (s1, s2 and s3) starting with AA were designed based on the complete genome of HBV subtype ayw (GenBank accession no. U95551) and analysed by BLAST to ensure that they did not have significant nucleotide sequence identity to other genes. The three target sequences are listed in Fig. 1(a). Small interfering RNAs (siRNAs) with two different chemical structures (Ts and cTs) were synthesized by GenePharma. The Ts oligonucleotides with the native RNA structure were synthesized by using 2'-OH tert-butyldimethylsilyl (TBDMS) chemistry. The cTs oligonucleotides were synthesized by replacing rU and rC with 2'-O-methyl rU and 2'-O-methyl rC, respectively, whilst leaving rA and rG unchanged. All oligonucleotides were purified by HPLC and annealed to form duplexes before use. For construction of the siRNA expression plasmids, pSilencer 2.1-U6 neo vector (Ambion) was linearized with BamHI and HindIII to facilitate directional cloning and purified to remove the digested insert. For each target gene, the designed complementary 64-mer oligonucleotide with a 5' single-stranded overhang for ligation into the pSilencer vector was synthesized, annealed and cloned into the linearized vector.
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RNAi construct administration in vivo.
For all in vivo experiments, we used 68-week-old female BALB/c mice. All animal experimental procedures were approved by the Shanghai Department of Experimental Animals Management. To test the inhibitory effect of RNAi constructs in liver, the RNAi constructs and/or pHBs were delivered to mice by hydrodynamic injection (Liu et al., 1999; Zhang et al., 1999
).
To test the inhibitory effect of RNAi constructs in muscle, the RNAi constructs and pHBs in 40 µl 0·9 % saline were injected into mouse gastrocnemius muscle. Immediately after injection, two stainless-steel electrodes were inserted around the injection site and six electric pulses with 120 V cm1 field strength and 60 ms pulse duration were applied.
HBV viral DNA quantification.
HBV viral DNA copy numbers were determined by using an HBV fluorescent quantitative PCR kit (Daan Gene Co.). HBV DNA in 40 µl culture medium and cell lysates were extracted and PCRs were prepared according to the kit's protocol. PCRs were carried out in an ABI Prism 7000 using the following program: pre-denaturation at 93 °C for 2 min; 10 cycles of 93 °C for 45 s and 55 °C for 60 s; and 30 cycles of 93 °C for 30 s and 55 °C for 45 s. Negative-control and serially diluted standard samples were processed synchronously (for the standard curve, r=0·9991). Results were expressed as copies (ml culture medium)1 (cell lysates).
RNA extraction and Northern blot analysis.
Total RNA was extracted from pooled frozen liver samples by using a guanidinium thiocyanate/phenol-based protocol (Sangon). For Northern blot analysis, 20 µg total RNA was denatured, electrophoresed in 1·0 % agarose gel containing 37 % formaldehyde and transferred onto a nylon membrane filter (Hybond-N+; Amersham Biosciences). The probe was an 880 bp EcoRI fragment of HBsAg cDNA in the pHBs plasmid, labelled with horseradish peroxidase (HRP) by using a North2South Direct HRP Labelling and Detection kit (Pierce). Labelling was carried out strictly according to the kit's protocol, followed by stringent washing and autoradiography.
HBsAg and HBV surface antibody (anti-HBs) assays.
Mouse blood samples were collected and sera were separated by low-velocity centrifugation. Levels of anti-HBs and HBsAg in the sera were determined by using the corresponding quantitative ELISA kits (Sino-American Biotechnology Co.). At the same time, for the detection of HBsAg concentration in tissues, mice were sacrificed at designated time points. The gastrocnemius muscle or liver was dissected and homogenized. The level of HBsAg in the tissue lysates was determined by using the quantitative ELISA kit (Sino-American Biotechnology Co.).
Statistical analysis.
The results are presented as mean values±SD and statistical analyses were carried out by using Student's t-test.
