HK1148311A

HK1148311A - Sirna sequence-independent modification formats for reducing off-target phenotypic effects in rnai, and stabilized forms thereof

Info

Publication number: HK1148311A
Application number: HK11102405.2A
Authority: HK
Inventors: N‧普瑞; I‧查尔斯; S‧马格达雷诺; A‧乌拉索夫; C‧博内特
Original assignee: 应用生物系统有限公司
Priority date: 2007-09-19
Filing date: 2008-09-17
Publication date: 2011-09-02

Description

Sequence-independent modified forms of siRNA and stable forms thereof for reducing off-target phenotypic effects in RNAi

FIELD

The present teachings relate generally to compositions, methods, and kits for reducing off-target phenotypical effects (off-target phenotypical effects) that are sequence independent in RNA interference.

Introduction to the design reside in

Short interfering RNA (siRNA) effectively induces cleavage of complementary mRNA, rendering the mRNA nonfunctional and resulting in loss of functional protein in the cell. The mechanism by which such molecules act has been characterized in part. Briefly, one of the 2 RNA strands of siRNA is incorporated into an enzyme complex called RISC, which is then able to bind and cleave mRNA containing complementary sequences, thereby eliminating the mature mRNA from translation into protein. Each of the two strands of the siRNA can be incorporated into the R I SC complex. However, strands with weaker base pairing at their 5' ends are preferred for integration. Thus, any siRNA will result in a mixture of activated RISC complexes (which can cleave the intended target RNA and non-targeted RNA). In biological applications, it is expected that most, preferably all, of the activated RISC complexes will contain siRNA strands complementary to the desired target. The present teachings provide methods, compositions, and kits useful not only for reducing cleavage of mRNA by undesired siRNA strands, but also for reducing off-target effects of interference of endogenous genes.

SUMMARY

Surprisingly, increasing or maintaining strand bias (strand bias), while necessary to maintain the potency of endogenous RNA interference, is not sufficient to reduce off-target effects in cell biology assays. The ability to reduce or minimize off-target effects while maintaining potency under conditions in which the target mRNA is provided exogenously and in which the target mRNA is endogenous, under conditions in which different types of modified nucleotides are introduced into the siRNA, and under conditions in which different modified forms are introduced into the siRNA is investigated herein. In addition, various siRNA modification forms were studied for each of many target RNAs. Thus, modified nucleotides and modified forms that reduce or minimize off-target events and maintain the potency of highly potent sirnas (as observed at the phenotypic and cellular biological levels) can be used for any siRNA sequence and are independent of the base sequence of the siRNA. That is, the modifications provided herein are sequence-independent modifications.

In some embodiments, a chemically synthesized passenger (sense) oligonucleotide is provided having a length of 15 to 30 nucleotides and comprising one of a sequence independent modification (1), a sequence independent modification (2), and a sequence independent modification (3) as follows:

(1)5′N_p-m-m-N_x-m-m-N_q-n_r3', wherein p is 0 or 1; x is 7, 8, 9, 10 or 11; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (p + x + r + 4);

(2)5′N_p-m-m-N_y-m-N_z-m-N_q-n_r3', wherein p is 0 or 1; y is 7, 8 or 9 and z is 1; or y is 7 and z is 2 or 3; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (p + y + z + r + 4);

(3)5′m-N_l-m-N_x-m-m-N_q-n_r3', wherein x is 8, 9 or 10; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (x + r + 5);

wherein each m is independently a bicyclic nucleotide or a tricyclic nucleotide; each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide; when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide, and when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide.

In some embodiments, provided is a chemically synthesized short interfering RNA comprising the passenger (sense) oligonucleotide described above and a guide (antisense) oligonucleotide having 15 to 30 nucleotides, a region of continuous complementarity to the passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand also has complementarity to at least a portion of the target mRNA.

In some embodiments, short interfering RNAs are provided comprising a passenger (sense) oligonucleotide having a length of 17 to 30 nucleotides and comprising one of a sequence-independent modified form (4), form (5), and form (6), and a guide (antisense) oligonucleotide having 17 to 30 nucleotides, a region continuously complementary to the passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand also has complementarity to at least a portion of the target mRNA,

(4)5′m_p-N_x-m-m-N_q-n_r3', wherein when p is 0, x is 12; when p is 1, x is 11; when p is 2, x is 10; when p is 3, x is 9; when p is 4, x is 8; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (p + x + r + 2);

(5)5′m-m-N_x-m-N_q-n_r3', wherein x is 11; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (x + r + 3);

(6)5′m_p-N_y-m-N_z-m-N_q-n_r3', wherein p is 3; y is 9 and z is 1; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (p + y + z + r + 2);

wherein each m is independently a bicyclic nucleotide, a tricyclic nucleotide, or a 2' -modified nucleotide; each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide and is an overhang nucleotide; when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide, when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide, and when m is a 2 '-modified nucleotide, each N is independently a nucleotide other than a 2' -modified nucleotide.

In embodiments of short interfering RNAs, the passenger (sense) oligonucleotide has a sequence-independent modification (4), p is 2, x is 10, r is 2, each n is an overhang nucleotide, and each n is independently a modified nucleotide; the guide (antisense) oligonucleotide comprises 2 nucleotides modified by 3' -overhangs; and each modified nucleotide is independently a bicyclic nucleotide, a tricyclic nucleotide, or a 2' -modified nucleotide. In one embodiment, at least one of the 3 'penultimate and 3' penultimate positions of the passenger oligonucleotide is a modified nucleotide. The 3' penultimate site is the third site from the last site, i.e., the site preceding the penultimate site (e.g., form DH47 of fig. 1H). The 3' penultimate site is the fourth site from the last site, i.e., the site preceding the penultimate site (e.g., form DH29 of fig. 1H). Such siRNA is particularly stable against nucleases, e.g. in the presence of serum or plasma.

In some embodiments of short interfering RNA, when 17 nucleotides in length, at least one of positions 2 and 3 of the guide oligonucleotide is a modified nucleotide; when 18 nucleotides in length, at least one of positions 2, 3 and 4 of the guide oligonucleotide is a modified nucleotide; when 19 nucleotides in length, at least one of positions 2, 3,4 and 5 of the guide oligonucleotide is a modified nucleotide; when 20 nucleotides in length, at least one of positions 2, 3,4, 5 and 6 of the guide oligonucleotide is a modified nucleotide; and when the length is 21 to 30 nucleotides, at least one of positions 2, 3,4, 5, 6 and 7 of the guide oligonucleotide is a modified nucleotide. Form DH1 and form DH39-DH43 of fig. 1K provide examples of siRNA modified forms having a length of 21 nucleotides and at least one of positions 2, 3,4, 5, 6 and 7 of the guide oligonucleotide is a modified nucleotide. In some embodiments, only one of positions 2, 3,4, 5, 6 and 7 (depending on the length cited above) of the guide oligonucleotide is a modified nucleotide. Such siRNA is also particularly stable against nucleases, for example in the presence of serum.

A method of reducing or minimizing off-target events with respect to inhibition of expression of a target gene by RNA interference is provided, the method comprising contacting a cell containing the target gene with the chemically synthesized short interfering RNA described above in an amount sufficient to reduce off-target events while maintaining potency, wherein each m is independently a bicyclic or tricyclic nucleotide. The phrase "while maintaining potency" is used herein in the context of using a sufficient amount of siRNA to reduce off-target events while maintaining potency. In this context, the phrase refers to the tolerance to a decrease in inhibitory (knockdown) activity of the siRNA at the time of a reduced off-target event, as indicated by whether the decreased inhibitory activity causes a gene-associated phenotype. In general, "while maintaining potency" means that inhibition is reduced to 80% of the normal level of inhibition is acceptable. However, tolerance to reduced inhibition of a particular gene may range from 50% to 95% of normal inhibition.

