despite availability of a vaccine to prevent hepatitis b virus ( hbv ) transmission , complications arising from chronic infection with the virus remain an important global health problem . about 700,000 people die each year as a result of hbv - related cirrhosis and hepatocellular carcinoma ( hcc ) . currently , licensed treatments rarely eliminate hbv and development of durable and effective anti - hbv therapy is a priority to limit mortality in the world s 240 million chronic carriers.3 , 4 , 5 , 6 the genome of hbv has a remarkably compact arrangement . open reading frames ( orfs ) overlap with each other and encompass the entire genome ( figure  1a ) . there are four orfs : surface ( s ) , polymerase ( p ) , core ( c ) , and x. cis - elements that regulate transcription and viral replication are embedded in the protein coding sequences . following hepatocyte infection , encapsidated relaxed circular dna ( rcdna ) is transported to the nucleus where it is released and  repaired  to form covalently closed circular dna ( cccdna ) . this stable replication intermediate7 , 8 is the template for transcription of pregenomic rna and protein coding mrna . persistence of cccdna has been an obstacle to curing hbv carriers from the infection and durable inhibition of hbv replication is desirable to eliminate the virus . the highly economical use of its genetic material limits sequence plasticity of hbv . this feature has been exploited to advance therapy that is based on nucleic acid hybridization . several studies have shown that hbv is susceptible to rna interference ( rnai)-mediated silencing ( reviewed in ivacik et  al . ) . this is despite some evidence that the viral x protein is capable of countering the cellular silencing mechanism . both synthetic and expressed rnai activators are capable of reprogramming the rnai pathway to silence hbv replication in  vivo . a  combinatorial approach has previously been shown to knock down hiv-1 replication and prevent emergence of escape mutants.11 , 12 , 13 , 14 the compact arrangement of the hbv genome restricts plasticity , and the number of rnai effectors necessary to prevent escape is likely to be fewer than the four that are required for hiv-1 . use of synthetic rnai activators against hbv has now reached a stage of evaluation in clinical trials . the formulation termed arc-520 , which includes cholesterol - conjugated sirna and a hepatocyte - targeting peptide , showed promise in early clinical trials ( https://www.clinicaltrials.gov/ct2/show/nct02065336 ) . however , the candidate drug has been placed on hold by the united states food and drug administration after concerns relating to evidence of non - clinical toxicology were raised . a drawback of using synthetic sirnas as therapy for chronic hbv infection is that efficacy against the virus is usually short lived and repeat administrations are required . lasting renewable transcription of expressed artificial micrornas ( amirs ) , produced from stable dna templates within transduced cells , is thus attractive . expression cassettes encoding the rnai activators also have the advantage of compatibility with highly efficient recombinant viral vectors . short hairpin ( sh)rna - encoding sequences have commonly been used as artificial mimics of precursor microrna ( pre - mir ) . typically , these sequences are transcribed from constitutively active pol  iii promoters , such as u6 or h1.16 , 17 a drawback of pol iii shrna expression cassettes , elucidated while investigating use of expressed rnai activators to silence hbv gene expression , is that overexpression of the pre - mir mimics from a u6 promoter may disrupt natural mir processing to result in lethal toxicity.12 , 18 research aimed at advancing therapeutic expressed sequences has thus been focused on improving regulation of transcription of rnai activators in transduced cells . pol ii transcriptional regulatory elements have been favored as they are capable of precise and tissue - specific control of expression of rnai effecters in targeted cells . to  enhance compatibility of pol ii promoters with rnai activators , primary mir ( pri - mir ) mimics rather than shrnas have been incorporated into artificial expression cassettes.13 , 19 , 20 the design takes advantage of the natural transcription of pri - mirs from pol ii promoters . the polycistronic nature of natural pri - mir expression also enables incorporation of multiple antiviral sequences into an expression cassette to improve efficacy . simultaneous targeting of different viral sequences is also desirable to suppress emergence of viral escape mutants . using natural human pri - mir-31 as a scaffold , efficient anti - hbv artificial mono- and trimeric pri - mir expression cassettes have been generated in our laboratory.14 , 20 recombinant lentivirus - mediated delivery of artificial pri - mirs ( apri - mirs ) to neonatal hbv transgenic mice resulted in durable suppression of markers of hbv  replication . transcription of the anti - hbv apri - mirs from a liver - specific murine transthyretin receptor ( mttr ) promoter , was sustained over a 3  month period of analysis without evidence of toxicity . a recent study demonstrated that a monomeric apri - mir , expressed from a liver - specific promoter , was capable of silencing hbv replication in transgenic mice over a period of 84  days . to evaluate the utility of anti - hbv mono- and trimeric apri - mirs over a longer time period under conditions that would resemble treatment in a clinical setting , we have generated self - complementary adeno - associated viral vectors ( scaavs ) that encode the anti - hbv expression cassettes . the safety and versatility of aavs makes them attractive for use in experimental and clinical settings . methods for preparing aavs are now well established and features such as hepatotropism may be conferred by pseudotyping with the serotype 8 capsid to generate scaav8 vectors . moreover , an interesting feature that is relevant to use of the vectors to treat hbv infection is that hbv replication in hepatocytes renders the cells more susceptible to transduction by aavs . we show that  systemic administration of preparations of scaav8 encoding apri - mirs achieve sustained knockdown over a period of 32  weeks . inhibition of viral replication was more efficient in animals treated with vectors encoding the trimeric cassette . this durable silencing with a safe vector that has potential clinical utility is an important feature . apri - mir expression cassettes were based on previously characterized hbv - targeting sequences.14 , 20 , 21 the natural pri - mir-31 sequence was adapted to serve as a scaffold for monocistronic ( mir-31/5 ) and tricistronic ( mir-31/5,8,9 ) antiviral sequences that target sites within the x orf ( figure  1a ) . the x sequence is multifunctional and conserved in hbv isolates,26 , 27 which makes it a good target for rnai - mediated silencing . moreover , x is contained within all hbv transcripts , enabling simultaneous cleavage of all the viral rnas . to control expression in a liver - specific manner , expression cassettes , comprising the promoter , an intron , mir-31/5 or mir-31/5,8,9 , and a transcription termination signal were incorporated into scaavs ( figure  1b ) . according to the intended design , primary transcripts of mir-31/5 and mir-31/5,8,9 are processed using the canonical mir pathway to generate pre - mir intermediates and guide rnas of approximately 21 nt in length ( figure  1c ) . although repetitive sequences , derived from the mir-31 scaffold , were present in the vectors , rearrangement was not observed ( data not shown ) . this was in accordance with our similar previous findings when using recombinant adenoviral or lentiviral vectors . liver - derived huh7 cells were transduced with scaavs pseudotyped with serotype 2 capsid ( scaav2 ) , which encoded mir-31/5 , mir-31/5,8,9 , or the enhanced green fluorescent protein ( egfp ) . after infecting cells with the egfp reporter - encoding vectors at a multiplicity of infection ( moi ) of 100,000 , fluorescence imaging showed that all cells were transduced ( data not shown ) . to confirm processing of the apri - mirs , putative guide sequences of 2022 nt were detected after hybridization to probes complementary to anticipated mature mir-31/5 and mir-31/8 sequences ( figure  2a ) . guide 5 was only detectable in rna extracts of cells treated with scaav2 mir-31/5 and scaav2 mir-31/5,8,9 . a comparison of bands corresponding to guides 5 , 8 , and 9 showed variation in quantities of the individual mature mirs . the guide 5 sequence was present in high amount in transduced cells , followed by guide 8 , while the guide 9 sequence was least abundant and usually undetectable . despite the guide 9 sequence being undetectable on northern blot , functional analysis in transfected cultured cells and in mice subjected to hydrodynamic injection indicated that the guide 9 sequence was indeed active ( figures s1 and s2 ) . these data also demonstrated that the trimeric expression cassette is effective against a target with a mutation in the cognate of guide 5 , which suggests that potential for viral escape would be limited . we have made similar observations following transient transfection of huh7 cells