alzheimer 's disease ( ad ) is a degenerative disorder of the human central nervous system ( cns ) .
its clinical and neuropathological features are defined by a progressive loss of cognitive function and by the onset of a slowly progressive impairment of memory during mid- to late - adult life .
the neuropathological hallmarks of ad include the accumulation and aggregation of amyloid- peptide ( a ) , neurofibrillary tangles , astrocytic gliosis , and reductions in the numbers of both neurons and synapses in many areas of the brain , particularly in the cerebral cortex and hippocampus . strong evidence from multiple studies suggests that defects in a regulation are one of the central biochemical events leading to the development of ad .
the neurotoxic a fragment originates from the amyloid protein precursor ( app ) following sequential cleavages by  ( bace ) and -secretases ( presenilin complex ) .
observations on the physiological processing of app and on the effects of pathogenic mutations in the app and/or the presenilin genes have led to the hypothesis that aberrant processing of app into a peptides is linked to ad .
we have previously reported strong evidence indicating that the amyloid cascade is an early and critical event in the neurodegeneration associated with ad .
for example , cell lines and/or transgenic mice expressing mutant presenilin 1 ( ps1 ) , presenilin 2 ( ps2 ) , or app exhibit an accelerated rate of neurotoxic a formation .
moreover , the 4 allele of apolipoprotein e , a strong genetic risk factor for the development of ad , has been linked to either enhancing a aggregation or decreasing its clearance in brain tissue [ 5 , 6 ] .
altogether , these observations strongly suggest that targeting a metabolism is a worthwhile therapeutic approach and that reducing its level in the brain may block both the neurodegenerative process and cognitive decline .
most likely , an approach that reduces either the level of a or the rate of its aggregation and deposition in the brain would be beneficial for patients with ad . targeting the secretases may be risky because they appear to have multiple roles in cells .
we have decided to address the problem with a new generation of ribozymes ( rz ) targeting the first step in the amyloid cascade , specifically , the app mrna .
hepatitis delta virus ribozyme ( hdv rz ) is an interesting potential tool for the development of a gene - inactivation system because it is well adapted to the human cell environment .
in fact , this ribozyme offers several unique properties , including the natural ability to function in the presence of human proteins and at physiological magnesium concentrations as well as outstanding molecular stability ( i.e. , it has a long half - life ) .
recently , a novel target - dependent ribozyme that increases hdv rz fidelity was engineered .
this new ribozyme possesses a module ( the sofa , for specific on / off adaptor ) that switches the cleavage activity from off to on when in the presence of the appropriate substrate ( figure 1 ) .
specifically , this module is composed of three domains : a blocker , a biosensor , and a stabilizer .
the blocker sequence inhibits the cleavage activity of the ribozyme by intramolecularly binding the recognition domain of the rz , which was limited to only 7 nucleotides before the addition of the module .
binding of the blocker switches the ribozyme domain to an inactive state , namely , the off conformation .
upon addition of the substrate , the biosensor binds its complementary sequence on the substrate and unlocks the sofa module , thereby permitting a switch of the ribozyme into the active fold , namely , the on conformation .
the sequences of the substrate binding of both the ribozyme recognition and biosensor domains are not contiguous , but rather are separated by a small region called the spacer that varies from 4 to 7 nucleotides for optimal design . finally , the presence of a stem ( namely , a stabilizer ) that brings together both the 5 and 3 extremities has no effect on the cleavage activity but stabilizes the sofa - hdv rz in vivo against ribonucleases .
a proof of concept of this man - made ribozyme has been demonstrated both in vitro and in vivo using ribozymes that cleaved various mrna and viral rna [ 1113 ] .
the fact that the sofa - hdv rz is activated by its mrna substrate greatly diminishes its nonspecific effects ; consequently , it displays significant potential for applications in both functional genomics and gene therapy . in this study
, we evaluated the potential of the new sofa - hdv ribozymes as an rna silencing tool in mammalian cells . in cell culture , we demonstrated the effects of sofa - hdv rz targeting app mrna on a production .
sofa - hdv ribozymes were constructed using a pcr - based strategy that included two complementary and overlapping oligonucleotides . briefly , two dna oligonucleotides were synthesized and annealed with the reverse primer ( 5-ccagctagaaagggtcccttagccatccgcgaacggatgccca(n)6(p1)accgcgaggaggtggaccctg(n)4(bl ) ) and the sense primer ( 5-ttaatacgactcactatagggccagctagttt(n)12(bs)(n)4(bl)cagggtccacc ) , where n is a , c , g , or t , and p1 , bs , and bl indicate the p1 , biosensor , and blocker sequences , respectively .