In additional embodiments, a method of reducing or minimizing cleavage by a passenger strand in the inhibition of expression of a target gene exogenously provided by RNA interference is provided, the method comprising contacting a cell containing the target gene with a chemically synthesized short interfering RNA in an amount sufficient to reduce or minimize cleavage by the passenger strand while maintaining the potency of the guide strand, the passenger strand of the short interfering RNA comprising one of sequence-independent modified forms (1), (2), (3), (4), (5), or (6), wherein each m is independently a 2' -modified nucleotide; each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide; and each N is independently an unmodified nucleotide. The reduction in cleavage was compared to the reduction in cleavage of unmodified siRNA.

In some embodiments, the contact is containing cells in cell culture or containing cells of tissue and chemical synthesis of short interfering RNA in vitro contact. In other embodiments, the contact is containing cells of the tissue, containing cells of the body fluid or containing cells of the organ and chemical synthesis of short interfering RNA contact ex vivo. In other embodiments, the contact is containing cells of the organ or animal with chemical synthesis of short interfering RNA in vivo contact. In some embodiments, the contact is with the nuclease stability of the modified form of short interfering RNA to the subjects in need of in vivo intravascular administration. As shown by the data of example 8, a greater amount of stable short interfering RNA is present in vivo after administration when compared to the amount of control short interfering RNA lacking modified nucleotides present in vivo at the same post-administration time point.

RNA interference assays provided by embodiments herein and performed on exogenously provided genes show that: many types of modified nucleotides in different modification formats provide enhanced strand bias while maintaining potency. See fig. 4A and 4B for data using the bicyclic nucleotide forms H, K and O and fig. 6A and 6B for data using the 2' -O-methyl nucleotide forms H, K and O.

Surprisingly, such modified nucleotides and modified forms differ in their ability to reduce or minimize off-target events in RNA interference of endogenous genes. Microarray analysis was performed to determine the number of differentially expressed genes, which is a measure of off-target effects caused by the introduced siRNA. Differentially expressed genes are defined as genes that have at least a 2-fold change (p value less than 0.001) due to RNA interference. Compared to the number of differentially expressed genes caused by unmodified form a sirnas having the same siRNA sequence,the nucleotide modified form H siRNA produced a gene with reduced differential expression of 38% to 68%. In contrast, the 2' -O-methyl modified form HsiRNA produced genes with reduced differential expression by 0% to 22% when compared to the unmodified form AsiRNA. Therefore, the temperature of the molten metal is controlled,nucleotide modified sirnas were more effective in reducing off-target effects than 2' -O-methyl modified sirnas, as determined by whole gene profiling analysis. See fig. 9A to 9C.

These unexpected findings were confirmed using cell biology studies as illustrated in example 6. When the results of siRNA interference analysis on endogenous genes were pooled, it was evident that 2' -O-methyl modified nucleotides in the various modified forms shown in fig. 11C did not eliminate the "off-target" phenotype to a statistically significant degree compared to the unmodified forms. These results are in contrast to the results of fig. 10E, which shows the statistically significant ability of sirnas with form H to eliminate off-target effects, as evidenced by lower apoptotic signals compared to unmodified form a.

In some embodiments, the chemically synthesized short interfering RNA also binds to a cell-targeting ligand (cell-targeting ligand) as described further below.

Embodiments herein relate to compositions comprising a passenger oligonucleotide, a guide oligonucleotide, or a chemically synthesized short interfering RNA, and a biologically acceptable carrier. In addition, embodiments herein also relate to kits comprising, for example, a passenger oligonucleotide, a guide oligonucleotide, or a chemically synthesized short interfering RNA, and a transfection agent. The short interfering RNAs of embodiments having modified forms containing modified nucleotides provide improved efficacy, thereby saving time in designing and testing sirnas, improved specificity, thereby saving time and money spent on spurious results, improved potency, thereby allowing the desired effect to be achieved using less material. The improved efficacy and specificity also allows for individual gene and library screening studies using fewer sirnas per gene.

In another embodiment, a method of reducing off-target effects of RNA interference comprises obtaining an siRNA having a passenger sense strand (which comprises a region homologous to an RNA transcribed from a target) and having a guide antisense strand complementary to the sense strand, the siRNA having a modified form of H, Q, V-2, Y, Y +1, JB1, JB2, JB3, JB4, or JB5, and contacting the siRNA with a cell. The reduction in off-target effect is measured by comparison to the off-target effect caused by unmodified siRNA, which has the same sequence as the modified formatted siRNA.

In additional embodiments, a method of increasing the stability of an siRNA in a nuclease-rich environment comprises obtaining an siRNA having a passenger sense strand (which comprises a region homologous to an RNA transcribed from a target), a guide antisense strand complementary to the sense strand, and having a 3' overhang nucleotide, the siRNA having a modified form of DH47, DH29, DH1, DH39, DH40, DH41, DH42, or DH43, and contacting the siRNA with a nuclease-rich environment. Increased stability of the siRNA is measured by comparison to the stability of an siRNA having form F, the same type of modified nucleotide, and the same sequence.

Additional embodiments include sirnas having the modified forms provided herein for use in RNA interference to inhibit expression of a target gene in vitro, ex vivo, or in vivo applications. Where expression of a target gene is known to be associated with a disease, the sirnas herein can be used for screening, diagnosis, or treatment of the disease.

A surprising aspect of the siRNA performance studies described herein is that reporter-based strand assays (reporter-based strand assays) cannot predict the performance of modified formatted sirnas at the cellular phenotypic level. Differences in performance at the phenotypic level were observed using microarray whole gene profiling or cell-based assays described below.

These and other features of the present teachings will become more apparent in light of the description herein.

Drawings

Those skilled in the art will appreciate that the following figures are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

Fig. 1A to 1K provide modified forms for siRNA studies. The numbers above the nucleotides indicate the position of the nucleotides in the siRNA relative to the 5' end of the form. m-modified nucleotide, N-nucleotide or nucleotide having modification other than the modification of m, N-nucleotide or modified nucleotide, and P-phosphate group.

FIGS. 2A-2B provide schematic illustrations of expression reporter vectors for different working examples. FIG. 2A shows a pMIR-REPORT comprising a firefly luciferase reporter gene^TMmap of miRNA expression reporter vectors (Ambion/Applied Biosystems). Coding regions of different genes of interest (GOI)Inserted between the HindIII (nucleotide position 463) and SpeI (nucleotide position 525) restriction endonuclease sites in the Multiple Cloning Site (MCS) at the 3' end of the luciferase gene. FIG. 2B shows a pMIR-REPORT comprising a beta-galactosidase reporter gene^TMMap of beta-gal control vector (Ambion/Applied Biosystems). Comparison vectors with GOI-carrying pMIR-REPORT^TMThe miRNA expression reporter vector was co-transfected and the control vector was used as a transfection normalization control.

FIGS. 3A-3B provide a depiction of pMI R-REPORT^TMSchematic representation of the directional relationship between mi RNA of cloned GOI and mRNA of luciferase gene in mi RNA expression vector. FIG. 3A provides forward orientation (forward orientation), i.e., the 5 '-end of the GOI mRNA is immediately adjacent to the 3' -end of the fLuc mRNA. Fig. 3B provides the inverted orientation, i.e., the 5 '-end of the GOI mRNA is distal to the 3' -end of the ffluc mRNA.

Fig. 4A to 4E provide the results of the strand assays described in examples 2 and 3 performed on sirnas having unmodified form a and the specified modified form.

Figure 4A provides the amount of normalized reporter protein present after RNAi studies with siRNA in the specified modified form were performed. Dark bars indicate the inhibitory activity of the guide strand, light bars indicate the inhibitory activity of the passenger strand. To show the distribution of the strand pattern results of the modified siRNA strands, the data of fig. 4B to 4E are provided in the form of box plots (box plot), also referred to as "box whisker" plots. The dark horizontal bars of the "box whiskers" plot represent the median of the dataset, the boxes represent the distribution of data over the lower and upper quartiles of the dataset, the dashed lines (whiskers) represent the minimum and maximum values of the dataset, and the ellipses represent outliers in the dataset for each modification.

