Patent Application: US-40218106-A

Abstract:
the present invention provides plant - derived agents to interfere with the nonstructural ns1 gene from the influenza a virus subtype h1n1 . more particularly , the sirnas that exhibit strong inhibitory activity towards ns1 , which effectively suppress replication of the influenza virus in mammalian cells . the invention further provides methods for production of sirnas for the suppression of a broad range of influenza viral subtypes with sequence homologies .

Description:
the invention provides a method of producing an agent comprising the steps of : ( a ) amplifying a region of the h1n1 ns1 gene by pcr ; ( b ) cloning the pcr product in pgem - t vector ; ( c ) digesting the pgem - t easy derivative with restriction enzymes ; ( d ) cloning the resulting fragments into corresponding sites in pbluescript skii (−) derivatives to form a “ 35s - s 5mns1 - tga1 intron - as 5mns1 - nos ” cassette ; ( e ) digesting the cassette with restriction enzymes ; ( f ) cloning of the digested cassette into the t - dna in a pbi101 backbone plasmid derivative ; and ( g ) mobilizing the binary vector into agrobacterium tumefaciens strain gv3101 / mp90 for transformation of tobacco cultivar samsun nn . the agent may be small interfering rna or sirna , and the pcr may be performed by using two specific primers . the primers are represented by one of the following sequences : i .) seq id 1 : 5 ′- gggcggccgcggatcc atggacccaaacactgtg - 3 ′; or ii .) seq id 2 : 5 ′- caactagt atttcgctttcagtatga - 3 ′. the restriction enzymes may be bamhi , spei , noti , and kpni . the invention also provides a method of suppressing virus propagation in mammalian cells infected with the influenza virus , comprising the steps of : ( a ) harvesting 5mns1 sirnas from the leaves of transgenic plants ; and ( b ) transfecting confluent madin - darby canine kidney ( mdck ) cells with plant - derived 5mns1 sirnas . preferably , the influenza virus is h1n1 , and the plant - derived 5mns1 sirnas have anti - viral efficacy . the invention further provides a set of 5mns1 sirna which bind to and deactivate mrna of nonstructural protein ns1 from influenza a virus subtype h1n1 , and a “ 35s - s 5mns1 - tga1 intron - as 5mns1 - nos ” cassette . in another embodiment , the invention provides a vector comprising a plasmid and the cassette and a cell or cell line incorporating or transfected with the plasmid and the cassette . the following experimental section is intended to be illustrative , and not limiting of the scope of the invention . described herein are plant - derived sirnas against the influenza virus could inhibit viral replication . the sirnas are derived from a 0 . 4 - kb fragment representing the 5 ′- portion of the ns1 gene in strain a / wsn / 33 , subtype h1n1 . this invention also provides cost - effective technique in utilizing transgenic plants for large - scale sirna production . influenza a viruses are medically - important viral pathogens that cause significant mortality and morbidity throughout the world . their easy transmission , antigenic shift and drift have made current methodology of vaccination and therapy limited in efficacy [ 22 ]. inhibitors of the anti - m2 ion channel and neuraminidase are common drugs for influenza , but both have their drawbacks . the anti - m2 ion channel inhibitors ( e . g ., amantidine ) induce viruses to develop drug - resistant mutations , while the neuraminidase inhibitors ( e . g ., tamiflu ), though very potent , are effective only at early disease onset . to investigate if plant - derived sirnas against the influenza virus could inhibit viral replication , the inventors selected a 0 . 4 - kb fragment representing the 5 ′- portion of the ns1 gene in strain a / wsn / 33 , subtype h1n1 . the ns virion rna ( vrna ) consists of about 890 nucleotides and encodes two nonstructural proteins , ns1 and ns2 . the sequence of this vrna is highly - conserved among different subtypes of influenza viruses [ 23 ]. the ns1 protein has not only been proposed to regulate viral replication cycle , splicing and translation of mrnas [ 24 ], but also been shown to have inhibitory effect on cellular mrna maturation and cellular antiviral response [ 25 ]. thus , the ns1 gene plays an important role in virus replication and virus - host interactions . the chosen 0 . 4 - kb fragment was amplified by polymerase chain reaction ( pcr ) using forward primer 5 ′- gggcggccgcggatcc atggacccaaacactgtg - 3 ′ with noti ( in italics ) and bamhi ( in bold ) sites incorporated at its 5 ′- end , and reverse primer 5 ′- caactagt atttcqctttcaqtatga - 3 ′ with an added spei site ( in italics ). the underlined nucleotides represent ns1 sequences . the pcr product was initially cloned in pgem - t easy vector ( promega ) for verification of dna sequence . subsequently , the pgem - t easy derivative was digested with either bamhi and spei or noti and spei . the 0 . 