Study On Hepatitis B Gene Therapy Based On AAV-mediated Artificial MiRNA

Hepatitis B is an infectious disease caused by hepatitis B virus (HBV). Hepatitis B virus infection is one of the major infectious diseases hazard to human health, which caused serious global public health problem. Nearly 350 million people were chronic HBV infections and 1 million people died from HBV-related diseases every year, including hepatitis, liver cirrhosis, liver failure and hepatocellular carcinoma, according to global statistics. China has a heavy disease burden of HBV infections, the prevalence of HBV infection in some special areas reach 35%, which result in heavy social and economic burden. There are more than 93 million HBV carriers in China, including 25 million patients with chronic hepatitis B infection. Hepatitis B has become one of the three major infectious diseases in China, as well as the main cause of hepatocellular carcinoma (HCC). Current antiviral therapies involving PEGylated interferon (IFN) and nucleos(t)ide analogs (NUCs), only a minority of patients with hepatitis B can produce long-term answer to those drugs, and can’t completely clear virus from the body. Moreover, high recurrence rate, multiple side-effects and exorbitant price are the main disadvantages of these medication. Therefore, new therapeutic strategies are urgently needed. Gene therapy is a new milestone in biotechnology, and will become one of the main directions of bio techno logy industry. In recent years, great progress has been made in gene therapy, it was recognized by Science as one of the top 10 scientific breakthroughs of 2009. Notably breakthrough has been made in treatment of congenital amaurosis disease and hemophilia B in clinical trials by using recombinant adeno-associated virus (AAV) vector. AAV has been considered as one of the most promising viral vectors in gene therapy, with low pathogenicity, low immunogenicity and the capability of mediating long-term expression of exogenous gene, etc. More than 10 serotype of AAV has been identified, and AAV-8 is one of the most commonly used AAV vectors for liver gene transduetion. Found in plants, animals and some viruses, miRNA is a small non-coding RNA molecule (containing about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression. Artificial miRNAs (amiRNA) are designed from a precursor of endogenous miRNA, used as structural support and in which the region of the mature miRNA is replaced with a specific amiRNA sequence complementary to the desired target sequence. Due to the pathway of miRNA natural production, amiRNA has better interference effect and lower in vivo toxicity than shRNA and has become a research focus. The main aim of our study was to evaluate the anti-HBV effect of amiRNA mediated by recombinant AAV-8.In the first part, we explored anti-HBV gene therapy in HBV transgenic mice by AAV-8-mediated amiRNA. In previous study, we have designed 13 amiRNAs, which targeting different gene sequence of HBV conservative region without any non-specific sequences of human and mouse. By in vitro pharmacodynamic evaluation we have screened out 3 amiRNAs with significant inhibition of HBsAg and HBeAg. In consideration of HBV virus resistance, we have constructed three amiRNA tandem expression vectors driven by liver-specific promoter (pAAV-liver-amiRNA135), which has been confirmed to be more effective than single amiRNA driven by the CMV promoter in in vitro and in vivo pharmacodynamic evaluation. For in vitro study, pAAV-liver-amiRNA135 and different genotype HBV expression plasmids were cotransfected into HepG2 cells, HBsAg/HBeAg relative levels and HBV DNA copy numbers in cell supernatant were measured by ELISA and QPCR, respectively. The results showed that amiRNA135 significantly inhibited the expression of HBsAg/HBeAg and the replication of HBV DNA, compared to negative control (p<0.05). For in vivo study, pAAV-liver-amiRNA135 plasmid was packaged into recombinant adeno-associated virus 8 (AAV8-amiRNA135), vector efficacy and safety were evaluated in HBV C genotype transgenic mice. AAV8-amiRNA135 was injected into mice via the tail vein at different dose (5×1011vg、5×1010 vg,5×109 vg and 5×108 vg,6-8 mice/group). Mice injected with 5×1011 vg of AAV8-luc and AAV8-Neg were as blank control group and negative control group, respectively. Venous blood samples were collected at different time points after vector injection, serum HBsAg and HBeAg levels were measured by quantitative ELISA. The results