Figure 4B provides the inhibitory activity of siRNA passenger strand measured by the following formula: passenger strand activity P ═ (remaining fhuc activity/β gal activity from the reverse clones treated with test siRNA)/(remaining fhuc activity/β gal activity from the reverse clones treated with Neg control siRNA). The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form E, 0.1434; form F, 0.1011; form G, 0.0002052; form H, 0.0001755; form J, 0.0002052; form K, 0.006516; form M, 0.002246; form O, 0.0009626.

Fig. 4C provides the inhibitory activity of the guide strand as measured by the following formula: guide strand activity G ═ (remaining fhuc activity/β gal activity from forward clones treated with test siRNA)/(remaining fhuc activity/β gal activity from forward clones treated with Neg control siRNA). The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form E, 0.0001271; form F, 0.8774; form G, 0.0006498; form H, 0.8115; form J, 0.000006557; form K, 0.01051; form M, 0.7898; form O, 0.8774.

Fig. 4D provides the difference in the fhuc activity between the passenger and guide strands of the sirnas of fig. 4B and 4C. The difference in activity was calculated by subtracting the normalized reverse fLUC activity from the normalized forward fLUC activity, or difference ═ P-G. Thus, if the passenger strand activity is less (lower inhibition, i.e., higher luciferase activity) than the guide strand (higher inhibition, i.e., lower luciferase activity), the value of the activity difference is expected to be greater than 0. Values below 0 indicate that the passenger strand is more active than the leader strand. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form E, 0.00792; form F, 0.06043; form G, 0.9664; form H, 0.0014; form J, 0.008715; form, 0.6634; form M, 0.002516; form O, 0.0006498.

FIG. 4E shows the calculation as (log)₂(P)-log₂(G) Fold activity change) that provides a measure of the effect of the modified form on the guide strand compared to the effect of the modified form on the passenger strand of the s i RNA of fig. 4D. Thus, the greater the fold change in activity, the greater the guide strand bias of the siRNA. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form E, 0.0002781; form F, 0.08937; form G, 0.2076; form H, 001944; form J, 0.0003223; form K, 0.8553; form M, 0.002516; form O, 0.003504.

Fig. 5A to 5C provide the results of the study described in example 3 with respect to the modified form in which the chain conversion was performed (compare the study of fig. 4A to 4E). That is, for example, for modification F, the 1, 20, 21 modification of the passenger (sense) strand is introduced into the guide (antisense) strand and the 20, 21 modification of the guide (antisense) strand is introduced into the passenger (sense) strand.

Figure 5A provides the normalized amount of reporter protein present after RNAi studies performed on 12 different sirnas with strand switch modifications. Dark bars indicate the inhibitory activity of the guide strand, light bars indicate the inhibitory activity of the passenger strand.

Fig. 5B provides the difference in activity between the passenger strand and the guide strand of the siRNA of fig. 5A in a boxed line graph. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form were identical for all forms, 0.03125.

Fig. 5C provides fold-changes in activity between the passenger and guide strands of the siRNA of fig. 5B. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form were identical for all forms, 0.03125.

Fig. 6A to 6C provide the results of the strand assay described in example 3 for the study in which the modified form of the modified nucleotide was 2' -O methylated.

Figure 6A provides the normalized amount of reporter protein present after RNAi studies performed on 6 different sirnas with the specified modified forms. Dark bars indicate the inhibitory activity of the guide strand, light bars indicate the inhibitory activity of the passenger strand.

Fig. 6B provides the difference in the fl activity between the passenger and guide strands for the siRNA group of fig. 6A and the group of additional 18 different sirnas described in example 3 in a boxed line graph. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H-1, 0.001918; form H, 0.00001574; form H +1, 0.001091; form V-2, 0.0002131; form K, 0.0003619; form O, 0.406.

Fig. 6C provides fold-changes in activity between the passenger and guide strands of the siRNA of fig. 6B. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H-1, 0.02514; form H, 0.00001574; form H +1, 0.007443; form V-2, 0.0001464; form K, 0.0003619; form O, 0.5446.

Fig. 7A-7B provide the results of the strand assays described in example 3 for the study in which the modified form of the modified nucleotide is a 2 ', 5' -linked nucleotide.

FIG. 7A provides the difference in inhibitory activity of the guide strand and passenger strand for siRNA with unmodified form A and siRNA with modified forms H-1, H, H +1, V-2, K and O. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H-1, 0.3594; form H, 0.07422; form H +1, 0.5703; form V-2, 0.4258; form K, 0.2031; form O, 0.01953.

Fig. 7B provides fold changes in inhibitory activity between the passenger strand and the guide strand of the siRNA of fig. 7A. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H-1, 0.3008; form H, 0.01953; form H +1, 0.2031; form V-2, 0.4961; form K, 0.1921; form O, 0.00909.

Figure 8 provides the inhibitory activity data of 6 sirnas for each of the 8 endogenous targets. Each of the 48 sirnas had a designated modification form incorporating the bicyclic modified nucleotides described in example 4. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form E, 0.000000008657; form F, 0.4631; form G, 0.000009698; form H, 0.5569; form J, 0.00000000191; form K, 0.00001336; form M, 0.7269; form O, 0.0593.

Fig. 9A to 9C provide venn maps of the intersection of 2-fold differentially expressed genes between unmodified siRNA, 2' -O-methyl-siRNA or dicyclo-modified siRNA as determined by microarray analysis and described in example 5. For each figure, the lower left group (red if colored) represents genes that changed 2-fold or more in the unmodified siRNA treated sample, the upper group (blue if colored) represents genes that changed 2-fold or more in the 2' -O-methyl form H modified siRNA treated sample, and the lower right group (green if colored) represents genes that changed 2-fold or more in the bicyclic modified nucleotide treated sampleForm H modified siRNA treated samples changed 2-fold or greater for the genes. FIGS. 10A-10E are provided in connection withData on the effect of modified forms on-target and off-target phenotypes in cell biology studies. The negative control (Neg) was a scrambled (scrambled) non-targeting siRNA for which the quantification of mitosis or apoptosis was normalized to a value of 1.0. See example 6. FIG. 10A provides a block diagram of a computer system withA boxplot of normalized mitotic cells resulting from the siRNA of modified forms E, F, G, H, J, K, M and O silencing a subset of gene targets for which inhibition is expected to increase mitosis, for which inhibition is expected to have no effect on mitosis, and for which inhibition is expected to decrease mitosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form E, 0.805; form F, 0.5483; form G, 0.3215; form H, 0.004561; form J, 0.000952; form K, 0.01974; form M, 0.1007; form O, 0.9438.

Figure 10B provides a boxplot of a subset of genes whose inhibition is expected to reduce mitosis as in figure 10A. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form E, 0.3247; form F, 0.4683; form G, 0.1815; form H, 0.03423; form J, 0.01387; form K, 0.09874; form M, 0.5509; form O, 0.6705.

FIG. 10C provides a schematic view of a display device havingA boxplot of normalized apoptotic fragments resulting from the silencing of a panel of gene targets, a subset of which inhibition is expected to increase apoptosis, by the siRNA of modified forms E, F, G, H, J, K, M and O, and a subset of which inhibition is expected to have no effect on apoptosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form E, 0.2641; form F, 0.5366; form G, 0.0001888; form H, 0.0000102; form J, 0.00000263; form K, 0.000000553; form M, 0.5366; form O, 0.05753.