4 - kb bamhi - spei 5mns1 fragment was cloned into corresponding sites in a pbluescript skii (−) derivative that contains the arabidopsis tga1 intron [ 19 ] inserted at its spei - xbai site . next , the 0 . 4 - kb noti - spei 5mns1 fragment from the pgem - t easy derivative was cloned in the noti - xbai site of the pbluescript sk ( ii )(−) derivative containing the dna fusion of “ sense 5mns1 - tga1 intron ,” to generate a dsrna cassette “ sense ( s ) 5mns1 - tga1 intron - antisense ( as ) 5mns1 .” this cassette was then released by noti and bamhi digestion , and , with the help of a noti / xbai adaptor ( upper strand , 5 ′ ggccgagttgtta3 ′; lower strand , 5 ′ ctagtaacaactc3 ′), was cloned in the bamhi - xbai site between the camv 35s promoter and the nos terminator , in another pbluescript skii (−) derivative . the resulting vector therefore contains a cassette of “ 35s - s 5mns1 - tga1 intron - as 5mns1 - nos ”( fig1 c ). this cassette was further digested with noti and kpni , and was cloned into corresponding sites within the t - dna in a pbi101 backbone plasmid derivative ( clontech , palo - alto , usa ). the binary vector was then mobilized into agrobacterium tumefaciens strain gv3101 / mp90 for transformation of tobacco cultivar samsun nn by the leaf - disk procedure [ 20 ]. hence , transgenic plants obtained in agrobacterium - mediated plant transformation from this binary vector should produce hairpin dsrna , which would subsequently be processed into sirnas by the ptgs machinery . reports have shown that “ sense - antisense ” cassettes can be transcribed to produce sirnas after transfection of host cells [ 11 , 26 , 27 ]. to investigate if the construct generated in this study ( fig1 c ) could produce sirna in transgenic tobacco , rnas were extracted from tobacco leaves using trizol ( invitrogen ). twenty μg of total rna were separated on a 15 % polyacrylamide gel containing 7 m urea , and were electroblotted onto a nitrocellulose membrane ( genescreen plus ®, perkinelmer life sciences , inc .). the blot was then hybridized overnight at 42 ° c . to 32 p - utp - labeled 5mns1 riboprobes generated using the riboprobe ® in vitro transcription systems ( promega ), in a solution of 50 % ( v / v ) formamide , 250 mm nacl , 7 % sds and 125 mm phosphate buffer , ph 7 . 0 . after hybridization , the blot was washed twice with 2 × ssc plus 0 . 5 % sds , and was then analyzed using a phospho - imager . the volumes of the synthetic sirna and of the sirna from transgenic plants were measured using an imagequant software ( molecular dynamics ), and the amount of sirna in the plant rna sample was calculated based on its volume relative to that of synthetic , known amount of rna oligos . of the 21 independent transformants screened , 13 showed obvious sirna production . the levels of sirna accumulation in different lines varied , some produced obvious signals after an overnight exposure using a phospho - imager , while others barely yielded visible signals ( data not shown ). the sirna signals in selected transgenic lines are shown in fig2 . transgenic lines 1 and 2 had apparent accumulation of 5mns1 sirnas , while lines 8 and 9 produced much lower levels of the same sirnas . in line 10 , the sirnas were barely detectable . a variation in sirna levels may be due to several reasons . first , t - dna location in the genome could affect expression . in agrobacterium - mediated plant transformation , t - dna is transferred from the bacterium to the eukaryotic host cell and further integrated into the host genome [ 28 ]. if the transgenes were inserted in the genome where active transcription occurs , the transgene would be active . otherwise , it would be less active or even silent . second , the copy number of the transgene may be a contributing factor in expression levels , although in some cases , transgene activity may not be directly proportional to its copy number due to co - suppression . third , methylation of transgene may occur , especially at or near the promoter , if it is considered foreign . as a safeguard , the host generally has a mechanism to methylate and inactivate the transgene . this has been reported with foreign dna expressing dsrna in ptgs [ 29 , 30 ], and is supported by a requirement of dna methylase in initiating rna - dependent dna methylation [ 31 ]. as revealed by an increasing number of reports , sirna is the hallmark in triggering