or infection of these cells with mir-31/5,8,9-expressing lentiviral vectors . variability in processing of the components of the tricistronic apri - mirs is likely to be the cause of differences in the concentrations of guide rnas . detection of the mature mirs did not appear to be influenced by the melting temperature of the guide and probe hybrids and the concentration of each mature guide sequence correlated with silencing efficacy . evaluation of silencing efficacy of the scaav2-delivered mirs was also carried out in cultured huh7 cells . hbv surface ag ( hbsag ) was measured in the culture supernatant of huh7 cells after transfection with an hbv replication - competent plasmid and transduction with the scaav2s containing monocistronic and tricistronic pri - mir cassettes ( figure  2b ) . when compared to the cells treated with the scaav2s lacking mir cassettes , hbsag was significantly reduced in cells infected with the vectors containing mir-31/5 or mir-31/5,8,9 . the hbv transgenic mice used to assess efficacy of scaavs contain an integrated replication - competent greater - than - genome - length viral sequence . long term replication of hbv occurs in hepatocytes of these animals , but formation of cccdna and infection of the murine liver cells does not occur . nevertheless , the animals are useful as the long term replication of hbv in the transgenic mice simulates chronic hbv infection of humans.30 , 31 following injection of 1   10 scaav8 particles into a tail vein of  each animal , hbsag and circulating viral particle equivalents ( vpes ) were measured as indicators of viral replication ( figures 3a and 3b ) . scaav8 mir-31/5 and scaav8 mir-31/5,8,9 both caused rapid and significant reduction in hbsag and circulating vpes by 2  weeks after the injection . values for hbsag decreased to approximately 5% of the controls , while the circulating vpes diminished to 1%2% of the values obtained for animals treated with saline or the control scaav8 . these low levels of hbsag and circulating vpes were sustained over 32 of the 40  weeks of the analysis . declining efficacy against markers of replication correlated with the concentrations of aav genome equivalents that were detectable in hepatocytes ( figure  s3 ) . by 40  weeks , the average hbsag concentrations and circulating vpes in the serum of control animals also had diminished . in our hands , steady decline and variability in markers of hbv replication over a period of approximately 40  weeks is a feature of the transgenic mice used here . normalizing the data to values obtained from animals treated with the vector lacking mir revealed similar efficacy and the inhibitory effect had diminished by 40  weeks following injection of the scaavs ( figures s4a and s4b ) . average values for hbsag and vpes , although not statistically significant , were lower in animals treated with scaav8 mir-31/5,8,9 than they were for mice receiving scaav8  mir-31/5 . intrahepatic hbv core antigen ( hbcag ) , detected  using immunohistochemistry , was carried out on mice that were killed at 2  weeks and 40  weeks after injection with scaav mir-31/5 and scaav8 mir-31/5,8,9 ( figures 3c and 3d ) . this analysis revealed decreased numbers of positive cells in sections obtained from animals that had received the scaav8 mir-31/5,8,9 vector for both time points . a decreased number of hbcag - positive cells from livers of animals treated with the scaav mir-31/5 monomeric cassette was only observed at the later time point of 40  weeks , but not at 2  weeks . this better efficacy of the trimeric cassette correlates with our previous observations.20 , 21 although hbv core particles in the hepatocytes of hbv transgenic mice are stable , the diminished number of cells that were positive for the viral antigen at 40  weeks suggests that in this model , hbcag may be a more sensitive indicator of hbv replication than are hbsag and vpes . northern blot hybridization was carried out to evaluate processing of apri - mirs in the livers of hbv transgenic mice . animals were killed at 2 , 8 , 28 , and 40  weeks after they had received the scaavs . probes complementary to guides 5 , 8 , and 9 were again used to detect the matured amirs ( figure  4 ) . as with the data obtained from analysis of cultured cells , the guide 5 sequence was most abundant and the rna was present for the entire 40  weeks of the analysis . guide 8 rna was also detectable for 40  weeks in hepatic extracts from animals that had received scaav8 mir-31/5,8,9 vectors . as expected , this sequence was not found in rna isolated from mice treated with scaav8 mir-31/5 . undetectable guide 9 sequences