Patent Application: US-75458804-A

Abstract:
a method for utilizing sirna for the detection of optimal sirna targeting sites capable of affecting the level of a target nucleic acid , comprising the targeting of one or more sites on an mrna with one or more sirna molecules and observing the level of the target nucleic acid .

Description:
the present invention will now be described in more detail with reference to the examples . the examples serve only as a representative group of the various embodiments of the present invention . the list of examples is thus not meant to be exclusive . the term “ sirna ” or “ sirna molecule ” as used herein means — definition : double stranded rna molecules in which each strand comprises 19 - 29 nucleotides that may or may not be chemical modified . the term “ chemical modification of the sirna ( molecule )” or “ chemical modified sirna ( molecule )” as used herein means any chemical modification of said rna sequence . non - limited examples of such chemical modifications are 2 ′- oh - modification , e . g . alkylation such as methylation ; 3 ′ or 5 ′ end modifications such as fluorescent labels , non - standard nucleotides , lipophilic linker molecules or peptides ; or modification or exchange of the phosphodiester bond , e . g . with phosporothioates , methylphosphonates , or polyamide . the term “ suitable range of sirna ” as used herein means any set of unique sequences or a mixture thereof , including a randomized collection of such molecules , provided by any of the means previously stated . there is a commonly held belief that mrna is generally accessible to sirna . however , the results presented herein demonstrate the clear differences in activity of various sirna molecules . also , even if most sirnas have some activity , some applications may require the identification of the best sirna . this would be of particular importance for therapeutic applications requiring modifications for example increasing the in vivo stability of sirna or for delivery of sirna in vivo . since such modifications gradually reduce activity ( 11 ), only the most effective sirna would have the necessary excess capacity to tolerate modification and still retain sufficient activity for mrna targeting . according to another aspect of the invention , various steps in the method can automated . an automated search for good target positions would be significantly cheaper and simpler to perform in the case of chemically synthesized sirnas , automation can be accomplished through streamlining of the following technologies : 2 . automated transfection by robotic mixing of rna , transfection agent and cells 4 . automated determination of target mrna expression by quantitative real - time rt - pcr ( ref qiagen or perkin elmer ) because the sirna is double stranded , an automated method would require the annealing of the separately - synthesized sense and anti - sense strands . this step could be automated as well , for example , by providing robotic means to pipette the sense and anti - sense rna from separate solutions into a new receptacle , wherein annealing occurs through controlled heating and gradual cooling under conditions that prevent evaporation . the screening strategy can also be performed with in vitro transcribed rna ( resulting in sense and anti - sense strands that afterward are annealed as described above ), starting with the synthesis of a dna strand consisting of the complementary strand of the particular strand of the sirna in conjunction with the sequence of the minus strand of a phage ( t7 , t3 , sp6 ) rna polymerase promoter . annealing of the above dna strand with the sense strand of the promoter would yield a template for in vitro transcription of rna . following in vitro transcription ( and preferably a purification step ), transfection and further analysis would proceed as described for chemically synthesized rna . a mixture of sirna can be obtained by enzymatic digestion ( by either dicer or rnaseiii ) of in vitro transcribed longer dsrna . in this case , following transfection with the rna mixture and total rna isolation , a different analysis step would be required to detect the most effective sirna molecules . the preferable way to do this is by primer extension analysis , which identifies the most prominent cleavage positions within the target mrna . from the known sequence of target mrna , the sequence of sirna causing the most prominent cleavage events can be inferred . the invention will now be described by way of examples . although the examples represent preferred embodiments of the present invention ( best mode ), they are not to be contemplated as restrictive or limiting to the scope of the present invention and the enclosed claims . 21 - nucleotide rnas according to seq id &# 39 ; s 1 - 41 were chemically synthesized using phosphoramidites ( pharmacia and abi ). deprotected and desilylated synthetic oligoribonucleotides were purified by reverse phase hplc . ribonucleotides were annealed at 10 μm in 500 μl 10 mm tris - hcl ph 7 . 5 by boiling and gradual cooling in a water bath . successful annealing was confirmed by non - denaturing polyacrylamide gel electrophoresis . sirna species were designed targeting sites within human protein serine kinase hi ( pskh1 ) ( acc . no . aj272212 ) and human tissue factor ( tf ) ( acc . no . m16553 ) mrnas . hela , cos - 1 and 293 cells were maintained in dulbecco &# 39 ; s minimal essential medium ( dmem ) supplemented with 10 % foetal calf serum ( gibco brl ). the human keratinocyte cell line hacat was cultured in serum - free keratinocyte medium ( gibco brl ) supplemented with 2 . 