| Hepatitis B is one of the most common liver diseases in the world, which is caused by hepatitis B virus (HBV) infection and may progress to cirrhosis (scarring) and hepatocarcinoma. The current treatments for chronic hepatitis B are to administer interferon, antiviral drugs such as lamivudine, or their combinations. However, all these drugs could not eradicate the cccDNA within infected cells, and the recurrence of viremia after cessation of therapy and development of escape mutants are always big concerns. Therefore, new strategies for the better treatment of the disease are urgently needed.Most recently, RNA interference (RNAi), a process of sequence specific post-transcriptional gene silencing initiated by double-stranded RNA (dsRNA), emerged as not only an extremely powerful tool for functional genomic studies but also a potentially useful method to develop specific gene-silencing therapeutics, especially, the strategy is considered enormously useful for antiviral therapy with many advantages. Besides that it can work in the absence of active viral replication, RNAi activity is sequence specific hence minimizing many undesirable side effects as those observed with conventional drug therapies. Another important advantage of the RNAi approach is that there are numerous potential targets for RNAi along the viral genome, and RNAi cocktails targeted towards different genes may be even more efficacious. Thus the RNAi may be provides an effective method for chronic HBV treatment. However, for advancing such a strategy towards clinical use, there are still many aspects need to be well defined, including validate whether the RNAi can interference the replication and expression of HBV, select effective target sequence and suitable siRNA constructs, test their RNAi efficiency in vitro and in vivo, analyze theirpharmacokinetic/pharmacodynamic properties, and look for effective method to target delivery siRNA into the liver. These parameters are essential for the development of therapeutic applications based on RNAi mechanism. We aimed at the HBV surface antigen encoding region and screened three different sequences, presented a detailed comparison study of the in vitro and in vivo activities and their pharmacodynamic behaviors of several different RNAi molecular constructs. We found that they all had specific inhibitory effects in vitro and in vivo, but their stability, dose responsiveness, and effective duration were distinctively different. The detail results were summarized as following:1. We selected the HBV subtype ayw as research object according to HBV epidemiology in China and experimental model we have, confirmed their genome sequence via sequencing, and then chose three target sequences in S open reading domain gene as RNAi target sites. In addition to the natural DNA duplexes, we also made the modified siRNA oligos by changing the native rU and rC with 2'-O-Methyl rU and 2'-O-Methyl rC respectively, while the native rA and rG remain the same. The shRNA expression vector was constructed by inserting the specific DNA segment into a plasmid vector which contained human U6 promoter..2. The three different sequences were screened for their inhibitory effects in human hepatoma cell line Huh-7 cotransfected with pHBS. All the three RNAi sequences in either short strand RNA duplex form or in plasmid vectors resulted in significant reduction of HBV mRNA and HBsAg expression (about 70-90 %), and the effect was dose and time depend. In HepG2.2.15 cell model, the RNA duplex can significantly interfere with the transcription of HBV and reduce virus DNA copy numbers both in culture medium and in cell lysates (about 50-70 % reduction). The inhibition efficiency of the chemically modified siRNA was lower than that of the unmodified version. However, the inhibition effect of modified siRNA lasted longer than that of natural siRNA. Furthermore, in our studies, better inhibitory efficacies were observed when the three RNAi sequences were used in combination at a relatively high dose.3. In in vivo test, various RNAi constructs were cotransfected into liver together with pHBS by hydrodynamical injection. The results showed that the siRNA can significantly reduced the amount of intact mRNA and HBsAg in mice liver, and the interference effects were clearly dose dependent for all constructs. The native siRNA duplex had the best inhibition effect and culminated quickly, but can only maintain its inhibition activity for less than 5 days, while the methyl modified siRNA duplexes were more stable inside cells and can exert its inhibitory effect over a longer duration time. Most interestingly, the effect of siRNA expression plasmid was so long lasting that even at 15 days after its administration it could still interfere with freshly injected pHBS transcription and maintain 50 % inhibition of the protein expression. In a mice model with pre-existing HBsAg expression, the RNAi administration can effectively clear some of the HBsAg in liver (60-70%), and the reduction of HBsAg levels in serum was more significant than in liver samples. We also found the specific siRNA can reduced HBsAg level to about 20 % of the original amount in muscle, and also affected the induction of antibodies against the antigen.4. In the development of therapeutic applications of RNAi, the delivery of siRNA is still the biggest and most pressing issue. In in vitro test, we found that it is difficult to transfect the siRNA expression plasmid into the HepG2.2.15 cell using lipofectamine and DOTAP liposomes. So we developed liposome formulations encapsulating high density solution such as Dimeglnmine Gadopentetate and Iohexol. We found these two kinds of liposomes can enhance the transgene expression 4-25 fold. The denser the liposome encapsulates, the higher the transfection efficiencyies. We also tested a few more new formulations and physical method for siRNA liver targeted transfection, including multi-vesicular liposomes, liposomes containing gas bubbles combined with ultrasound, intraarterial injection, liver direct injection combined with electroporation, and hydrodynamical injection. All these methods resulted improvements in transfection efficiencies at various degrees. But only the hydrodynamic injection method can delivery the plasmid DNA direct into hepatocytes and got a significantly higher level of transgene expression in the liver.5. DNA vaccines against hepatitis B virus (HBV), with which the antigen is synthesized in vivo after direct introduction of its encoding sequences, can induce strong and long-lasting humoral and cell-mediated immune responses. It has been shown that DNA vaccines can overcome tolerance in a transgenic mouse model of the HBV genome, opening up the possibility of an effective therapeutic DNA vaccine to treat chronic carriers of HBV. We developed a electroporation mediated DNA vaccination procedure and tested the immune responses (anti-HBs in the serum and the HBs specific IFN-γ) and the immune elimination of HBsAg in vivo. Electroporation mediated immunization in mice resulted high serum antibody (300mIU/mL) as well as strong cellular responses four weeks after the immunization. Then the HBV preS2-S encoding plasmid was delivered directly to mouse liver using hydrodynamic injection to imitate a psedo existing injection model. The HBsAg levels expressed in liver and secreted in serum were significantly lower than that in unimmunized group. The immunohistochemical analysis of liver tissues also showed that the HBsAg was cleared quickly in the experimental mice. The results of HE staining showed that there were no visible necrosis, apoptosis and inflammatory cell infiltration in liver sections. The level of ALT was seen to become elevated transiently (up to 2000U/mL) in the wake of hydrodynamic injection, but there was no significant difference between two groups, and then it fell down to the normal range (20-40 U/mL) 3 days post the injection. The results show that in vivo electroporation could get more rapid, stronger, and longer-lasting immune responses and the immune responses can eliminate the HBsAg expressed in hepatocytes.
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