in extracts from mice treated with scaav8 mir-31/5,8,9 correlates with data from previous studies20 , 21 and observations in cultured cells ( figure  2a ) . steady - state levels of guide 5 appeared to correlate with the intrahepatic copies of scaav ( figure  s3 ) . however , guide 8 has a more constant concentration , despite the decrease in vector copy number over time . this apparent anomaly may result from greater sensitivity of the guide 8 probe for the mature mir . rna species that are larger than the guides were absent in the blots that encompassed larger areas of the northern hybridization ( figure  s5 ) . this observation indicates that processing of the artificial cassettes is efficient and intermediates of the mature mirs comprising apri - mirs and apre - mirs exist transiently . toxicity caused by overexpression of rnai activators may be significant and is potentially lethal.12 , 18 several analyses were therefore carried out to exclude toxicity of scaav8 mir-31/5,8,9 and scaav8 mir-31/5 . the average mass of all groups of mice steadily increased for the duration of the 40  weeks of the study ( figure  s6 ) . no differences in weight gain between animals from each of the control and mir - treated groups were observed . sections stained with hematoxylin and eosin or sirius red were within normal limits and there were no differences between groups ( figure  s7 ) . measurement of alanine transaminase ( alt ) activity in the serum also indicated that the scaavs containing the mttr mir-31-derived cassettes did not cause hepatotoxicity ( figure  5a ) . all groups of animals had average alt activities that were below the upper limit of normal ( < 100 u / l ) for a period of 8  weeks after receiving the vectors . innate immunostimulation following in  vivo administration of exogenous rnai activators is also potentially toxic . to evaluate activation of expression of genes that participate in the ifn response , rna was extracted from mice 6  hr after they were injected with scaav8 mir-31/5,8,9 , scaav8 mir-31/5 , scaav8 lacking mir , poly(i : c ) , adenoviral vectors expressing mir-31/5,8,9 , or saline . quantitative reverse transcription pcr was carried out to measure mrna derived from ifn- , ifn - induced protein with tetratricopeptide repeats ( ifit)-1 , and oligoadenylate synthase-1 ( oas-1 ) genes relative to the amount of gapdh mrna ( figures 5b5d ) . mrna concentrations of the ifn response genes were similar in liver cells of mice that had received saline , the scaav8 without mir , scaav8 mir-31/5,8,9 , and scaav8 mir-31/5 . these values were lower than those obtained from samples extracted from mice that had received poly ( i : c ) , a positive control for stimulation of the innate immune response . animals receiving recombinant adenoviruses ( ads ) expressing mir-31/5,8,9 were also used as a control group . concentrations of transcripts from the ifn response genes in the mice receiving scaav mir-31/5,8,9 and scaav8 mir-31/5 were consistently lower than those detectable in mice receiving poly ( i : c ) . the ad mir-31/5,8,9 vector induced oas-1 expression that was similar to that of the poly ( i : c)-treated animals , but the stimulatory effect on ifit-1 and ifn- was minimal . effects of the scaavs on release of pro - inflammatory cytokines were assessed as further assessment of innate immunostimulation . this was performed using a cytometric bead array ( cba ) ( figures 6 and s8 ) . concentrations of interleukin 6 ( il-6 ) , interleukin 10 ( il-10 ) , monocyte chemoattractant protein-1 ( mcp-1 ) , interferon gamma ( ifn- ) , tumor necrosis factor alpha ( tnf- ) , and interleukin 12p70 ( il-12p70 ) were measured in the serum of mice 6  hr after they had received the scaavs , poly ( i : c ) , ad mir-31/5,8,9 , or saline . il-6 , il10 , mcp-1 , and tnf- were significantly elevated in the animals that received poly ( i : c ) . in animals receiving saline and the scaavs , serum concentrations of each of the cytokines were similar and low . collectively , our findings indicate that the hbv - silencing effected by scaav8 mir-31/5,8,9 and scaav8 mir-31/5 mediates prolonged knockdown of markers of hbv replication without evidence for toxicity and activation of an immunostimulatory response . available therapies have modest curative efficacy and consequently the risk of complicating liver cancer and cirrhosis remains high in carriers of the virus . to limit the life - threatening complications of the infection , there is a need to improve on current clinically useful anti - hbv agents . availability of treatments that have a durable and safe effect against the virus is desirable