Fig. 10D provides a boxplot of a subset of genes whose inhibition is expected to increase apoptosis as described in example 6, as in fig. 10C. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form E, 0.2188; form F, 0.4375; form G, 0.3125; form H, 0.2188; form J, 0.03125; form K, 0.03125; form M, 1; form O, 0.2188.

Fig. 10E provides a boxplot of a subset of genes whose inhibition is expected to have no effect on apoptosis as described in example 6, as in fig. 10C. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (with increased median and larger data distribution compared to Neg control). The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form E, 0.5303; form F, 0.804; form G, 0.00005488; form H, 0.00001347; form J, 0.0001253; form K, 0.00003023; form M, 0.441; form O, 0.1551.

FIGS. 11A through 11F provide data on the effect of 2' -O-methylated modifications on the on-target and off-target phenotypes in cell biology studies. Negative control (Neg) is a scrambled non-targeting siRNA for which the quantification of mitosis or apoptosis is normalized to a value of 1.0. See example 6.

Figure 11A provides a boxplot of normalized mitotic cells resulting from silencing of a set of gene targets, a subset of which is expected to increase mitosis, by sirnas having unmodified form a and having 2' -O-methyl modified forms H and K, a subset of which is expected to have no effect on mitosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.3529; form K, 0.3289.

Figure 11B provides, as figure 11A, a boxplot of a subset of genes whose inhibition is expected to increase mitosis. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.3125; form K, 0.5469.

Figure 11C provides a boxplot, as in figure 11A, of a subset of genes whose inhibition is expected to have no effect on mitosis. However, siRNAs that have been empirically demonstrated to have mitotic off-target effects are specifically selected for study to determine whether such siRNAs, when modified to formalize, abrogate or reduce off-target effects. Off-target efficacy of siRNA can be demonstrated by a boxplot of unmodified form a (which has a larger data distribution compared to Neg control). The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.9102; form K, 0.4961.

Figure 11D provides a boxplot of normalized apoptotic fragments resulting from silencing of a panel of gene targets, a subset of which inhibition is expected to increase apoptosis, by sirnas with 2' -O-methyl modified forms H and K, and a subset of which is expected to have no effect on apoptosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.3529; form K, 0.3529.

Fig. 11E provides a boxplot of a subset of genes whose inhibition is expected to increase apoptosis as described in example 6, as in fig. 11D. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.3125; form K, 0.5469.

Fig. 11F provides a boxplot of a subset of genes whose inhibition is expected to have no effect on apoptosis as described in example 6, as in fig. 11D. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.9102; form K, 0.7344.

FIG. 12A-1 provides the difference in activity between passenger and guide chains, calculated as P-G, where the modifications are for unmodified form A and for test forms H, H +1, H-2, V, V-1, V-2, K and Q (where the modification isResidue) defines P and G. Wilcoxon test comparing each modified form to the unmodified form (to)Pair) the results were as follows: form H, 0.04199; form H +1, 0.123; form H-1, 0.1748; form H-2, 0.2402; form V, 0.06738; form V-1, 0.05371; form V-2, 0.5771; form K, 0.4648; form Q, 0.1016.

FIG. 12A-2 provides fold-changes in activity between the passenger strand and the guide strand of the siRNA of FIG. 12A-1. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H, 0.3652; form H +1, 0.2783; form H-1, 0.2402; form H-2, 0.4131; form V, 0.2061; form V-1, 0.1016; form V-2, 0.7002; form K, 0.6377; form Q, 0.7002.

FIG. 12B provides siRNA with unmodified form A and siRNA withData for inhibitory activity of modified forms H, H-2, H-1, H +1, Q, K, V, V-1 and V-2 of the siRNAs against endogenous targets, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H, 0.029598160; form H-2, 0.004296849; form H-1, 0.000833165; form H +1, 0.052779900; form Q, 0.011454170; form K, 0.745852000; form V, 0.005660834; form V-1, 0.998100800; form V-2, 0.374661100.

FIG. 12C provides a graph showing the results of having unmodified form A and havingBoxplots of normalized mitotic cells resulting from the silencing of a panel of gene targets by the siRNAs for modified forms H, H +1, H-2, K, Q, V, V-1, and V-2, the inhibition of which is expected to increase mitosis, as described in example 6. Negative control (Neg) was a scrambled non-targeting siRNA for which the quantification of mitosis was normalized to a value of 1.0. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: shape ofFormula H, 0.01563; form H +1, 0.1953; form H-1, 0.07813; form H-2, 0.07813; form V, 0.05469; form V-1, 0.1953; form V-2, 1; form K, 0.1953; form Q, 0.1953.

FIG. 12D provides a graph showing the results of having unmodified form A and havingBoxplots of normalized apoptotic fragments resulting from silencing of a panel of gene targets for which inhibition was expected to have no effect on apoptosis by the siRNAs of modified forms H, H +1, H-2, K, Q, V, V-1 and V-2. The negative control (Neg) was a scrambled non-targeting siRNA for which quantification of apoptosis was normalized to a value of 1.0. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (with increased median and larger data distribution compared to Neg control). The calculation of the statistical significance of the Wilcoxon test (paired) comparing the unmodified form a control with the individual modified forms (calculated as a percentage of control for normalized apoptotic fragments) was: form H, 0.04199; form H +1, 0.2061; form H-1, 0.2783; form H-2, 0.6377; form V, 0.05371; form V-1, 0.4648; form V-2, 0.006836; form K, 0.01855; form Q, 0.024.

FIG. 13A-1 provides the difference in activity between the passenger and guide strands, calculated as P-G, where P and G are defined as for siRNAs with unmodified form A and modified forms H, H +1, H-1, V-2, K, and O (where the modification is a 2' -O-methyl modified nucleotide) as in FIG. 4B and FIG. 4C. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H, 0.00001574; form H +1, 0.001091; form H-1, 0.001918; form V-2, 0.0002131; form K, 0.0003619; form O, 0.406.

FIG. 13A-2 provides fold-changes in activity between the passenger strand and the guide strand of the siRNA of FIG. 13A-1. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H, 0.00001574; form H +1, 0.007443; form H-1, 0.02514; form V-2, 0.0001464; form K, 0.0003619; form O, 0.5446.

FIG. 13B provides data on the inhibitory activity of siRNAs with unmodified form A and modified forms H, H +1, H-1, K, V-2 and O (where the modification is a 2' -O-methyl modified nucleotide) against endogenous targets. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H, 3.47E-06; form H +1, 0.001957517; form H-1, 8.79E-09; form K, 0.005446013; form V-2, 1.95E-09; and form O, 0.9972392.

Figure 13C provides a boxplot of normalized mitotic cells resulting from sirnas with unmodified form a and with modified forms H, H-1, K, and V (with 2' -O-methyl modified nucleotides) silencing a panel of gene targets whose inhibition is expected to increase mitosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form to the unmodified form are as follows: form H, 0.1953; form H-1, 0.6406; form V, 0.1953; form K, 0.6406.

Figure 13D provides a boxplot of normalized apoptotic fragments resulting from siRNA with unmodified form a and with modified forms H, H-1, V, and K (with 2' -O-methyl modified nucleotides) silencing a panel of gene targets whose inhibition is expected to have no effect on apoptosis, as described in example 6. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by boxplot of unmodified form a (which has a larger data distribution compared to Neg control). The results of the Wilcoxon test (paired) comparing unmodified form a with each modified form are: form H, 0.9102; form H-1, 0.25; form V, 0.6523; form K, 0.7344.

FIG. 14A provides a polypeptide having unmodified form A and havingBoxplot of the mRNA inhibitory activity of the siRNAs of modified forms H, M, W, W +1, W-1, Y, Y +1 and Y-1 against endogenous targets. The result of the calculation of the statistical significance of the Wilcoxon test (paired) comparing unmodified form a with each modified form is: form H, 0.005302; form M, 0.02475; form W, 1; form W +1, 0.8423; form W-1, 0.5096; form Y, 0.932; form Y +1, 0.887; form Y-1, 0.5891.