5 ng / ml epidermal growth factor and 25 μg / ml bovine pituitary extract . all cell lines were regularly passaged at sub - confluence and plated one or two days before transfection . lipofectamine - mediated transient co - transfections were performed in triplicate in 12 - well plates with 0 . 40 μg / ml plasmid ( 0 . 38 μg / ml reporter and 20 ng / ml control ) and typically 30 nm sirna ( 0 . 43 μg / ml ) essentially as described ( 29 ). luciferase acitivity levels were measured in 25 μl cell lysate 24 h after transfection using the dual luciferase assay ( promega ). serial transfections were performed by transfecting initially with 100 nm sirna , followed by transfection with reporter and internal control plasmids 24 h before harvest time points . for northern analyses and coagulation assays , hacat cells in 6 - well plates were transfected with 100 nm sirna in serum - free medium . for endogenous targets , lipofectamine2000 ™ was used for higher transfection efficiency . poly ( a ) mrna was isolated 24 h after transfection using dynabeads oligo ( dt ) 25 ( dynal ). isolated mrna was fractionated for 16 - 18 h on 1 . 3 % agarose / formaldehyde ( 0 . 8 m ) gels and blotted on to nylon membranes ( magnacharge , micron separations inc .). membranes were hybridised with random - primed tf ( position 61 - 1217 in cdna ) and gapdh ( 1 . 2 kb ) cdna probes in perfecthyb hybridisation buffer ( sigma ) as recommended by the manufacturer . for tf activity measurements hacat cell monolayers were washed thrice with icecold barbital buffered saline ph 7 . 4 ( bbs , 3 mm sodium barbital , 140 mm nacl ) and scraped into bbs . immediately after harvesting and homogenisation the activity was measured in a one - stage clotting assay using normal citrated platelet poor plasma mixed from two donors and 10 mm cacl 2 . the activity was related to a standard ( 29 , 30 ). one unit ( u ) tf corresponds to 1 . 5 ng tf as determined in the tf elisa ( 30 , 31 ). the activity was normalised to the protein content in the cell homogenates , as measured by the biorad bradford or dc assays . tf antigen was quantified using the imubind tissue factor elisa kit ( american diagnostica , greenwich , conn ., usa ). the samples were left to thaw at 37 ° c . and homogenised . an aliquot of each homogenate ( 100 μl ) was diluted in phosphate - buffered saline containing 1 % bsa and 0 . 1 % triton x - 100 . this sample was then added to the elisa - well and the procedure from the manufacturer followed . the antigen levels were normalised to the total protein content in the cell homogenates . analysis of htf sirna efficacy in cells transiently cotransfected with htf - luc and htf sirna ( i . e . analysis of rnai by sirna ( s ) in cotransfection assays ) the initial analysis of tf sirna efficacy was performed in hela cells transiently cotransfected with htf - luc ( fig1 b ) and htf sirna ( fig1 a ) using the dual luciferase system ( promega ). ratios of luc to rluc expression were normalised to levels in cells transfected with a representative irrelevant sirna , protein serine kinase 314i ( psk314i ). the sirnas had potent and specific effects in the cotransfection assays , with the best candidates , htf167i and htf372i , resulting in only 10 - 15 % residual luciferase activity in hela cells ( fig1 c ). furthermore , also a positional effect was found , as htf562i showed only intermediate effect , and htf478i had very low activity . this pattern was also found in 293 , cos - 1 and hacat cells ( fig1 c ), and with sirnas from different synthetic batches and at various concentrations ( the sirnas caused the same degree of inhibition over a concentration range of 1 - 100 nm in cotransfection assays ; data not shown ). coculturing sirna transfected cells with reporter plasmid transfected cells , both in hela cells and in the contact - inhibited growth of hacat cells , gave no indication of sirna transfer between cells ( data not shown ), despite the medium - mediated transfer previously reported by other investigators ( 25 ). the accessibility of the region surrounding the target site of the best sirna ( i . e . htf167i ) at a higher resolution was investigated . sirnas ( htf158i , htf161i , htf164i , htf170i , htf173i and htf176i ) were synthesized which targeted sites shifted at both sides of htf 167i in increments of 3 nts , wherein each of them shared 18 out of 21 nts with its neighbours ( see fig1 c ). surprisingly it was found that despite the minimal sequence and position - differences between these sirnas , they displayed a wide range of activities ( fig2 ). there was a gradual change away from the full activity of htf167i that was more pronounced for the upstream sirnas . the two sirnas htf158i and htf161i were shifted only nine and six nucleotides away , respectively , from htf167i , yet their activity was severely diminished . these results suggest that local factor ( s ) caused the positional effect . the results of cotransfection assays involving the use of forced expression of reporter genes as substrates may