it is important to note that both the p1 and bs segments were varied to correspond to specific app mrna sequences and that the bl was complementary to the first 4 nucleotides on the 5 end of the rz 's recognition domain .
for in vitro synthesis of the ribozymes , the sense primer also included the sequence of the t7 rna polymerase promoter at the 5 end .
the filling reaction was performed in a 100-l volume containing 20  mm tris - hcl ( ph 8.8 ) , 10  mm kcl , 10  mm ( nh4)2so4 , 2  mm mgso4 , 0.1% triton x-100 , 2  m of each dntp , 1  m of each dna oligo , and 5  u of pwo dna polymerase ( roche diagnostics ) . the reactions were ethanol precipitated and washed and the dna pellets resuspended in 56  l of deionized water .
the resulting pcr products were directly used for in vitro transcription reactions ( see below ) . for the in cellulo experiments ,
the sofa - hdv - rz cassettes were removed by digestion with bamhi and subcloned into prnat - u6.1/lentivector ( genscript ) under control of the u6 snrna promoter .
the resulting plasmids were named prnat - sofa - hdv - rz - appx , where x represents the app cleavage position .
rna transcriptions were performed as previously described . in the case of the sofa - hdv ribozymes ,
the resuspended dna pellets were used in 100-l transcription reactions containing 80  mm hepes - koh ( ph 7.5 ) , 24  mm mgcl2 , 2  mm spermidine , 40  mm dtt , 5  mm of each rntp , 0.01  u of pyrophosphatase ( roche diagnostics ) , 24  u of rnaguard ( amersham biosciences ) , and 10  g of purified t7 rna polymerase and allowed to proceed for 4  h at 37c .
the reactions were then treated with 4  u of rq1 dnase ( promega ) , phenol - chloroform extracted , ethanol precipitated , and washed . following these steps , the rna pellets were resuspended in 40  l of deionized water .
one volume of loading buffer ( 97.5% formamide , 0.05% bromophenol blue , 0.05% xylene cyanol , 10  mm edta ) was added , and the samples were fractionated by 8% denaturing ( 8  m urea ) polyacrylamide gel electrophoresis ( page , 19  :  1 ratio of acrylamide to bisacrylamide ) , using 45  mm tris - borate ( ph 7.5 ) and 1  mm edta .
the rna bands were visualized by uv shadowing , and the gel slices were cut out and eluted overnight using 500  mm ammonium acetate , 1  mm edta , and 0.1% sds .
after ethanol precipitation , the rna transcripts were resuspended in deionized water and quantified by uv absorbance at 260  nm .
the plasmid papp12 ( containing a full - length copy of the app mrna ) was used as a template .
after linearization by the stui restriction enzyme , mrna was synthesized as described above and purified using 5% page .
after purification , the transcripts ( 40  pmol ) were dephosphorylated in a final volume of 50  l containing 200  mm tris - hcl ( ph 8.0 ) , 10  u rnaguard , and 0.2  u of calf intestinal alkaline phosphatase ( amersham biosciences ) at 37c for 30  min .
the reactions were purified by extracting twice with phenol  :  chloroform , and the mrna was then precipitated with ethanol , washed with 70% ethanol , and dried .
dephosphorylated rna ( ~6  pmol ) was 5-end - labeled in a final volume of 10  l containing 3.2  pmol of [ -p]atp ( 6000  ci / mmol , new england nuclear ) , 10  mm tris - hcl ( ph 7.5 ) , 10  mm mgcl2 , 50  mm kcl , and 3 u of t4 polynucleotide kinase ( united states biochemicals ) at 37c for 90  min .
the reaction was stopped by the addition of formamide dye buffer ( 5  l ) , and the reaction mixtures were fractionated through denaturing 5% page gels and recovered as described above .
ribonuclease h ( rnase h ) reactions were performed with a library of randomized oligonucleotides ( 5-n6cd-3 , where n is for any a , c , g , or t residue and d is for any a , t , or g residue ) . specifically , nonradioactive app mrna ( 0.5  m ) and randomized oligonucleotides ( 5  m ) were preincubated for 10  min at 25c in a final volume of 8  l containing 20  mm tris - hcl ( ph 7.5 ) , 20  mm kcl , 10  mm mgcl2 , 0.1  mm edta , and 0.1  mm dtt .
rnase h ( 0.5 u , united states biochemicals ) was then added , and the samples were incubated at 37c for 30  min . after the incubation , 90  l of water was added , and the mixture was phenol  :  chloroform extracted .
four dna oligonucleotides complementary to the app rna were purchased from invitrogen ( 5-gttcctcagcctcttcct-3 ( position 928 - 911 ) , 5-tcagccagtgggcaacac-3 ( position 719 - 702 ) , 5-gtcaggaacgagaagggc-3 ( position 540 - 523 ) , and 5-ctgaatcccacttcccat-3 ( position 310 - 293 ) ) .
the p - end - labeled oligonucleotides were purified with denaturing 20% page , and the relevant bands were excised from the gel and eluted overnight at 25c , passed through a g-25 spun column , ethanol precipitated , washed , dried , and dissolved in deionized water ( 60  l ) .