FIG. 14B provides a sample having unmodified form A and havingBoxplot of the mRNA inhibitory activity of the sirnas of modification forms H, M, JB1, JB2, JB3, JB4 and JB5 on endogenous targets. The result of the calculation of the statistical significance of the Wilcoxon test (paired) comparing unmodified form a with each modified form is: form H, 0.005302; form M, 0.02475; form JB1, 0.009613; form JB2, 0.7259; form JB3, 0.4213; form JB4, 0.04904; form JB5, 0.972.

FIGS. 15A to 15D are provided for having unmodified form A and havingData on the effect of modified forms H, M, W, W +1, W-1, Y, Y +1 and Y-1 of siRNA on the on-and off-target phenotypes in cell biology studies. See example 6.

Figure 15A provides a boxplot of normalized mitotic cells described in example 6 for a set of gene targets whose inhibition is expected to reduce mitosis, as described in example 6. Negative control (Neg) was a scrambled non-targeting siRNA for which the quantification of mitosis was normalized to a value of 1.0. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.4961; form M, 1; form W, 0.01953; form W +1, 0.5703; form W-1, 0.07422; form Y, 0.03906; form Y +1, 0.4961; form Y-1, 0.09766.

Figure 15B provides a box plot as in figure 15A of a set of gene targets whose inhibition is not expected to affect mitosis. However, siRNAs that have been empirically demonstrated to have mitotic off-target effects are specifically selected for study to determine whether such siRNAs, when modified to formalize, abrogate or reduce off-target effects. Off-target effect of siRNA was demonstrated by a boxplot of unmodified form a (which has a median value of approximately 0.7 compared to Neg control). The statistical significance of the Wilcoxon test (paired) comparing unmodified form a control to each modified form was calculated as: form H, 0.5; form M, 0.75; form W, 0.25; form W +1, 0.25; form W-1, 0.25; form Y, 0.25; form Y +1, 0.25; form Y-1, 1.

Fig. 15C provides a boxplot of normalized apoptotic fragments for a panel of gene targets whose inhibition is expected to increase apoptosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.75; form M, 0.25; form W, 0.25; form W-1, 1; form W +1, 0.25; form Y, 0.25; form Y-1, 0.25; form Y +1, 0.25.

Figure 15D provides a boxplot of a subset of gene targets whose inhibition is expected to have no effect on apoptosis as in figure 15C. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (with a median value greater than 3.5 and larger data distribution compared to Neg control). The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.01221; form M, 0.8501; form W, 0.07715; form W +1, 0.06396; form W-1, 0.1763; form Y, 0.03418; form Y +1, 0.021; form Y-1, 0.2036.

FIGS. 16A to 16D are provided for having unmodified form A and havingData on the effect of modified forms H, M, JB1, JB2, JB3, JB4 and JB5 on the on-and off-target phenotype in cell biology studies. See example 6.

Figure 16A provides a boxplot of normalized mitotic cells for a set of gene targets whose inhibition is expected to reduce mitosis. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.4961; form M, 1; form JB1, 0.3594; form JB2, 0.6523; form JB3, 0.02734; form JB4, 0.1641; form JB5, 0.2031.

Figure 16B provides a box plot as in figure 16A of a set of gene targets whose inhibition is not expected to affect mitosis. However, siRNAs that have been empirically demonstrated to have mitotic off-target effects are specifically selected for study to determine whether such siRNAs, when modified to formalize, abrogate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (which has a median value of approximately 0.65 compared to Neg control, showing a reduction in mitosis). The results of the Wilcoxon test (paired) comparing unmodified form a control to each modified form are as follows: form H, 0.5; form M, 0.75; form JB1, 0.25; form JB2, 0.25; form JB3, 0.75; form JB4, 0.25; form JB5, 0.25.

Figure 16C provides a boxplot of normalized apoptotic fragments resulting from silencing a panel of gene targets whose inhibition is expected to increase apoptosis, as described in example 6. The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.75; form M, 0.25; form JB1, 0.25; form JB2, 0.25; form JB3, 0.25; form JB4, 0.5; form JB5, 0.75.

Fig. 16D provides a box plot of a panel of gene targets whose inhibition is expected to have no effect on apoptosis as in fig. 16C. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effects of siRNA can be demonstrated by a boxplot of unmodified form a (with an increased median value of greater than 3 and larger data distribution compared to Neg control treated samples). The results of the Wilcoxon test (paired) comparing each modified form with the unmodified siRNA form are as follows: form H, 0.01221; form M, 0.8501; form JB1, 0.0009766; form JB2, 0.0004883; form JB3, 0.0004883; form JB4, 0.003418; form JB5, 0.003418.

FIG. 17 provides an assay for serum stability as a function of time in 90% serum at 37 ℃ for unmodified siRNA. The percentage of remaining full-length siRNA duplexes of 8 different sirnas at 0, 5, 10, 15, 30, 60, 120 min incubation is shown.

Fig. 18A provides a boxplot of the percentage of siRNA that retained full length when treated with 90% serum under the conditions described in example 7. Provided are sirnas with respect to unmodified forms of AsiRNA, siRNA modified form F (which form is referred to as sinna 5 by Elmen et al (Nucleic acids research 33: 1, 439-447, 2005) and used herein as a reference control for stability) and sirnas with modified forms H, DH21, DH20, DH3, DH30, DH35, DH6, DH34 and DH2 (wherein the modification isResidue). The results of the Wilcoxon test (paired) comparing form F with each modified form are: form H, 0.007813;form DH21, 0.007813; form DH20, 0.007813; form DH3, 0.007813; form DH30, 0.007813; form DH35, 0.01563; form DH6, 0.007813; form DH34, 0.02488; form DH2, 0.2049.

FIG. 18B provides siRNA presented by forms A, F, H, DH21, DH20, DH3, DH30, DH35, DH6, DH34, DH2 (as described in example 7) relative to Neg control siRNABoxplot of fraction of mRNA inhibition by modified siRNA. The statistical significance of the Wilcoxon test (paired) comparing unmodified form a control to each modified form was calculated as: form F, 0.05469; form H, 0.3828; form DH21, 0.007813; form DH20, 0.007813; form DH3, 0.007813; form DH30, 0.007813; form DH35, 0.01563; form DH6, 0.007813; form DH34, 0.03906; form DH2, 0.6406.

Fig. 19A provides a boxplot of the percentage of siRNA that retained full length when treated with 90% serum under the conditions described in example 7. Data are provided for modified forms F, DH2, DH19, DH4, DH31, DH27 and DH25, wherein the modification isAnd (c) a residue. The result of the calculation of the statistical significance of the Wilcoxon test (paired) comparing form F with each modified form is: form DH2, 0.2049; form DH19, 0.1953; form DH4, 0.02249; form DH31, 0.01563; form DH27, 0.1094; form DH25, 0.1094.

Figure 19B provides siRNA against Neg control by presence of form F, DH2, DH19, DH4, DH31, DH27 and DH25 (as described by example 7)Boxplot of fraction of mRNA inhibition by modified siRNA. Comparison of unmodified form A with the respective modificationsThe result of the calculation of the statistical significance of the Wilcoxon test (paired) with parenthetical comparisons is: form F, 0.05469; form DH2, 0.6406; form DH19, 0.01563; form DH4, 0.007813; form DH31, 0.01563; form DH27, 0.01563; form DH25, 0.007813.

Fig. 20A provides a boxplot of the percentage of siRNA that retained full length when treated with serum under the conditions described in example 7. Data are provided for modified forms F, DH2, DH47, DH29, DH28 and DH18, wherein the modification isAnd (c) a residue. The result of the calculation of the statistical significance of the Wilcoxon test (paired) comparing form F with each modified form is: form DH2, 0.2049; form DH47, 0.03906; form DH29, 0.07813; form DH28, 0.01415; form DH18, 0.06836.