be difficult to interpret . the effect of sirna was therefore also measured on endogenous mrna targets in hacat cells ( fig3 a ) which express tf constitutively . the two best tf sirnas , htf167i and htf372i , showed strong activity also in this assay , as normalised tf mrna was reduced to 10 % and 26 %, respectively ( fig3 a ). interestingly , cleavage products , whose sizes were consistent with primary cleavages at the target sequences , were clearly visible below the depleted main band , though cleavage assays of mrna based on rnai have so far failed in mammalian systems ( 26 ). thus , the present invention also relates to sirna which is able to cleave mrna in mammalian cells . furthermore , the observed effect suggests that risc may be active also in mammals . the third best sirna in cotransfection assays , htf256i , also resulted in significant depletion of tf mrna levels ( 57 % residual expression , data not shown ). the remaining tf sirna did not show any activity as measured by northern assays ( fig3 b ), nor did they stimulate tf expression , a point of some interest , as transfection with chemically modified ribozymes can induce tf mrna three - fold ( data not shown ). thus , this relative inertness of irrelevant sirnas ( i . e . sirnas with & lt ;& lt ; non - specific & gt ;& gt ; effects ) further enhances the promise of sirna - based drugs . the coagulation activity in the hacat cells was reduced 5 - fold and 2 - fold , respectively , in cells transfected with htf167i and htf372i , compared to mock - transfected cells ( fig3 b and fig5 b ). the effect of sirnas on total cellular tf protein was also measured ( fig3 b ), and demonstrated an inhibitory effect that was generally greater than the observed effect on procoagulation activity . thus , according to the present invention , we conclude that the sirnas htf167i and htf372i display specificity and potency in a complex physiological system , and that we have demonstrated positional effects , as other sirna molecules against the same target mrna are basically inactive . data from a new series of tf sirna are in support of this conclusion ( data not shown ), and this inactivity of certain sirnas might be due to mrna folding structure or blockage of cleavage sites by impenetrable protein coverage ( 6 ). the time - course of mrna silencing was measured , and northern analysis of cells harvested 4 , 8 , 24 and 48 hours after start of transfection showed maximum sirna silencing after 24 hours ( fig5 a ). there seemed to be a difference in the apparent depletion rate , as htf167i reduced the mrna level more than htf173i at each time - point . similar observations were made for modified versions of htf 167i , in which the induced mutations ( m1 and m2 ) resulted in reduced inhibitory activity . mutations in the anti - sense strand had a more pronounced effect than the corresponding mutations in the sense strand . the fact that sirna - induced target degradation was incomplete ( a level of approximately 10 % of the target mrna remained even with the most effective sirnas ), may be due to the presence of a fraction of mrna in a protected compartment , e . g . in spliceosomes or in other nuclear locations . however , in view of the above data and data from competition experiments , a more likely possibility may be a kinetically determined balance between transcription and degradation , the latter being a time - consuming process . experiments in plants ( 27 ) and nematodes ( 12 , 13 ) have suggested the existence of a system whereby certain sirna genes are involved in the heritability of induced phenotypes . to investigate the existence of such propagators in mammalian cell lines , the persistence of the sirna silencing in hacat cells transfected at a very low cell density was measured . in an experiment based on serial transfection of reporter constructs there was a gradual recovery of expression between 3 and 5 days post - transfection , and the time - dependence of the sirna effect on endogenous tf mrna was similar ( fig5 b ). the level of tf mrna in mock - transfected control cells declined gradually during the experiment , in what appeared to be cell expansion - dependent down - regulation of expression . interestingly , the procoagulant activity showed little indication of recovering to control levels in transfected cells ( fig5 b , columns ). similar observations were made with htf372i and with a combination of htf167i , htf372i and htf562i ( data not shown ). as mentioned previously , the present sirna were mapped more systematically in order to determine whether mutations were equally tolerated within the whole sirna . a total of 8 different new single - mutant sirna were designed and named according to the position ( starting from the 5 ′ of the sense strand ) of the mutation ( s1 , s2 , s3 , s4 , s7 , s11 , s13 , s16 , i . e . seq id no 9 - 17 ). the previously described central single - mutant m1 ( eksempel 4 ) was included in this analysis and renamed s10 . all sirnas were analysed for productive annealing by denaturing page ( 15 %). all the various mutant sirnas were analysed for depletion of endogenous tf mrna in hacat cells , 24 h after lipofectamine2000 - mediated transfection , as previously described . a summary of the data is shown in fig7 . the wild type sirna , and the mutant s10 , included as