showed that AAV8-amiRNA135 significantly inhibited HBsAg and HBeAg in HBV transgenic mice and the effects were dose-dependent. Above all, we found that a single intravenous injection of AAV8-amiRNA135 could effectively inhibit HBV replication for up to 15 months. After that, mice were euthanized, copy numbers of HBV DNA in liver was evaluated by QPCR. Data showed that liver HBV DNA copy numbers was significantly reduced, which indicated that amiRNA135 expression could effectively inhibit the replication of HBV DNA. Additionally, liver tissue HE stain and HBsAg/HBcAg immunohistochemical stain were conducted. Through HE staining, obvious inflammation and disordered structure of hepatic lobule were detected in control group, while in those injected with 5x 1010 vg or 5x1011 vg vectors, normal hepatocytes and hepatic plates were observed and no significant inflammatory cell infiltration and fibrosis was found. Thus, we came to a conclusion that high doses of AAV8-amiRNA135 can effectively inhibit the occurrence of hepatitis. Liver immunohistochemistry staining showed that HBsAg and HBcAg positive cells decreased with the increasing of vector doses, indicating that AAV8-amiRNA135 significantly inhibited the expression of HBsAg and HBcAg in a dose-dependent manner. We further evaluated the anti-HBV effect of AAV8-amiRNA135 in several different strains of high-level HBV replication transgenic mice, named C16-4(?),2Cd(?) and 2B(?)2B(?).5×1011vg AAV8-amiRNA135 was injected into different mice via tail vein respectively. Venous blood samples were collected and serum HBsAg and HBeAg were detected by quantitative ELISA at 1,2,3 weeks after vector injection. The results showed that HBsAg and HBeAg were significantly inhibited by AAV8-amiRNA135 in those HBV transgenic mice, the inhibition ratio of HBsAg were 80% to 90%, and 40% to 70% of HBeAg. Euthanization was carried out at 4th weeks after vector injection, copy numbers of HBV DNA and HBV RNA in livers were evaluated by QPCR. Results showed that HBV DNA and RNA significantly reduced, which indicated that amiRNA135 can effectively inhibit the replication of HBV DNA and RNA in liver tissue in different HBV transgenic mice. In summary, AAV8-amiRNA135 could broadly and significantly inhibit the replication of HBV and our study led to a new clue of anti-HBV gene therapy.In order to further improve the efficiency of AAV mediated gene therapy for liver disease, the next main objective of this study is to explore a more efficient liver-targeting recombinant adeno-associated viral vector. Previous studies demonstrated that AAV-8 is one of the most commonly used vectors for liver gene delivery. AAV-DJ is a variant generated from the libraries of AAV hybrids of eight serotypes by DNA shuffling method. It is able to efficiently transduce a broad range of cell types in vitro and has specific liver targeting in vivo. AAV-DJ has been considered as a clinical candidate in the potential gene therapy of human liver diseases, owing to its lowest neutralization by pre-existing antibody levels. Nevertheless, there exist some challenges for AAV-mediated gene transduction in liver, for example, AAV can be degraded in cell through ubiquitination proteasomal degradation machinery, which result in reducing of gene delivery efficiency. In order to minimize AAV immunogenicityand its intracellular degradation, many efforts have been explored for further improvement of AAV transduction efficiency. Among these, generating point mutations at specific tyrosine, serine, threonine or lysine on virus capsid seems to be the simplest and universal method, allowing vectors to evade intracellular phosphorylation and subsequent ubiquitination and proteasome-mediated degradation. In this study, we have constructed three single-tyrosine mutants of AAV8 (Y447F, Y703F and Y708F) and three mutants of AAV-DJ (K137R, T251A and S503A), all these mutants were selected by using the online Phosphorylation and Ubiquitination Sites Prediction tools and constructed by over-lapping PCR. Recombinant AAV vectors were generated by polyethyleneimine (PEI)-based triple transfection of 293T cells. First, recombinant AAV8 and its three tyrosine mutants (Y447F, Y703F and Y708F) containing dual luciferase gene (Glue-Fluc) were injected into male C57BL/6 mice of 6-8 weeks, with 1×1010 vg virus each in the same volume via the tail vein (TV). At 7,14,21,28 days after vector injection, venous blood samples were collected to evaluated the relative light unit (RLU) emitted