5-p - labeled primer ( 6  l ) and 10x reverse transcriptase buffer ( 0.6  l of 500  mm tris - hcl ( ph 8.3 ) , 800  mm kcl , and 100  mm mgcl2 ) were used to resuspend the pellets resulting from the rnase h hydrolysis .
the primer annealing step was performed by successively incubating the samples at 65c for 2  min followed by 2  min on ice .
the reactions were initiated by adding 0.8  mm of each dntp , 3.3  mm dtt , and 100  u of superscript ii reverse transcriptase ( invitrogen ) in a final volume of 12  l .
the samples were incubated at 45c for 30  min and then ethanol precipitated and analyzed by 5% sequencing page .
dna sequencing reactions using the same primer were migrated on the same gels to allow for identification of the primer extension stops .
cleavage reactions were carried out under single turnover conditions ( [ rz ]  [ s ] ) , as previously described .
specifically , p - end - labeled app mrna ( 50  nm ) was mixed with sofa - hdv ribozymes ( 1  m ) in a 10-l mixture containing 50  mm tris - hcl ( ph 7.5 ) and 10  mm mgcl2 and then incubated at 37c for 1  h. the reactions were stopped by the addition of loading buffer , rna fractionated with denaturing 5% page , and analyzed with a phosphorimager .
mohamed chahine , laval university ) and human neuroblastoma sh - sy5y cells ( atcc ) were cultured in dulbecco 's modified eagle 's medium ( dmem , gibco ) supplemented with 10% ( v / v ) fetal bovine serum ( biomedia ) .
the hek-293 cells were transiently transfected with prnat - sofa - hdv - rz - appx ( rz - app - x ) plasmid using the calcium phosphate procedure . the empty prnat - u6.1 vector ( genscript )
this system consists of the multiply deleted packaging construct pcmvr8.91 ( which encodes gag , pol , tat , and rev ) , the pmd.g expressing vesicular stomatitis virus ( vsv - g ) surface glycoprotein ( g ) , and prnat - u6 ( either with or without rz - appx ) . to produce the infectious virions , hek-293 cells ( 2  10 ) were plated on 5 dishes ( 10  cm ) and transfected the next day with 20  g of rz - appx , 15  g pcmvr8.91 , and 5  g pmdg using the calcium phosphate procedure .
conditioned medium was harvested at 48  hr after transfection , cleared of debris by low - speed centrifugation , and filtered through 0.45  m filters ( sarstedt ) .
the filtrate containing the virions was concentrated by ultracentrifugation at 71,000  g for 90 minutes at 16c using a sw-40 beckman rotor , followed by a second cycle of centrifugation for the collected and resuspended pellets at 84,000  g for 90  min ( using a 4-ml centrifuge tube ; sw60 beckman rotor ) .
virions pellets were then resuspended in 0.5  ml of phosphate buffered saline ( pbs ) .
shsy-5y cells were infected with 0.2  ml of the virions expressing sofa - hdv - rz - appx in the presence of 6  g / ml polybrene ( hexadimethrine bromide , sigma ) .
three days postinfection , the medium was replaced with medium containing 600  g / ml g418 for selection .
the culture medium was changed every 3 - 4 days for the duration of the experiment . to test the expression of the rz - appx
, total rna was extracted from transduced cells using the trizol reagent according to the manufacturer 's recommendations ( invitrogen ) .
briefly , the primers , corresponding to the 3 complementary sequence of either sofa - hdv - rzx ( 5-gggtcccttagccatgcgcgaacg-3 ) or u6 rna ( 5-ggccatgctaatcttctctg-3 ) , were 5-end - labeled with [ -p]atp ( 6000  ci / mmol ; new england nuclear ) , as previously published , annealed to 10  g of total rna by a 5  min incubation at 65c and immediately chilled on ice .
the reactions were initiated with the addition of 0.4  mm of dntps , 10  mm dtt , and 200 units of superscript ii reverse transcriptase ( invitrogen ) in a buffer containing 50  mm tris - hcl ( ph 8.3 ) , 75  mm kcl , and 3  mm mgcl2 in a final volume of 50  l .
the samples were incubated at 42c for 50  min ; the reactions were stopped by heating the samples to 70c for 15  min and then fractionated through 10% denaturing polyacrylamide gel electrophoresis .