rnai . therefore , the accumulated 5mns1 sirnas in the transgenic plants should initiate degradation of ns1 transcripts or endogenous tobacco transcripts with sequences complementary to 5mns1 . a blast analysis was performed with 5mns1 as query sequence for such complementation in transcripts of tobacco or species evolutionarily close to tobacco , but no match was identified . northern blot analysis of tobacco total rna with the 5mns1 probe also did not yield any obvious bands . therefore , 5mns1 does not seem to share homology to any tobacco transcripts , and would not cause unintended degradation of rna transcribed from endogenous genes . this is consistent with the fact that no abnormal phenotypes were observed in all the transgenic lines ( data not shown ). to test if the plant - derived 5mns1 sirnas were functional in degrading ns1 transcripts specifically , 5mns1 - and eyfp - t2m - expressing binary vectors were introduced into agrobacterium cells by inoculating in an induction solution containing 1 g / l nh 4 cl , 0 . 3 g / l mgso 4 . 7h 2 o , 0 . 15 g / l kcl , 0 . 01 g / l cacl 2 , 0 . 0025 g / l feso 4 . 7h 2 o , 2 mm phosphate , 1 % glucose , 20 mm 2 -( n - morpholino ) ethanesulfonic acid ( mes , ph5 . 5 ), 100 μm acetosyringone , 50μg / ml kanamycin and 50 μg / ml gentamycin . the eyfp - t2m contains eyfp fused in - frame to a mutant version of the arabidopsis tga2 gene ( t2m ), and is used as an expression reference after agroinfiltration [ 21 ]. following overnight culture at 28 ° c ., the cells were collected by centrifugation at 3 , 000 × g for 15 min , and then resuspended in an infiltration solution containing 10 mm mes ( ph5 . 5 ), 10 mm mgso 4 and 100 μm acetosyringone . the resuspended agrobacterium cells were adjusted to an od 600 of 0 . 8 with the same solution before infiltration of tobacco leaves using a 1 - ml syringe . after two days , total rna was extracted from the infiltrated leaf areas for northern blot analysis . five μg of total rna , extracted from the agroinfiltrated and non - infiltrated leaf areas , were separated on a 1 . 2 % agarose gel , blotted with 20 × ssc onto a nitrocellulose membrane , and hybridized to 32 p - dctp labeled dna probes generated from 5mns1 and eyfp dna fragments using a rediprime ™ ii random prime labelling system ( amersham , uk ). hybridization was performed at 65 ° c . overnight in a buffer containing 250 mm nacl , 7 % sds and 125 mm phosphate , ph 7 . 0 . after hybridization , the blot was washed twice at room temperature in 2 × ssc plus 0 . 5 % sds , then at 65 ° c . for 15 min in 0 . 2 × ssc plus 0 . 1 % sds . the blot was analyzed using a phospho - imager . as shown in fig3 b , all the 3 transgenic lines 1 , 9 and 16 , representing high , middle and low accumulation of 5mns1 sirna , respectively , had reduced 5mns1 rna levels , indicating that plant - derived 5mns1 sirnas indeed triggered ptgs of ns1 in vivo . a negative correlation was observed between the levels of 5mns1 sirnas and 5mns1 transcripts in infiltrated tobacco leaves . to obtain a percentage of the 5mns1 transcript level in the transgenic lines relative to that of wild - type , volumes of each 5mns1 band and of the reference eyfp band were determined using the imagequant software , and percentage was calculated using the formula described in the fig3 legend . in transgenic tobacco line 1 , which had the highest level of 5mns1 sirna accumulation , the percentage was only 4 . 7 ± 3 . 0 %, demonstrating high efficiency of this line in 5mns1 - specific rna degradation ( fig3 c ). in rnai studies , synthetic or in vitro expressed sirnas have been used in transfection of target cells [ 26 ], and injection of worms [ 32 ] and animals [ 33 ], for evaluation of sirna efficacy . to test if the 5mns1 sirnas produced in transgenic tobacco could be potentially used in suppressing viral propagation in mammalian cells infected with the influenza virus , 5mns1 sirnas were harvested from the leaves of transgenic plants for transfection of mdck cells . confluent madin - darby canine kidney ( mdck ) cells grown in a t - 175 flask were washed twice with phosphate - buffered saline ( pbs ) and trypsinized for 10 min in 10 ml trypsin solution at 37 ° c . after termination of trypsinization with 20 ml pbs , the cells were collected by centrifugation for 5 min at 1 , 500 rpm with an eppendorf 5810r centrifuge , and were washed twice in 30 ml cold pbs , followed by one wash in 30 ml cold rpmi1640 medium ( gibco ), before resuspension in cold rpmi1640 to a density of 1 × 10 7 cells / ml . the amount of sirna in total rna was quantified by sirna analysis ( fig4 a ). the sirna oligo ns - 128 , ( 5 ′- cggcuucgccgagaucagadadt - 3 ′) was used as a positive control , since it has been proven best of three ns1 - targeting sirna oligos [ 8 ]. cells transfected with rna from non - transformed plants were the negative control , and cells transfected with water constituted the mock transfection . subsequently , 500 μl resuspended cells were transferred into a 0 . 