to counter the stable replication intermediate comprising cccdna . several different approaches are being employed to eliminate hbv infection , and these include use of new nucleoside and nucleotide analogs , inhibitors of viral capsid assembly , immunomodulators , gene editing strategies , and silencing by harnessing the rnai pathway . harnessing gene therapy to treat hbv infection offers advantages , such as rational drug design , and the field is now advancing impressively . advances with hepatotropic delivery of therapeutic nucleic acids have also been remarkable . evidence now indicates that gene therapy - based treatment , alone or in combination with other therapeutics , is feasible and may lead to complete or functional cure from hbv infection.9 , 35 , 36 renewable production of therapeutic gene silencers from stable dna  templates is useful to achieve sustained silencing . this feature is particularly important to inhibit gene expression of pathogens , such as hbv , that cause chronic infection . pol iii promoters , such as the u6 small nuclear ( sn)rna and rnase p h1 , have been widely used to encode shrnas that mimic pre - mirs . small size and constitutive transcription of short sequences in most tissues are useful features of the pol iii promoters . compatibility of dna expression cassettes with viral vectors , such as aavs , is valuable for delivery of therapeutic sequences . aavs are well characterized , have a good safety profile , and use of the vectors has extensively been evaluated in clinical trials . the utility of aavs was exploited here to generate hepatotropic self - complementary particles that delivered silencing sequences to the liver . demonstration that expression of hbv - targeting shrnas from a u6  promoter may cause lethal toxicity in  vivo was an important development.12 , 18 the limited range of transcriptional control by pol iii promoters is a drawback for their therapeutic application . pol ii promoters have greater flexibility and ability to regulate transcription in a tissue - specific manner and respond to subtle regulatory signals . we harnessed this property to transcribe trimeric pri - mir mimics from the liver - specific mttr pol ii promoter . sustained expression from the mttr pol ii promoter - containing cassettes enabled silencing of hbv replication over a prolonged period . this inhibitory action following single administration of the recombinant scaavs has potential therapeutic utility and was an improvement over what has previously been described . michler and colleagues also demonstrated good silencing of hbv in transgenic mice following administration of 1   10 scaavs expressing a single apri - mir from a liver - specific promoter . this effect , which was observed after administration of a similar dose of scaav to what we report here , accords with our observations . chen and colleagues reported on inhibition of markers of hbv replication for 22  weeks following a single systemic administration of 1   10 scaavs to hbv transgenic mice , which is 10-fold higher than the number of vector particles that was injected in our study . liver - specific expression of potent gene silencers that are more lastingly effective at a lower dose of vector particles are useful to limit side effects . sequential administration of scaavs pseudotyped with different capsids is useful to avoid a host s immune response to the vectors and prolong anti - hbv efficacy . safety of exogenous rnai activators is vitally important for their use in a therapeutic context . unintended side effects may derive from immunostimulation by duplex rna , silencing of host cellular sequences , disruption of natural rnai mechanisms , and effects of the vectors . our data have shown that scaavs pseudotyped with capsid 8 to achieve hepatotropic delivery of apri - mirs do not have discernible toxic effects . since the apri - mirs are transcribed within the nucleus , and unlike synthetic sirnas delivered with non - viral vectors do not traverse the endosomal compartment , toll - like receptor ( tlr ) activation is not expected . although pattern recognition receptors may be activated in the cytoplasm , our observations show no evidence for stimulation of the ifn response by apri - mir - expressing scaavs . moreover , non - specific indicators of hepatotoxicity , including alt and histological evaluation , were also not perturbed following scaav administration . markedly elevated alt was a notable feature of the liver injury caused by expression of anti - hbv u6 shrnas.12 , 18 collectively , our data indicate that scaavs encoding mono- and trimeric hbv - targeting sequences safely