first - strand cdna synthesis was performed using 2  g of total rna in the presence of poly dt primers and 200 units of superscript ii reverse transcriptase .
aliquots of 2  l from the resulting single - stranded cdna products were used along with the appropriate primers ( see below ) for app and gapdh .
amplifications were performed for each sample from each separate well in a total volume of 25  l containing 1x sybr green universal pcr master mix and 400  nm of specific forward and reverse primers .
the primers were designed to overlap the boundaries of two exons ( to avoid amplification of genomic dna ) , using the primer express software v2.0 ( applied biosystems ) .
the first pair ( sense primer 5-ggcggtgttgtcatagcga-3 and antisense primer 5-tgcatcttggacaggtggc-3 ) provided an amplicon of 136 base pairs ( bp ) , whereas the second pair ( sense primers 5-aacgaagttgagcctgttgatg-3 and antisense primer 5-aacgaaggctggcacaac-3 ) amplified a 67-bp fragment .
amplification of gapdh mrna using the sense primer 5-cgacacttccagctctttgct-3 and antisense primer 5-gaatcagggttatctggtcatcg-3 , which produces an amplicon of 131  bp , was also performed .
the pcr amplifications were performed on an abi prism 7000 sequence detector system ( applied biosystems ) , according to the manufacturer 's instructions and using the following conditions : 1 cycle at 95c for 10  min , followed by 40 cycles at 95c for 15  s , 58c for 10  s , 72c for 20  s , and a final step at 60c for 60  s. the control samples were amplified without the reverse transcription step .
native and transfected cells were rinsed twice with ice - cold pbs and then lysed for 30  min on ice in cell lysis buffer containing 50  mm tris ( ph 7.6 ) , 150  mm nacl , 2  mm edta , 1% np-40 , 20  mm pmsf , and minicomplete protease inhibitors ( roche - diagnostic ) .
insoluble material was removed by centrifugation at 13,000  g for 15  min at 4c .
finally , the proteins were quantified using a standard bradford assay ( bio - rad ) .
briefly , 20  g of total protein from each sample was mixed with novex 2x reducing sample buffer containing 500  mm tris - hcl ( ph 6.8 ) , 20% glycerol , 10% sds , 0.1% bromophenol blue , and 5% -mercaptoethanol .
the samples were then boiled for 5  min and subjected to sds - page . following the migration ,
proteins were transferred onto a pvdf membrane ( millipore ) according to the manufacturer 's protocol .
the membranes were probed with an anti - app c - terminal antibody ( a8717 , sigma - aldrich ) and -tubulin specific antibody ( antibody e7 for -tubulin , developmental studies hybridoma bank ) .
the blots were revealed using a chemiluminescence detection system ( immobilon western , millipore ) according to the manufacturer 's recommendations .
the intensity of the signals was analyzed using image densitometry software ( imaging densitometry , bio - rad ) .
the level of -tubulin was used to normalize the levels of app ( i.e. , the ratio of app versus -tubulin ) to control for differences in the loading of total proteins .
modulations of the app levels in the cells treated with rz - app - x were expressed as a percentage of the level in the control cells ( empty vector ) .
following sh - sy5y transduction and during the selection , media was collected , preserved , and frozen at 80c .
following , secreted a was measured by sandwich elisa , according the manufacturer 's protocol ( human amyloid  ( a 1-x ) assay kit , ibl ) .
for the in vitro data , the results from several experiments were analyzed using student 's t - test .
the first step of this study consisted of designing a collection of ribozymes that produced in vitro cleavage of the app mrna . due to unfavorable competition with intramolecular base pairing , the target sequences located in single - stranded regions of an mrna are potentially more accessible for rz binding than those in double - stranded regions .
it has been demonstrated that both target site accessibility and the ability to form an active ribozyme - substrate complex constitute interdependent factors that can be addressed using a combinatorial library of oligonucleotides or ribozymes . to identify the cleavage sites with the greatest potential for targeting
, we adopted a procedure based on the use of a library of partially randomized oligonucleotides mimicking the interaction with the recognition domain of the target   ( figure 2 ) . in principle , all of the accessible sites within the app mrna should be specifically bound by an oligonucleotide and the resulting rna - dna heteroduplex subsequently hydrolyzed by rnase h. the resulting cleavage sites were identified by primer extension reactions using 5-end - labeled primers , and the most potent sofa - hdv ribozymes were tested for cleavage activity .