4 - cm pre - chilled cuvette , and were mixed with 10 μl water , 10 μl water with 42 ng ns - 128 , 10 μl wild - type rna sample or 10 μl rna sample containing 42 ng sirnas from transgenic plant . equal amounts of total rna from wild - type or transgenic plants were used . the cuvette was kept on ice for 10 min , before electroporation at 0 . 4 kv and 960 μf using a gene pulser system ( bio - rad ). cells were then transferred into 5 . 6 ml of pre - warmed mdck medium ( mem , 10 % cow serum , 1 % penicillin and 1 % streptomycin ). three ml were transferred into a 6 - well plate and incubated at 37 ° c . for 24 hr before infection with the influenza virus . twenty four hr after transfection , cells in each well were washed twice with pbs , and 300 μl diluted influenza virus strain a / wsn / 33 ( moi = 0 . 001 in pbs ) was added into the well . after shaking the mixture for 1 hr , the viruses in the supernatant were discarded , and 2 ml infection medium ( 0 . 5 μ / ml tpck - trypsin ( sigma ), 0 . 5 % fcs ( gibco ), 1 % ps with mem ( gibco )) were added into the well . the cells were then incubated at 37 ° c . supernatants were collected at different post - infection time points for the ha titre test as described [ 8 ]. the ha titre , which is an indicator of virus replication , was determined at 24 , 36 and 48 hr post - infection . the mock - transfected and the negative control cells showed similar ha titre , indicating that rna from wild - type tobacco plants did not suppress viral replication . though the ha titre values varied in 3 separate sets of transfection and infection studies , a phenomenon unavoidably associated with conditions of the cells e . g . passage history , both plant - derived and synthetic sirnas significantly reduced h1n1 viral replication . the antiviral effect of sirna was most prominent at 36 hr post - infection ( fig4 c ). in one set of experiments , plant - derived sirna proved superior to the ns - 128 oligo ( fig4 c , experiment a ). these results strongly support the conclusion that 5mns1 sirna from transgenic plants can effectively suppress replication of the influenza virus in mammalian cells . in addition , plant sirnas showed similar suppression ability as the synthetic sirna ns - 128 , demonstrating that plant - derived sirnas confer the same efficacy . given the fact that transgenic plants can generate sirnas targeting different areas of the 5mns1 transcript , and that ns1 sequences are highly - conserved among influenza viruses [ 23 ], 5mns1 sirnas from transgenic plants should suppress the replication of a broad range of influenza viral subtypes with sequences homologous to the 5mns1 . while these results clearly indicate antiviral effects of plant - derived 5mns1 sirnas , the invention is primarily focusing on developing a strategy for economical and sustainable production of sirnas . besides using transgenic technology described herein , a pool of sirnas can also be generated with dicer - dependent kits . when compared with the transgenic approach , the later method is much more expensive , since it involves more expensive reagents ( i . e . ntps , dicer , rna polymerase ), more complicated steps ( i . e . in - vitro transcription , in - vitro cleavage of dsrna , clean - up ) and a more experienced technician / scientist . the high cost not only limits production scale , but also requires repetition of the production process if the sirnas are to be used over and over again . therefore , the current proof - of - concept invention demonstrates that transgenic plants are superior to the commercial kits for sirna production , and the time taken for generating them would be well compensated . though the ns1 - targeting sirnas possesses anti - viral effects , those against the np genes may be more potent in suppressing viral replication , as suggested by studies using mammalian cells [ 8 ] and animals [ 34 ]. in both studies , one of the np - targeting sirnas , np - 1496 , significantly reduced the virus titres . these observations indicate that mrna of the np gene might be a better target of sirna , if positional effects on sirna accessibility could be faithfully addressed . in our future study of using transgenic plant - derived sirnas for viral suppression , generating np - 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