achieve lasting silencing of the virus in  vivo . developing new approaches to affect a cure from hbv infection has become a very active field of investigation ( reviewed in revill et  al . ) . advances with improved understanding of the basic biology of hbv replication have facilitated identification of new targets that may be used to inactivate the virus . improved insights into the deficiencies of innate , cell - mediated , and humoral immunity that occur in hbv chronic carriers have also led to new approaches to treatment . demonstration that toll - like receptor 7 stimulation and ifn - induced degradation of cccdna by apobec3a and 3b , are active against the virus are interesting new advances . use of gene editors to mutate hbv cccdna specifically has also showed promise ( reviewed in ely et  al . ) . as with rnai - based therapies , ensuring specificity of the therapeutic for the intended target is important . ultimate successful treatment is likely to use combination strategies , and rnai - based therapeutic approaches such as are described here may be a useful component of future therapies . anti - hbv sequences targeting x were extracted from previously described ptz57r plasmid containing mir-31/5 or mir-31/5,8,9 sequences which were each linked to the mttr promoter.21 , 28 both mir-31/5 and mir-31/5,8,9 mttr cassettes were excised with  asc i , blunt ended , and then inserted into eco rv sites of the scaav - generating transgene plasmid ( pbs - h1-rsv - gfp ) . to limit risk of recombination during scaav propagation , this plasmid contains itrs from serotype 2 ( itr2 ) and serotype 4 ( itr4 ) that flank the transgene . insertion of mttr mir-31/5 and mttr mir-31/5,8,9 resulted in substitution of the antiviral sequences for the rsv - gfp cassette to create paavmttr31/5 and paavmttr31/5,8,9 . to create the empty plasmid ( paav ) , the gfp sequence was deleted from pbs - h1-rsv - gfp by digestion with nhe i and bam hi , blunt ending , and then self - ligation of the backbone . plasmids encoding the individual apri - mir cassettes under control of the cmv promoter / enhancer , together with mutant and wild - type targets have been described previously . hydrodynamic injection with the pch-9/3091 replication competent sequence , plasmids containing antiviral cassettes , and targets was carried out as described . scaav vectors produced in this study were pseudotyped with serotype 2 ( scaav2 ) or serotype 8 ( scaav8 ) capsids for use in cell culture or in  vivo , respectively . large scale production , amplification , and purification of scaavs were carried out according to published methods with minor modifications.43 , 44 briefly , hek293 cells were co - transfected with recombinant scaav genome - containing plasmid ( paavmttr31/5 or paavmttr31/5,8,9 , or paav , or pbs - h1-rsv - gfp ) , the plasmid carrying aav2 or aav8 rep and cap genes , and plasmid encoding adenoviral helper genes , pxx-6 and pacg , respectively . after 72  hr of incubation at 37c , cells were harvested and lysed using three cycles of freeze thawing . to digest the host dna and rna , lysates were incubated with 50  u / ml of benzonase at 37c for 1  hr . there were 50  l aliquots of the pure virus that were stored at 80c . titers of viral particle preparations were determined using quantitative pcr ( qpcr ) with primer sets targeting the promoter regions of  the vectors ( mttr forward : 5 gcactgggaggatgttgagt 3 and mttr reverse : 5 cccctgttcaaacatgtcct3 or rsv forward : 5tctgaggggactagggtgtg3 and rsv reverse : 5ggactcctaaccgcgtacaa3 ) . preparation of the adenoviral vectors used as controls for some of the investigations has been described previously . to assess inhibition of hbv gene expression in huh7 cells , cultures were initially transfected with an hbv replication competent plasmid containing a greater - than - genome length viral sequence ( pch-9/3091 ) . after 5  hr , the transfected cells were infected with aav at an moi of 1,000 to 1,000,000 . hbv replication inhibition in  vivo was assessed by injecting six hbv transgenic mice per group with 1   10 viral particles via a tail vein . hbsag was measured using the monolisa ag hbs plus immunoassay kit ( bio - rad ) . assays were carried out on culture supernatants at 48 and 72  hr post aav infection or in serum of mice over a period of 40  weeks . serum hbv particle equivalents were measured using real - time qpcr with faststart sybr green master mix ( roche diagnostics ) . primers specific for the hbv surface orf were used as previously reported . for hbv core antigen staining , aav - infected mice were sacrificed 2 or 40  weeks after injection and livers