positive controls , depleted tf mrna to ca 10 % and 20 % residual levels , as expected and previously reported . the activities of the other mutants fall in three different groups depending on their position along the sirna . mutations in the extreme 5 ′ end of the sirna ( s1 - s3 ) were very well tolerated , exhibiting essentially the same activity as the wild type . mutations located further in , up to the approximate midpoint of the sirna ( s4 , s7 , s10 , s11 ), were slightly impaired in their activity , resulting in depletion of mrna to 25 - 30 % residual levels . both the mutations in the 3 ′ half of the sirna , however , exhibited severely impaired activity . this suggested to us a bias in the tolerance for mutations in the sirna . the activities of several double mutants , in which the central position ( s10 ) was mutated in conjunction with one additional position ( s7 , s11 , s13 , s16 ), were also analysed . the bias in mutation tolerance was also evident for these double mutants , as the rank order of their activity mirrored that of the activity of the single mutants of the variant position . this observation strengthens the proposition that the differential activity of mutants is due to an intrinsic bias in the tolerance for target mismatches along the sequence of the sirna . the reason for such a bias might be linked to the proposed existence of a ruler region in the sirna which is primarily used by the risc complex to “ measure up ” the target mrna for cleavage ( 28 ). a series of sirnas with one modification each in the extreme 5 ′ and 3 ′ ends of the sirna strands [ p1 + 1 , m1 + 1 , a1 + 1 , i . e . seq id no 22 ( 32 ), 26 ( 36 ) and 30 ( 40 ), respectively ( the numbers in parentheses represent the seq id of the complementary second strand )] was initially synthesized . the 5 ′ end of the chemically synthesized sirnas might be more sensitive to modification since it has to be phosphorylated in vivo to be active ( 12 ). we therefore also included sirnas with two modifications only in the non - base pairing 3 ′ overhangs ( sirnas p0 + 2 , m0 + 2 and a0 + 2 , i . e . seq id no 23 ( 33 ), 27 ( 37 ) and 31 ( 41 ), respectively , cf . fig6 ), which were known to be tolerant for various types of modifications ( 33 , 32 , 7 ). northern analysis of transfected hacat cells demonstrated essentially undiminished activity of all the modified sirnas , with the exception of the sirna with allylation at both ends ( fig8 ). allyl - modification in the 3 ′ end only had no effect on activity . the presence of a large substituent in 2 ′- hydroxyl of 5 ′ terminal nucleotide might interfere with the proper phosphorylation of the sirna shown to be necessary by nykanen et al ( 12 ). we next wanted to know if any of these mutations were sufficient to increase the persistence of sirna - mediated silencing . endogenous tf mrna recovers gradually 3 - 5 days after transfection with wild type sirna targeting htf167 . transfecting hacat cells with active and chemically modified sirna in parallel , we could not demonstrate any significant difference in the silencing activities 3 and 5 days post - transfection ( data not shown ). the moderate modifications we had introduced , although exhibiting full initial activity , were therefore clearly not sufficient to substantially stabilize the sirnas in vivo . with the activity of the sirna still intact after our initial moderate modifications , the degree of modifications was extended to include either two on both sides or two on the 5 ′ end in combination with four in the 3 ′ end . due to the less promising results with the allylated versions from the first series , and the higher cost associated with these modifications , we decided to restrict ourselves to phophorothioate modifications and methylations for the next series . the new set of sirnas were likewise analysed for initial activity 24 h following transfection into hacat cells . normalized expression levels in cells transfected with modified sirnas were slightly elevated , at 16 - 18 % residual levels compared to 11 % in cells transfected with wild type . the most extensively phosphorothioated sirna proved to be toxic to cells , resulting in approximately 70 % cell death compared to mock - transfected cells ( measured as the expression level of the control mrna gapdh ). due to these complications , this sirna species was not included in further analysis . the remaining sirna species were evaluated for increased persistence of silencing by analysing tf mrna expression 5 days after a single transfection of 100 nm sirna . at this point , tf expression in cells transfected with wild type sirna had recovered almost completely ( 80 % residual expression compared to mock - transfected cells ) ( fig9 a ). in cells transfected with the most extensively modified sirna ( m2 + 4 ; seq id no 29 ( 39 )), however , strong silencing was still evident ( 32 % residual expression ). the less extensively modified sirna species ( p2 + 2 , m2 + 2 ; seq id no 24 ( 34 ) and seq id no 28 ( 38 ) respectively ), although less effective than me2 + 4 , consistently resulted in lower tf expression 5 days post - transfection ( 55 - 60 %) than the wild type . time - course experiments demonstrated that the wild type sirna was still the most effective 3 days post - 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