the library was composed of oligonucleotides that were 8 nucleotides in length corresponding to one residue before the cleavage site ( i.e. , position -1 ) , which had to be single stranded for cleavage to occur , and the 7 residues of the recognition domain of the ribozyme . it is important to note that this experiment considered only the binding domain of the ribozyme and not the sofa module . it would be irrelevant to perform rnase h assays using long oligonucleotides that included the complementary sequence of the ribozyme 's recognition domain , spacer and biosensor sequences . in that case
, the spacer would also be bound , leading to significant formation of the duplexes and the introduction of an important bias .
the library was designed while taking into consideration the sequence specificities of the hdv ribozyme .
specifically , the nucleotide in position 1 can not be a guanosine ; therefore , the 3 end residue of the oligonucleotide can not be a cytosine .
moreover , the first base between the ribozyme 's recognition domain and the target must be a gu wobble base pair .
this constraint led to a library of 12,288 different variants corresponding to the 5-n6cd-3. the action of a ribozyme within the 5-end of an mrna region should enhance the probability that the cleavage product results in an rna that can not encode an active protein .
because each primer produced a readable sequence of 200 to 300 bases , 4 different oligonucleotides were designed for the reverse transcriptase reaction to analyze the first 900 nucleotides of the app transcripts corresponding to the 1040 nucleotides of the 5 end ( see section 2 ) .
the relative level of accessibility in function of the intensity of the primer extension products is compiled in table 1 .
this analysis led to the identification of 10 potential sites , located from positions 276 to 885 , of the app transcript .
seven of these sites appeared to be highly accessible , including 5 that were located near position 450 .
a high concentration of such sites in the same area is indicative of a single - stranded region , although it may also result from a synergetic effect of several oligonucleotides binding the same rna transcript , resulting in unfolding of that region and increasing the possibility that additional oligonucleotides can also bind .
subsequently , hdv - rz with the appropriate recognition sequences was designed . to increase specificity ,
the resulting ribozymes were named sofa - hdv - rz - appx , where x represents the app cleavage position .
the ability of these ribozymes to cleave the 5-end - labeled app transcripts was tested under single - turnover conditions ( [ rz ]  [ s ] ) and analyzed via page ( figure 2 inset ) . clearly , all of the sofa - hdv ribozymes exhibited cleavage activity , although at different levels .
moreover , all of the ribozymes exhibited a specific cleavage at only the expected site .
the 4 sofa - hdv ribozymes that exhibited a cleavage level higher than 30% were conserved for the subsequent step .
these ribozymes included sofa - hdv - rz - app276 , -app753 , -app756 , and -app885 , with cleavage activities of 62% , 36% , 36% , and 71% , respectively .
these 4 potential sofa - hdv ribozymes targeting the app mrna were tested with the ribosubstrates online software ( http://www.riboclub.org/ribosubstrates ) .
this integrated software searches selected cdna databases for all of the potential substrates for a given sofa - hdv ribozyme .
these potential substrates include not only mrnas with perfect matches with the catalytic rna tested , but also the wobble bp and mismatches .
interestingly , none of these 4 potential sofa - hdv ribozymes seemed to have the potential for off - target effects ( data not shown ) .
moreover , this analysis indicated that no other cleavage could occur within the app gene family . therefore , the chosen sequences were specific to app mrna .
in other words , the sofa - hdv ribozymes that exhibited significant cleavage activity in vitro against a derived app transcript appeared to be specific to the app mrna . in an attempt to achieve a high level of expression of sofa - hdv ribozymes that maintain their affinity for the targeted mrna , we adopted the prnat / u6 ( which employs the u6 rna polymerase iii promoter ) for a high level of small rna expression .
the advantage of this promoter is that rna transcription terminates with the addition of 4 or 5 uridines ( u ) at the 3-end , and this change has only a minimal effect on sofa - hdv ribozyme folding based on rna structure predictions .
this approach also avoids nonspecific effects that might be caused by the transcription of additional regions of the vector sequence . to determine whether the prnat / u6 sofa - hdv - rz - appx vector could express the anti - app sofa - hdv ribozymes , these constructs were transfected into hek-293 cells .
two days after transfection , total rna from transfected cells was subjected to primer extension analysis .
endogenously synthesized u6 snrna and sofa - hdv ribozymes transcribed from the u6 promoter were detected , respectively , by u6- and drz p - labeled primers ( see section 2 ) .
as indicated by figure 3 ( lanes 1 to 8) , the specific extension products corresponding to u6 sofa - hdv - rz - app276 , -app753 , -app756 , and -app885 were detectable in the transfected cells .
an expression vector lacking a sofa - hdv ribozyme coding sequence ( prnat / u6 ) was used as a negative control .
no detectable band of drz p - labeled primers was observed with the empty vector ( figure 3 , lane 9 ) .