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RESULTS |
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The three different sequences were screened for their inhibitory effect in the human hepatoma cell line HuH-7 co-transfected with pHBs (Fig. 1b). Cells treated with pHBs alone or with irrelevant siRNA or siRNA expression plasmid had substantial amounts of HBsAg (approximately 1·6 µg ml1) detectable in the culture medium. However, co-transfection of all three RNAi sequences in either short-stranded RNA duplex form (Ts) or in plasmid vectors (pS) resulted in a significant reduction in HBsAg expression (approx. 7090 %) compared with the respective irrelevant RNAi controls.
For in vivo tests, pHBs and the various RNAi constructs were co-transfected into mouse livers by hydrodynamic injection. The results showed that HBsAg expression was greatly reduced in Ts and pS co-transfected animals (Fig. 1c). HBsAg mRNA levels were also checked by Northern blot analysis and the amount of intact mRNA was reduced significantly in Ts and pS co-transfected liver tissues, indicating that the inhibitory effect was indeed mediated through the degradation of HBsAg mRNA (Fig. 1c
).
The actual inhibitory effects towards the three different target sequences were similar, so we selected the sequence S2 for subsequent studies to examine other parameters of the inhibitory effect.
Inhibitory effect of siRNA constructs in HEpG2.2.15 cell models
HEpG2.2.15 cells containing integrated HBV DNA were used as a cell model of HBV-infected hepatocytes. Cells were transfected, using Oligofectamine, with 80 nmol l1 of the RNA duplex Ts2 and methyl-modified RNA duplex cTs2 and compared with irrelevant siRNA. At 36 h post-transfection, HBV DNA copy numbers and the amount of HBsAg in the culture medium were assayed. The results (Table 1) showed that Ts2 can interfere significantly with the transcription of HBV and reduce viral DNA copy numbers both in culture medium and in cell lysates (approx. 5070 % reduction). Similar effects were found in the HBsAg level in culture medium. The efficiency of inhibition of the chemically modified siRNA (cTs2) was lower than that of the unmodified version (Fig. 2a
), indicating that the chemical modification in the backbone of siRNA may affect its activity to a certain extent. However, the inhibitory effect of cTs2 lasted longer than that of Ts2. The effect of Ts2 declined quickly and had disappeared completely by day 4, whilst the effect of cTs2 was maintained at approximately 50 % of the original level after 4 days (Fig. 2b
). The longer effective duration suggested that cTs2 is probably more stable inside cells.
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Doseresponse profiles of various RNAi constructs in vivo
It is important to determine the doseresponse properties of the various RNAi constructs before developing any in vivo application strategies. We explored the extent of inhibition after co-injection of pHBs with various amounts of Ts2, cTs2 and pS2 into mouse livers by using the hydrodynamic method. pHBs (5 µg) was co-injected with Ts2, cTs2 or pS2 at designated molar ratios. Irrelevant siRNA or siRNA expression plasmid was used as a control. HBsAg levels in the liver were examined 1 day after co-injection. The interference effects were clearly dose-dependent for all constructs: the higher the dose administered, the stronger the inhibitory effect exhibited (Fig. 3). Fifty per cent inhibition could be achieved by using the plasmid vector pS2 at 0·60·3 : 1 molar ratios and using Ts2 and cTs2 at a 1 : 1 molar ratio. The level of inhibition gradually reached a plateau at a 3 : 1 molar ratio for plasmid DNA vector and at a 10100 : 1 molar ratio for siRNA.
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Inhibition of HBsAg expression and anti-HBs induction in muscles in vivo
We also tested the inhibitory effects of siRNA constructs delivered intramuscularly by using electroporation. HBsAg expression in the muscles can reach up to 2·4 µg 1 day after injection of 10 µg pHBs by electroporation and can persist for several weeks. However, when the siRNA expression plasmids were delivered together, also by electroporation, expression levels of HBsAg were reduced significantly, to approximately 20 % of the original level. Again, the delivery of an irrelevant siRNA expression plasmid had no effect on the HBsAg expression level in the muscle (Fig. 5a).