FIG. 20B provides siRNA presented by forms F, DH2, DH47, DH29, DH28 and DH18 (as described in example 7) relative to Neg control siRNABoxplot of fraction of mRNA inhibition by modified siRNA. The statistical significance of the Wilcoxon test (paired) comparing unmodified form a control to each modified form was calculated as: f, 0.05469; form DH2, 0.6406; form DH47, 0.3125; form DH29, 0.02249; form DH28, 0.01563; form DH18, 0.01563. )

Fig. 21A provides a boxplot of the percentage of siRNA that retained full length when treated with serum under the conditions described in example 7. Data are provided for modified forms F, DH36, DH2, DH9, DH46, DH33, and DH10, wherein the modification isAnd (c) a residue. Statistical significance of Wilcoxon test (paired) comparing form F with each modified formThe calculation result is: form DH36, 0.1609; form DH2, 0.2049; form DH9, 0.5534; form DH46, 0.02071; form DH33, 0.1508; form DH10, 0.05469.

FIG. 21B provides siRNA presented by forms F, DH36, DH2, DH9, DH46, DH33 and DH10 (as described in example 7) relative to Neg control siRNABoxplot of fraction of mRNA inhibition by modified siRNA. The statistical significance of the Wilcoxon test (paired) comparing unmodified form a control to each modified form was calculated as: form F, 0.05469; form DH36, 0.01563; form DH2, 0.6406; form DH9, 0.1484; form DH46, 0.1484; form DH33, 0.007813; form DH10, 0.007813.

Fig. 22A provides a boxplot of the percentage of siRNA that retained full length when treated with serum under the conditions described in example 7. Data are provided for modified forms F, DH2, DH7, DH23, DH1, DH48, DH49, DH44, and DH45, wherein the modification isAnd (c) a residue. The result of the calculation of the statistical significance of the Wilcoxon test (paired) comparing form F with each modified form is: form DH2, 0.2049; form DH7, 0.3828; form DH23, 0.03461; form DH1, 0.1484; form DH48, 0.6406; form DH49, 0.1410; form DH44, 0.3125; form DH45, 0.1052.

FIG. 22B provides siRNA presented by forms F, DH2, DH7, DH23, DH1, DH48, DH49, DH44 and DH45 (as described in example 7) relative to Neg control siRNABoxplot of fraction of mRNA inhibition by modified siRNA. Calculation of statistical significance of Wilcoxon test (paired) comparing unmodified form A with each modified formThe results were: form F, 0.05469; form DH2, 0.6406; form DH7, 0.05469; form DH23, 0.007813; form DH1, 0.007813; form DH48, 0.1953; form DH49, 0.4609; form DH44, 0.007813; form DH45, 0.01563.

Figure 23A provides a boxplot of the percentage of siRNA that retained full length when treated with serum under the conditions described in example 7. Data are provided for modified forms F, DH2, DH38, DH1, DH39, DH40, DH41, DH42, and DH43, wherein the modification isAnd (c) a residue. The result of the calculation of the statistical significance of the Wilcoxon test (paired) comparing form F with each modified form is: form DH2, 0.2049; form DH38, 0.04206; form DH1, 0.1484; form DH39, 0.07813; form DH40, 0.2500; form DH41, 0.2620; form DH42, 0.1094; form DH43, 0.3621.

FIG. 23B provides siRNA presented by forms F, DH2, DH38, DH1, DH39, DH40, DH41, DH42 and DH43 (as described in example 7) relative to Neg control siRNABoxplot of fraction of mRNA inhibition by modified siRNA. The result of the calculation of the Wilcoxon test (paired) statistical significance comparing unmodified form A with each modified form is: form F, 0.05469; form DH2, 0.6406; form DH38, 0.007813; form DH1, 0.007813; form DH39, 0.007813; form DH40, 0.01563; form DH41, 0.007813; form DH42, 0.007813; form DH43, 0.02344.

Figure 24 provides quantification of stable siRNA present in vivo. Comparing the amount of siRNA present in the liver of a dosed control animal (spiked control animal) with that present in the liver injected with unmodified form A siRNA and having form DH1 or form DH47Modifying the amount of siRNA in the liver of a test animal that is formalized stable siRNA. CT is the cycle threshold.

Description of various embodiments

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, "at least one" means that there may be more than one. Likewise, the use of "including," comprising, "and" including, "or variations of such phrases, such as, but not limited to," including, "" containing, "and" including, "is not intended to be limiting, which means" including the following elements, but not excluding others. The term "consisting essentially of or" consisting essentially of, as used herein, does not include other elements having any substantial meaning in combination. Unless otherwise indicated, the use of "or" means "and/or. The term "and/or" means that the items preceding and following it can be taken together or separately. For purposes of illustration, and not by way of limitation, "X and/or Y" may mean "X" or "Y" or "X and Y".

When ranges of values are provided herein, unless expressly stated otherwise, the ranges include the starting and ending values and any value or range of values therebetween. For example, "0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; ranges therebetween such as 0.2 to 0.3, 0.3 to 0.4, 0.2 to 0.4; increments (increment) therebetween such as 0.25, 0.35, 0.225, 0.335, 0.49; the increment range therebetween is, for example, 0.26 to 0.39, etc.

All documents and similar materials cited in this application, including but not limited to patents, patent applications, treatises, books, treatises (treatises), and internet web pages, regardless of the form in which such documents and similar materials are expressly incorporated by reference in their entirety herein for any purpose. Where one or more of the incorporated documents and similar materials define or use a term in a manner inconsistent with the definition of that term in this application, the definition in this application controls. While the present teachings are described in conjunction with various embodiments, the present teachings are not intended to be limited to such embodiments. Rather, the present teachings encompass various alterations, modifications, and equivalents, as will be appreciated by those skilled in the art.

The term "or combinations thereof," as used herein, refers to all permutations and combinations of the items listed before the term. For example, "A, B, C or a combination thereof" is intended to include: A. b, C, AB, AC, BC, or ABC, and if the order is important in a particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. For this example, explicitly included are combinations containing one or more items or repetitions of an item, e.g., BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Those of skill in the art will understand that there is generally no limitation on the items or number of items in any combination, unless apparent from the context. The term "alternative", as used herein, means a product that indicates the presence of another product. For example, the amplification product is a surrogate for nucleic acid that has been amplified.

As used herein, a "modification format" of an siRNA refers to a pattern of modified nucleotides and "other-than modified" nucleotides, as exemplified by the patterns or formats shown in fig. 1A through 1K. Modified nucleotides are described below. "other than modified" refers to unmodified nucleotides or modified nucleotides having modifications other than those of the modified nucleotides in the specified form.

As used herein, "sequence-independent modifications" means that the modified nucleotides and modified forms provided by the embodiments can be used for any siRNA sequence regardless of the specific sequence of the siRNA.

An "on-target" event, as used herein, refers to the mRNA of a target gene being affected (i.e., inhibited) by an siRNA designed to target the gene, as evidenced by reduced expression, reduced mRNA levels, or loss or gain of a particular phenotype. For example, an "on-target" event can be verified by another method, e.g., using a drug known to affect the target, or by introducing a phenotype in which the target mRNA from the ortholog has been rescued from loss.

"off-target event," as used herein, means any event in RNA interference other than a desired event. An example of a off-target event is the mRNA of a gene that is not targeted being affected (i.e., inhibited) by siRNA. Such "off-target events" cannot be rescued by providing target mRNA from the appropriate relevant ortholog. An "off-target event" can be due to interaction with another mRNA, endogenous RNA, DNA, or protein in a manner that alters expression of non-targets. Such off-target events can be attributed to, for example, mismatched base pairing between the nucleotide sequence incorporated into the RI SC and the non-target mRNA or non-specific interactions in the cellular environment. Inappropriate RISC binding, inappropriate RISC-mediated interactions, and inappropriate RISC-mediated cleavage can contribute to off-target events. Off-target events can also be attributed to cytotoxic responses, interferon responses, microRNA effects, or interactions with non-coding RNAs. The most common off-target event may be the integration of the unwanted passenger (sense) strand into the RISC complex. However, off-target events affected by siRNA are not completely predictable and tend to be non-specific. Off-target events produce off-target effects, and the terms are used interchangeably herein.