the expression levels for sofa - hdv - rz - app753 and -app885 were among the highest , while two other ribozymes ( sofa - hdv - rz - app276 , and -app756 ) exhibited weaker expression . considering the close and positive correlation between the level of app mrna , protein , and a deposition in ad ,
previously , it has been shown that the sofa - hdv rz expressed in hek-293 cells could be a powerful and specific gene silencing tool .
therefore , sofa - hdv - rz - app was transiently transfected into hek-293 cells , which are well known for the expression of endogenous app mrna .
the total rna was extracted from cells 48  h after transfection , and app mrna levels were quantified by real - time quantitative pcr ( qpcr ) .
the gapdh mrna was used as a control for the qpcr to normalize the app mrna levels .
a significant effect of sofa - hdv - rz - app ribozyme expression on cellular app mrna levels was observed ( figure 4 ) .
sofa - hdv - rz - app276 and -app753 transfection led to a highly significant ( p < 0.001 ; student 's t - test ) decrease in app mrna steady - state levels ( 70% and 80% , resp .
conversely , both sofa - hdv - rz - app756 and -app885 expression did not show a significant decrease at the app mrna level , suggesting that cleavage sites at positions 756 and 885 may not be as accessible in cellulo as they were in the in vitro assays on partial mrna transcripts .
the transfection of cells with an empty vector resulted in a faint increase in app mrna compared with untransfected cells ( figure 4 , lanes 5 and 6 ) , but this effect was not significant ( p > 0.05 ; student 's t - test ) .
more importantly , this experiment provided physical evidence that the expression of both sofa - hdv - rz - app276 and -app753 in hek-293 cells resulted in an important decrease of the targeted app mrna levels and that nonspecific effects of vector transfection could not account for this decrease . because the correlation between the level of mrna and its concomitant protein is not always linear , the effect of app 's directed sofa - hdv ribozymes on app protein levels was then investigated to verify whether the decrease in app mrna level results in a reduction at the protein level .
sofa - hdv - rz - app ribozymes were transfected into hek-293 cells . as a control
, cells were transfected with a prnat - u6 vector expressing gfp protein . at 48  h after transfection
subsequently , a western blot was performed with a specific anti - app c - terminal antibody as a probe .
the level of app was estimated by densitometry and normalized using endogenous -tubulin ( figure 5 ) .
as expected , cells expressing both sofa - hdv - rz - app276 and -app753 showed a drastic decrease in app levels compared with untransfected cells , those transfected with empty vector or those transfected with sofa - hdv - rz - app885 ( figure 5(a ) ) .
the last construct exhibited a decrease estimated to be less than 20% , whereas the two other ribozymes led to reductions of over 70% relative to the controls .
all of the changes were highly significant ( p < 0.001 ) when compared with either untransfected cells or cells transfected with prnat / u6 .
thus , the decrease in app mrna resulting from the expression of sofa - hdv ribozyme is correlated with the change observed at the protein level .
moreover , these data are strongly consistent with the hypothesis that the expression level of app mrna is closely and positively correlated with its concomitant protein level . from the perspective of ad therapy , any attempts to decrease app mrna levels should also result in a decrease in a levels . to assess whether a decline of app in ribozyme - treated cells leads to a decline in total a levels , the level of secreted a following sofa - hdv - rz - app expression was determined by elisa . for this experiment ,
the sofa - hdv - rz - app276 and sofa - hdv - rz - app753 were selected as the two more active and powerful ribozymes .
because neurons will be the target of the ribozymes in the context of ad , this ribozyme was tested on a neuronal cell type , shsy-5y , using a lentiviral system of expression .
this system is essential for transducing neurons because post - mitotic cells can not be efficiently transfected by other vectors . following shsy-5y transduction , the sofa - hdv - rz - app753 expression was tested for its effect on the reduction of app at both the mrna and protein levels ( data not shown ) . to evaluate the a level ,
media samples were collected and analyzed for a 
 1-x species , as both a 
 40 and a 
 42 are associated with ad pathogenesis . knocking down app with
lentiviral sofa - hdv - rzapp276 or ofa - hdv - rzapp753 transduction of shsy-5y cells reduced the total level of a by more than 30% ( figure 6 ) .
this result indicates that a sofa - hdv ribozyme could be a potential means of targeting app .
in this study , we designed a new molecular tool to target the top of the amyloid cascade , namely , the app mrna .
the sofa - hdv ribozyme is based on a new synthetic hdv ribozyme harboring a biosensor module that activates mrna cleavage only in the presence of the specific rna target substrate .
this specific on / off adapter ( sofa module ) provides not only a higher specificity to the hdv rz toward its target but also a higher cleavage capacity .
an initial experiment to identify the most susceptible site within the 5 end region of the app mrna was performed based on the use of a randomized library of short oligonucleotides mimicking the recognition domain of the ribozyme .