fixed in 10% buffered formalin ( sigma ) . immunohistochemical staining of tissue sections was performed using anti - hbv core antigen primary antibody ( hbcag , abcam ) and an ultra - sensitive abc peroxidase staining kit ( pierce , thermo scientific ) . expression and processing of anti - hbv pri - mirs in  vitro was assessed by infecting liver derived huh7 cells with aavs at a moi of 100,000 . using tri reagent ( sigma ) , total rna was extracted from cells 3  days after the infection . to assess pri - mir expression and processing in  vivo , hbv transgenic mice were used according to a protocol approved by the university of the witwatersrand animal ethics screening committee . a dose of 1   10 viral particles / mouse was injected via a tail vein . livers were then harvested from mice killed at 2 , 8 , 28 , and 40  weeks after scaav administration . northern blot hybridization analysis of 30  g of total liver rna was performed according to previously described methods . radioactively labeled probes with the following sequences were used to detect mature guides : guide 5 probe , 5ccgtgtgcacttcgcttc 3 ; guide 8 probe , 5caatgtcaacgaccgacc 3 ; or guide 9 probe , 5 taggaggctgtaggcata 3. after stripping , blots were rehybridized to a u6 snrna probe , with sequence 5 tagtatatgtgctgccgaagcgagca 3 , to verify equal loading of rna in each of the lanes . to detect ifn- , oas-1 , and ifit-1 transcript concentrations , livers  from mice were harvested 6  hr after aav injection and rna isolated using tri reagent ( sigma ) . 1  g of rna was used for  reverse transcription using a quantitect reverse transcription kit ( qiagen ) . resultant cdna was then used for sybr green - based real - time qpcr using faststart sybr green master mix ( roche diagnostics ) . to assess induction of release of inflammatory cytokines by aavs , serum concentrations of il-6 , il-10 , mcp-1 , ifn- , tnf- , and assays using a mouse inflammation kit ( bd biosciences ) were carried out on serum taken immediately before and 6  hr after scaav administration to groups of mice , which each comprised four animals . cba reactions were run on a fortessa lsr flow cytometer using the bd facsdiva software . alt activity was measured in serum samples collected at weeks 1 , 2 , 3 , 4 , 6 , and 8 after scaav injections . assays were carried out using an  advia 1800 chemistry system ( siemens ) at the accredited facilities of the south african national health laboratory service ( nhls , johannesburg , south africa ) . to assess for change in liver tissue morphology after aav injection , livers were harvested from mice sacrificed at 2 or 40  weeks after injection with 1   10 aav viral particles and fixed in 10% buffered formalin ( sigma ) . the tissues were embedded in paraffin , sectioned , and then stained with hematoxylin and eosin ( h&e ) or sirius red according to standard procedures ( ampath laboratories , auckland park , south africa ) . differences were determined using the student s two - tailed t  test and were considered significant when p  0.05 . p.a . oversaw the research and wrote the manuscript with input from the other authors .
chronic infection with hepatitis b virus ( hbv ) remains a problem of global significance and improving available treatment is  important to prevent life - threatening complications arising in persistently infected individuals . 
 hbv is susceptible to silencing by exogenous artificial intermediates of the rna interference ( rnai ) pathway . 
 however , toxicity of pol iii cassettes and short duration of silencing by effectors of the rnai pathway may limit anti - hbv therapeutic utility . to advance rnai - based  hbv gene silencing , 
 mono- and trimeric artificial primary micrornas ( pri - mirs ) derived from pri - mir-31 were  placed under control of the liver - specific modified murine  transthyretin promoter . 
 the sequences , which target the x sequence of hbv , were incorporated into recombinant hepatotropic self - complementary adeno - associated viruses ( scaavs ) . 
 systemic intravenous injection of the vectors into hbv transgenic mice at a dose of 1   1011 per animal effected significant suppression of markers of hbv replication for at least 32  weeks . 
 the pri - mirs were processed according to the intended design , and intrahepatic antiviral guide sequences were detectable for 40  weeks after the injection . 
 there was no evidence of toxicity , and innate immunostimulation was not detectable following the injections . 
 this efficacy is an improvement on previously reported rnai - based inhibition of hbv replication and is important to clinical translation of the technology .