The term "potency", as used herein, is a measure of the concentration of an individual siRNA or mixture of sirnas required to inhibit its target mRNA to 50% of the starting mRNA level. Generally, IC50 (concentration of siRNA required for half maximal (50%) mRNA inhibition) is used to describe potency. Potency is determined herein by transfecting a series of siRNA concentrations (e.g., at least 6 concentrations in the 100nM to 10pm range) and then performing qRT-PCR analysis on the samples. The results are plotted and the IC50 determined by extrapolating the concentration at which 50% mRNA inhibition is obtained. Curve fitting procedures were used to determine the concentration of siRNA required to obtain 50% inhibition of the mRNA target. As used herein, the term "potency" is used interchangeably with the term "inhibitory activity" (which is a measure of the amount of mRNA decreased after exposure to a test siRNA compared to the amount of mRNA present after exposure to a negative control siRNA).

The term "efficacy", as used herein, is the percentage of siRNA that produces a minimum threshold of mRNA inhibition. The term efficacy is generally used for pooling of sirnas or for predictive power of siRNA design algorithms. Efficacy is measured by qRT-PCR or vector-based measurement of siRNA inhibition of the target mRNA, with the results expressed as the percentage of siRNA that produces the minimum threshold of inhibition, e.g., the percentage of siRNA that produces 70% or greater level of inhibition.

The term "specificity", as used herein, is a measure of the accuracy with which siRNA affects gene regulation at the mRNA level or protein level, or the true phenotype exhibited due to inhibition of target gene function. Specificity is measured by determining the number of genes that change relative to a certain threshold as a result of siRNA transfection that are not the desired target and are known not to be in the signal transduction pathway of the target gene. The industry standard threshold is a 2-fold change. In cell biology studies, highly specific sirnas directed to specific targets result in overlapping phenotypes with little or no unexpected phenotype. Less specific sirnas lead to phenotypes due to off-target effects.

By "increased" potency is meant an siRNA that has a lower IC50 or higher percent inhibition than another siRNA at the same concentration, by "increased" specificity is meant an siRNA that affects fewer genes than another siRNA, and by "increased" potency is meant a set of sirnas generated using an algorithm in which a greater percentage of sirnas in the set of sirnas produce a minimum specified amount of inhibition than another set of sirnas generated using a different algorithm.

For the studies herein, the test results were compared to control results, and all results were normalized against non-sense sirnas designed to be non-targeting. The Wilcoxon test for paired samples is a nonparametric equivalent of the paired sample t test. The Wilcoxon test arranges the absolute values of the differences between the paired data in sample 1 and sample 2 and calculates statistics on the number of positive and negative differences (differences are calculated as sample 2-sample 1). The software used is "R Package" available free from cran.

The term "nucleotide" generally refers to a phosphate ester of a nucleoside that is present in monomeric form or in a dinucleotide, oligonucleotide, or polynucleotide. Nucleosides are typically purine or pyrimidine bases linked to the C-1' carbon of a ribose (ribonucleoside) or deoxyribose (deoxyribonucleoside). Naturally occurring purine bases generally include adenine (A) and guanine (G). Naturally occurring pyrimidine bases generally include cytosine (C), uracil (U), and thymine (T). When the nucleobase is a purine, the ribose or deoxyribose is linked to the nucleobase at the 9-position of the purine (nucleobase), and when the nucleobase is a pyrimidine, the ribose or deoxyribose is linked to the nucleobase at the 1-position of the pyrimidine. Ribonucleotides are phosphate esters of ribonucleosides and deoxyribonucleotides are phosphate esters of deoxyribonucleosides. The term "nucleotide" is a generic term for ribonucleotides and deoxyribonucleotides. Dinucleotides generally have two nucleotides covalently bonded through a 3 '-5' phosphodiester linkage. An oligonucleotide typically has more than 2 nucleotides, and a polynucleotide typically refers to a polymer of nucleotide monomers. The applicants use the terms "nucleotide" and "nucleoside" interchangeably herein for convenience, although those skilled in the art will readily understand which term may be used in light of the context in which the term appears.

Nucleotide monomers are linked by "internucleoside-to-internucleoside linkage" such as phosphodiester linkage, wherein, as used herein, the term "phosphodiester linkage" refers to a phosphodiester linkage or comprises a phosphate analogue thereof (including an associated counterion such as H)⁺、NH₄ ⁺、Na⁺If such counter ions are present). Additional internucleoside or internucleotide linkages are described below. When the oligonucleotide is represented by a letter sequenceIt is to be understood that the nucleotides are arranged in 5 'to 3' order from left to right, unless otherwise indicated or otherwise evident to one of skill in the art in the reverse direction as the context dictates. Descriptions of methods for synthesizing oligonucleotides are found, inter alia, in U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319 and 5,262,530. The oligonucleotide may be of any length.

"base pairing," as used herein, refers to standard Watson-Crick base pairing. Common base pairing in double-stranded (duplex) nucleic acids is G: C, A: T and A: U. Such base pairs are referred to as complementary base pairs, with one base being complementary to the base to which it is paired. Nucleotide analogs described hereinafter are also capable of forming hydrogen bonds when paired with a complementary nucleotide or nucleotide analog.

As used herein, the term "complementary" or "complementarity" is used to refer not only to base pairs but also to antiparallel strands of oligonucleotides linked by Watson-Crick base-pairing rules, nucleic acid sequences capable of base-pairing according to standard complementarity rules or capable of hybridizing to a particular nucleic acid segment under relatively stringent conditions. For example, the sequence 5 '-AGTTC-3' is complementary to the sequence 5 '-GAACT-3'. The terms "perfect complementarity" or "100% complementarity" and the like refer to complementary sequences of base-perfect pairing between antiparallel strands (no mismatch in the polynucleotide duplex). The nucleic acid polymers may be complementary over only a portion of their complete sequence. The terms "partial complementarity," "incomplete complementarity," or "incomplete complementarity," and the like, refer to any alignment of bases between antiparallel polynucleotide strands that is less than 100% fully paired (e.g., there is at least one mismatch in the polynucleotide duplex). In addition, two sequences are considered complementary over a portion of their length if there are one or more mismatches, gaps, or insertions in their alignment.

Furthermore, a "complement" of a target polynucleotide refers to a polynucleotide that can bind to at least a portion of the target polynucleotide in an antiparallel association. Antiparallel associations can be intramolecular, e.g., in the form of a hairpin loop within a nucleic acid molecule, or intermolecular, e.g., when two or more single-stranded nucleic acid molecules hybridize to one another. The term "corresponding to," when referring to a nucleic acid, means that the particular sequence is sufficiently complementary to an antiparallel sequence that the two sequences will anneal and form a duplex under the appropriate conditions.

As used herein, a "passenger" strand, region, or oligonucleotide refers to a nucleotide sequence having a nucleotide sequence that is identical to the nucleotide sequence of at least a portion of a sense strand of DNA or at least a portion of an mRNA. The "passenger (sense) strand" includes the sense region of a polynucleotide that forms a duplex with the complementary guide (antisense) region of another polynucleotide. Hairpin single stranded oligonucleotides (shrnas) may also comprise within the same molecule a passenger (sense) region and a guide (antisense) region, which regions form a duplex structure connected by a loop sequence or loop linker moiety, as further described below. The hairpin may be in the left-hand orientation (i.e., 5 '-antisense-loop-sense-3') or in the right-hand orientation (i.e., 5 '-sense-loop-antisense-3').