the hydrolysis of the formed rna / dna heteroduplexes by the rnase h led to the identification of 10 potential sites ( table 1 ) . in vitro cleavage of a partial app transcript by the corresponding appropriate sofa - hdv ribozymes revealed that 4 of these sites could be cleaved at a significant level ( figure 2 ) .
interestingly , an analysis of the sequence and secondary structure of the sofa - hdv ribozymes that exhibited only moderate cleavage activity indicated that misfolding of 5 out of 6 of these ribozymes may explain their limited potential ( data not shown ) .
therefore , only one of the sofa - hdv ribozymes did not cleave efficiently for any specific reason .
this result is excellent , considering that the initial analysis was based on the hybridization of small oligonucleotides and that the sofa - hdv ribozyme is almost a magnitude larger in size but possesses a complex tertiary structure and two binding domains that interact with the substrate ( i.e. , the recognition and biosensor domains ) and undergoes conformation transition .  
the sofa - hdv ribozymes exhibiting the highest cleavage activity in vitro were further studied in cellulo .
these ribozymes were expressed from a u6 promoter for the rna pol iii because it allows efficient transcription of small rna molecules . according to the primer extension assays performed for these four sofa - hdv ribozymes
, they all exhibited good expression , although variable , in transfected hek293 cells ( figure 3 ) .
therefore , neither their ability to be expressed nor their stability accounted for their variable cleavage activity .
two of the sofa - hdv ribozymes exhibited equivalent and drastic reductions in app at both the mrna and protein levels ( figures 4 and 5 ; sofa - hdv rz - app276 and -app753 ) .
it is not surprising that only some of the ribozymes that showed excellent cleavage activity in vitro exhibited significant cleavage activity in cellulo .
several different factors in cells compared with the in vitro analysis may account for this result .
the in cellulo target is the full - length mrna , which may adopt a different structure , and cellular proteins may be bound to it and create steric hindrances that reduce the accessibility to some of the identified cleavage sites . to our knowledge
, this study is the first reporting acute silencing of app in human cells using an hdv ribozyme - based approach .
sofa - hdv ribozyme - based gene silencing constitutes an alternative to using small interfering rna , a method that faces several limitations .
one of the largest hurdles in rnai - based therapy is toxicity . in this context ,
independent off - target or nonspecific effects of sirna are a concern [ 23 , 24 ] .
side effects can result from unintended interactions between an sirna compound and an unrelated host gene .
moreover , shrna expression in neurons has been shown to interfere with dendritic spine structure and function , resulting in a decrease of synapses .
interferon response is the best known adverse effect in the viral - mediated transfection of sirnas .
similarly , synthetic sirnas formulated in nonviral delivery vehicles can also be potent inducers of interferons and inflammatory cytokines , both in vivo in mice and in vitro in human blood .
the most important difference between ribozyme technology and rnai technology is that rnai requires the recruitment of endogenous proteins , which are responsible for the high intracellular activity .
thus , problems of potency , specificity , and/or cell - type - dependent responses illustrate a lack of understanding of the intracellular mechanisms involved . by contrast
, the hdv rz , which derives from the hepatitis delta virus , possesses several unique features that are all related to the fact that it is the only naturally occurring catalytic rna discovered in humans [ 7 , 28 , 29 ] and that its action does not depend on intracellular factors .
in addition , it exhibits an outstanding stability ( i.e. , a half - life > 100  hr ) in cell culture .
moreover , a genome - wide search for innate ribozyme entities revealed the presence of hdv - like sequences in the human cpeb3 gene .
consequently , the hdv rz should not be recognized by the immune system as an external , invading rna .
several studies in human genetic and animal models support the notion that amyloid production or accumulation in the brain plays a central role in the pathogenesis of ad . lowering amyloid levels in different mouse models
multiple approaches aimed at interfering with a metabolism have been proposed as a therapy for ad .
first , compounds that aim to decrease the aggregation of a by blocking its oligomerization have been tested . although successful in mice models of ad , they failed in human trials .
second , lowering a levels by increasing its clearance using a vaccine was successful in animal models [ 35 , 36 ] , but the results from human clinical studies indicated important side effects , and there were concerns about safety in humans . finally , blocking the activity of the secretases (  and  ) is attractive because both of these enzymes participate in a production by cleaving app . however , because these secretases have numerous substrates essential for cellular functions , blocking their activity raises additional concerns .
the data from -secretase ( bace1 ) knockout animals have suggested potential liabilities with bace1 inhibition [ 3840 ] .
bace1 is also known to participate in myelination [ 4143 ] and the processing of sodium channels .