by the Gaussia luciferase using a luminometer, the results showed that the RLU in each animal reached a peak at day 21 and maintained at a level of 10,000-100,000 RLU, the Y703F mutant has significant gene expression enhancement, with 3-to 4-fold higher than wtAAV8 (P<0.01). In vivo bio luminescence was analyzed at 6 weeks post-vector injection. Results showed that all vectors have mainly liver tropism, just like wtAAV8. Statistical analysis revealed that the Y703F mutant elicited enhancement in liver luminescence level of 1.8-fold higher than wtAAV8 (P<0.05). For further evaluation, we compared the viral genome copy numbers in different tissue samples using real-time PCR. Data showed that, the Fluc DNA copy number of the four vectors maintained at a level of 1×106～1×107GC (genome copy)/100ng DNA in liver tissues. In kidney, heart and gastrocnemius tissues, the numbers dropped to 1×105～1×106GC (genome copy)/100ng DNA, which further illustrated that the four vectors have mainly liver tropism. The mutant Y703F showed significant enhancement in liver, kidney, and gastrocnemius tissues with 3,3 and 7-fold higher GC/100ng DNA than wtAAV8, respectively (P<0.05). We further evaluated the transduction efficiency of wtAAV8 and Y703F mutant carrying Angiotensin 1-7 (Ang-(1-7)) in C57BL/6 mice. Vectors were injected into male C57BL/6 mice of 6-8 weeks, with 1 ×1011vg virus each in the same volume via the tail vein (TV). At 7,14, 21,28 days after vector injection, venous blood samples were collected to evaluated the relative level of Ang-(1-7). Data showed that the Y703F mutant has significant gene expression enhancement, with 2-fold higher than wtAAV8 (P<0.01). Liver DNA copy number analysis revealed that the Y703F mutant showed significant enhancement in liver gene delivery, with 3-fold higher than wtAAV-8. Therefore, we came to a conclusion that the Y703F mutant of AAV8 led to significant improvement for in vivo gene delivery, which was mainly located in liver tissue.Similarly, recombinant AAV-DJ and its three mutants (K137R, T251A and S503A) containing dual luciferase gene (Gluc-Fluc) were injected into male C57BL/6 mice of 6-8 weeks, with 1×1011 vg virus each in the same volume via the tail vein (TV). Data showed that the RLU in each animal reached a peak at day 21 and maintained at a level of 100,000～1,000,000 RLU. The S503A mutant displayed significant enhancement of 2-to 3-fold at each time point (P< 0.001), and the T251A mutant showed～1.8-fold enhancement at day 21 and 28 (P< 0.01), while the K137R mutant showed no significant enhancement, compared with AAV-DJ. In vivo bioluminescence analysis showed that all vectors exhibited liver tropism. Fluc DNA copy number analysis revealed that the Fluc DNA copy number of the four vectors maintained at a level of 1×107～1×108GC (genome copy)/100ng DNA in liver tissues. In kidney, heart and gastrocnemius tissues, the numbers dropped to 1×105～1×107GC (genome copy)/100ng DNA, which further illustrated that the four vectors have mainly liver tropism. The K137R, S503A and T251A mutants led to significant enhancement in transduction in liver samples with 2.1-,3.5-,2.5-fold higher DNA copy numbers respectively, compared to AAV-DJ (P< 0.01). The S503A mutant resulted in 2-to 3-fold higher copy numbers in tibialis anterior (TA) and heart tissues while K137R showed 2.7-fold higher copy numbers in kidney tissue (P< 0.05). However, we found minimal enhancement for in vitro experiment. When AAV-DJ and its three mutants containing GFP gene were transduced to different cell line (293T, Hela, HepG2) in MOI=1000, the K137R and S503A point mutations led to 20-30% enhancement while the T251A mutation led to no significant enhancement of GFP expression when compared to AAV-DJ in these three cell lines. Therefore, we came to a conclusion that single point mutant of AAV-DJ led to significant improvement for in vivo gene delivery but had minimal enhancemtn for in vitro gene transduction.In summary, the main achievements of the present study are as follow:the amiRNA135 expressing AAV vector, AAV8-amiRNA135, was confirmed to be an efficient and extensive HBV inhibitor, which can stably and significantly inhibit the expression of HBsAg and HBeAg, and reduce HBV DNA, RNA level; by single point mutant of AAV8 and AAV-DJ capsid, we found out that the Y703F mutant of AAV8 and S503A mutant of AAV-DJ led to significant enhancement of AAV-mediated liver gene delivery. These findings provide new clues for gene therapy on liver diseases.