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DISCUSSION |
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In order to find an optimal vector for the RNAi sequence, we tested three different molecular structures carrying the same RNAi sequence. The plasmid vector encoded a hairpin RNA sequence that could fold into a duplex RNAi structure after transcription. The siRNA duplexes were made synthetically by using traditional 2'-TBDMS chemistry and the modified siRNA oligonucleotides were synthesized by changing the native rU and rC to 2'-O-methyl rU and 2'-O-methyl rC, respectively, whilst the native rA and rG remained the same. In both in vitro and in vivo experimental models, we showed that the pharmacodynamics of these different structures were considerably different. The native siRNA duplex had the greatest inhibitory effect, but culminated quickly, whilst the methyl-modified siRNA duplexes were more stable inside cells and exerted their inhibitory effect over a longer period. This is consistent with the report of Soutschek et al. (2004) on chemically modified siRNAs. In their study, partial phosphorothioate backbone and 2'-O-methyl sugar modifications were reported to stabilize siRNAs in vitro and in vivo. Most interestingly, we found that plasmid vectors were very stable once delivered inside liver cells and maintained a longer inhibitory effect. Surprisingly, even 30 days after a single hydrodynamic injection, the RNAi-encoding plasmids were still available and able to inhibit HBsAg expression from freshly administered pHBs to a considerable degree.
These different constructs also behaved differently with respect to their delivery in vivo. Plasmid DNA, with a high molecular mass and highly charged characteristics, was hard to deliver into cells in vitro, as well as in vivo. The siRNA constructs, on the other hand, were much smaller and entered the target cells more easily. Their structure could be further modified to shield the surface charge and make them more hydrophobic for easier transport across the cell membrane. Most impressively, Soutschek et al. (2004) conjugated cholesterol to the 3' end of the sense strand and reported improved delivery in vivo. However, the 2'-O-methyl modification of siRNAs tested in our experiment did not significantly improve the delivery by normal intravenous injection in vivo (data not shown). As a matter of fact, almost all earlier proof-of-concept studies in animals have had to use the hydrodynamic injection method, but the feasibility of using such an approach clinically is still not clear. Delivery is still the biggest and most pressing issue in the development of therapeutic applications of RNAi. In our study, in addition to applying a hydrodynamic method to test the RNAi efficacy in mouse liver, we used another highly efficient intramuscular gene-delivery method, electroporation (Aihara & Miyazaki, 1998
; Rols et al., 1998
), to verify the inhibitory effect of our RNAi constructs on HBsAg expression. Significant inhibition was apparent at all levels, including mRNA transcription, HBsAg expression and anti-HBs production. Unfortunately, electroporation is also very difficult to apply to hepatic cells for gene delivery. However, these data again suggest that RNAi therapeutics have great potential once an efficient delivery method is available.
Using the hydrodynamic delivery method, we also looked into the doseresponse and effective-duration characteristics of the different vectors. An equimolar ratio of siRNA to pHBs resulted in substantial inhibition of HBsAg expression. Increasing the dose further did not lead to significant improvement. However, as these siRNA constructs could only maintain their efficacy in vivo for less than 35 days, for therapeutic effects, they should probably be used frequently and repetitively. In contrast, for plasmid vectors, higher doses clearly resulted in better efficacy and, because these are stable inside liver cells and have a long duration of action, they could probably be used less frequently and at a higher dose.
In a mouse model with pre-existing HBsAg expression, we also demonstrated that RNAi administration could effectively clear some HBsAg from liver and serum. Interestingly, the reduction in HBsAg levels in serum was more significant than in liver samples. We suspect that this is because expressed HBsAg can accumulate in liver tissues, whilst secreted proteins are cleared quickly, and any reduction in supply would affect the serum concentration. We were also interested at looking into the possibility and effect of repeated RNAi administration in these models. However, because HBsAg expression resulting from pHBs hydrodynamic transfection was transient and usually disappeared within 5 days, it was difficult to observe the added effect of repeated RNAi administration in vivo.
To summarize, we have carried out detailed studies looking into several pharmacodynamic features of RNAi constructs for the treatment of HBV. We showed that RNAi sequences can be designed and screened to achieve effective inhibition in cell and animal models. Different constructs carrying the same RNAi sequences had different properties in terms of their activities in vitro and in vivo. Sequence, stability, method of delivery and several other issues are all important. These factors need to be taken into consideration when examining the clinical potential of these RNAi-based therapeutics.
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ACKNOWLEDGEMENTS |
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Received 9 May 2005;
accepted 6 September 2005.
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