nonetheless , bace1 inhibitors have been developed , but blood - brain - barrier penetration and limited access to cellular bace1 due to its major location in the endosome pose significant challenges that have yet to be overcome [ 44 , 45 ] .
however , recent clinical trials testing a very promising -secretase inhibitor have raised major safety issues about this route [ 46 , 47 ] .
this failure does not question the amyloid hypothesis , but instead the nonspecific targeting of an enzyme complex with so many cellular functions . because there have been many failures in targeting amyloid peptide metabolism , we believe that targeting the top of the cascade by decreasing app mrna would be a better way to decrease the overall amyloid level .
we do not expect a complete knock down of app mrna and a , but we are confident that we can significantly reduce app mrna levels ( and subsequently , a levels ) .
we believe that there is a threshold effect and that a modest reduction in a levels could shift the balance between toxicity and nontoxicity .
another advantage of specifically targeting app mrna is that the level of all forms of the peptide derived from app will also be decreased . to achieve this end , we engineered specific sofa hdv ribozymes , a new generation of catalytic rna tools , to decrease the app mrna level .
we demonstrated that a sofa - hdv ribozyme targeting app mrna is clearly effective for the reduction of a in neuron - like cells .
further analysis using an expression system based on the lentivirus indicated a significant decrease of ~30% in total a levels ( figure 6 ) .
therefore , this action could possibly affect downstream amyloid - related pathology . because only a 12% decrease in a levels in mice resulted in a dramatic reduction in a build - up and synaptic deficits , we are optimistic that the results obtained in our cellular model will allow for the development of an efficacious form of sofa - hdv ribozyme - based therapy . the exact role of a as a trigger of sporadic alzheimer 's disease is still a question of debate .
moreover it is actually not clear which a species is associated with the disease . over production of a is probably not the cause of amyloid accumulation in sporadic ad .
this is supported by the fact that the apoe4 allele , which is the major genetic risk factor for sporadic ad , slows down a clearance .
whatever the cause of a accumulation , we believe that decreasing a production by specifically targeting the app mrna will contribute to a decrease in the amyloid load to a non - toxic level . actually , all therapies targeting a  ( secretase inhibitors , vaccines , etc . ) aim to reach this nontoxic level .
one limitation of our gene silencing approach is the delivery of the ribozyme to the nervous system .
the presence of the blood - brain barrier limits the penetration of particle as large as a lentivirus into the central nervous system ( cns ) . to avoid this limitation local stereotaxic injections of lentiviruses
although this method is invasive , robust long - term and nontoxic lentiviral gene transfer is feasible in the rodent and nonhuman primate brains [ 51 , 52 ] .
expression over 3 to 8 months can be achieved and it has been demonstrated that up to 90% of cells from the central nervous system transduced by a lentiviral vector under the control of the nse promoter are neurons [ 53 , 54 ] . however the method of choice for lentiviral delivery is the i.v . or i.p . route .
this could be achieved by the fusion of the low - density lipoprotein receptor - binding domain of the apolipoprotein b to the therapeutic molecule .
successful application of this approach as a general method for the delivery of therapeutic molecules to the cns has been demonstrated .
moreover it remains possible that systemic presence of sofa hdv ribozymes will decrease a level in the periphery and concomitantly brain a levels due to the  sink hypothesis .
 evaluation of these delivery methods will involve extending our study to animal models of alzheimer 's disease , thus the exciting potential of this new treatment will be revealed in the future .
this development will involve extending our study to animal models of alzheimer 's disease , so the exciting potential of this new treatment will be revealed in the future .
we have presented an original and unambiguous demonstration that a sofa - hdv ribozyme can serve as an efficient gene silencing tool . moreover
, our results open the door to further evaluation of sofa - hdv ribozymes as potential therapeutic molecules , or at least to a study demonstrating whether a reduction in a levels is a viable therapy against alzheimer 's disease .
 alzheimer 's disease ( ad ) etiological studies suggest that an elevation in amyloid- peptides ( a ) level contributes to aggregations of the peptide and subsequent development of the disease . 
 the major constituent of these amyloid peptides is the 1 to 4042 residue peptide ( a 
 4042 ) derived from amyloid protein precursor ( app ) . 
 most likely , reducing a levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with ad . among the several possible ways to lower a accumulation in the cells , we have selectively chosen to target the primary step in the a cascade , namely , to reduce app gene expression . toward this end 
 , we engineered specific sofa - hdv ribozymes , a new generation of catalytic rna tools , to decrease app mrna levels . additionally , we demonstrated that app - ribozymes are effective at decreasing app mrna and protein levels as well as a levels in neuronal cells . 
 our results could lay the groundwork for a new protective treatment for ad . 
1. Introduction
2. Experimental Procedures
3. Results
4. Discussion