As used herein, "siRNA" refers to a short interfering RNA duplex that induces gene silencing via the RNA interference (RNAi) pathway. The short interfering RNA can vary in length and can contain at most one or two mismatched base pairs between the antisense and sense regions and between the antisense region and the target mRNA sequence. Each siRNA may comprise 15 to 30 base pairs, or 18 to 25 base pairs, or 19 to 22 base pairs or 21 base pairs or 19 base pairs. In some embodiments, the siRNA has 1, 2, 3,4, or 5 unpaired overhang nucleotides on the 5 'end, the 3' end, or both the 5 'end and the 3' end independently. In some embodiments, the siRNA has blunt ends. In addition, the term "siRNA" includes duplexes of two separate strands as well as single strands (shRNA) that can form a hairpin structure. The shRNA may have a nucleotide loop sequence of, for example, 4 to 30 or 6 to 20 or 7 to 15 nucleotides, or may comprise a non-nucleotide moiety such as a hydrocarbon linking region, or a combination thereof. The shRNA may comprise a mismatch or bulge (bulge).

In some embodiments, the present teachings include modified nucleotides that have enhanced base-pairing affinity or confer enhanced affinity to a nucleotide sequence of which the modified nucleotide is a part, as compared to unmodified nucleotides. The modified nucleotides, as referred to herein, shift the conformational equilibrium of the RNA to the nor thern (C3' -endo) conformation consistent with the a-type geometry (a-form geometry) of the RNA duplex. DNA-RNA duplexes may also be stabilized thereby. In some embodiments, enhanced base-pairing affinity is obtained by constructing a passenger (sense) oligonucleotide with bicyclic, tricyclic, or 2' -modified nucleotides.

In some embodiments, the modified nucleotides of the siRNA independently comprise a modified sugar moiety, a modified base moiety, a modified internucleoside moiety, or a combination of any of these modifications.

In some embodiments, modified nucleotides include modified sugar moieties, including, but not limited to, 2' -halo (where halo is chloro, fluoro, bromo, or iodo); arabinosyl (arabino) or 2' -Fluoro (FANA) in the arabinose conformation; 2' -H, -SH, -NH₂CN, -azide; OR-OR, -R, -SR, -NHR OR-N (R)₂Wherein R is alkyl, alkenyl, alkynyl, alkoxy (alkxy), oxyalkyl (oxyalkyllkyl), alkoxyalkyl, alkylamine, wherein the alkyl moiety is C1-C6.

Additional 2 'modifications include 2' -O- (CH)₂)₄NH₂(ii) a 2' -O-anthraquinonyl alkyl (anthrylakyl), wherein alkyl includes methyl or ethyl; 2' -O- (CH)₂)₂-OCH₃、2′-O-(CH₂CH₂O)_n-CH₃Wherein n is 1 to 4; 2' -O-CH₂-CHR-X, wherein X ═ OH, F, CF₃Or OCH₃And R is H, CH₃、CH₂OH or CH₂OCH₃(ii) a Peptide nucleic acid monomers or derivatives thereof (PNA); 2' modification ofThe 2' -position in the above group is an alkoxy group such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, phenoxy or the like.

In some embodiments, the modified nucleotide comprises a modified sugar moiety or pseudo sugar (pseudosugar), including but not limited to bicyclic nucleotide structure (a), tricyclic nucleotide structure (b), or bicyclic nucleotide structure (c):

wherein R1 and R2 are independently O, S, CH₂Or NR, wherein R is hydrogen or C_1-3-an alkyl group; r3 is CH₂、CH₂-O、CH₂-S、CH₂-CH₂、CH₂-CH₂-CH₂CH or CH₂-NR, wherein R is hydrogen or C_1-3-an alkyl group; r4 and R5 are independently an internucleoside linkage, a terminal group, or a protecting group; at least one of R4 and R5 is an internucleoside linkage; b is a nucleobase, a nucleobase derivative or a nucleobase analogue. In some embodiments, R4 and R5 are independently H, OH, phosphate, C_1-12Alkyl radical, C_1-12Alkyl amine, C_1-12-alkenyl, C_1-12-alkynyl, C_1-12-cycloalkyl, C_1-12-an aralkyl group, an aryl group, an acyl group, a silyl group, an oligonucleotide, a nucleoside bonded by an internucleoside linkage, or a combination thereof; and at least one of R4 and R5 is an oligonucleotide, or a nucleoside bonded by internucleoside linkage. Substituents such as phosphate, C can be used_1-12Alkyl radical, C_1-12Alkyl amine, C_1-12-alkenyl, C_1-12-alkynyl, C_1-12-cycloalkyl, C_1-12Aralkyl, aryl, acyl or silyl groups further derivatize the 5' end. In some embodiments, for example, C is utilized₁₂Or C₆Substituents or derivatizing the 5' terminus with phosphate.

Bicyclic nucleotides includeBicyclic nucleotides as described herein for structures (a) and (c). Bicyclic nucleotides of structure (a) are also known as locked nucleic acids (locked nucleic acids) orIncluding, for example, β -D, and α -L bicyclic nucleotides, bicyclic nucleotides such as xylose-locked nucleic acid (U.S. Pat. No. 7,084,125), L-ribose-locked nucleic acid (U.S. Pat. No. 7,053,207), 1 '-2' locked nucleic acid (U.S. Pat. Nos. 6,734,291 and 6,639,059), and 3 '-5' locked nucleic acid (U.S. Pat. No. 6,083,482). The synthesis of bicyclic nucleotides of structure (c) and oligonucleotides containing such bicyclic nucleotides is described, for example, in Ma ier et al (Nucleic Acids Research 2004, 32: 12, 3642-. The synthesis of tricyclonucleotides of structure (b) and oligonucleotides containing such tricyclonucleotides is described, for example, in Ittig et al (Nucleic Acids Research 2004, 32: 1, 346-353).

In some embodiments, the nucleotide includes a modified internucleoside linker moiety, including, but not limited to, phosphorothioate, phosphorodithioate, phosphoroselenoate (phosphoroselenoate), phosphorodiselenoate (phosphorodiselenoate), phosphoroanilothioate, phosphoranilidate, phosphoroamidate (phosphoroamidate), boronophosphate, thiophanate (-S-CH)₂-O-CH₂-), methylene (methylimino), dimethylhydrazino, phosphoryl-linked morpholino, -CH₂-CO-NH-CH₂-，-CH₂-NH-CO-CH₂-and any analogues of phosphate esters in which the phosphorus atom is in a +5 oxidation state and one or more oxygen atoms are substituted with a non-oxygen moiety such as sulphur. Peptide nucleic acids are nucleic acid analogs in which the backbone comprises synthetic peptide-like linkages (amide bonds), typically formed from N- (2-amino-ethyl) -glycine units, resulting in achiral and uncharged molecules. Exemplary phosphate ester analogs include bound counterions such as H⁺、NH₄ ⁺、Na⁺(if such counterions are present).

In some embodiments, nucleotides include modified base moieties including, but not limited to, pyrimidine and purine nucleobases and derivatives and analogs thereof, including, but not limited to, pyrimidines and purines substituted with one or more of alkyl, carboxyalkyl, amino, hydroxyl, halo (i.e., fluoro, chloro, bromo, or iodo), mercapto, or alkylmercapto moieties. Alkyl (e.g., alkyl, carboxyalkyl, etc.) moieties contain 1 to 6 carbon atoms. Additional modified nucleotides referred to herein include oxetane modified bases (see U.S. published patent application 20040142946 for a discussion of bases comprising oxetane modifications).

Pyrimidines, pyrimidine derivatives and pyrimidine analogs include, but are not limited to, bromoethylamine (bromothionine), 1-methylpseudouracil, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 2-thiopyrimidine, 2-hydroxy-5-methyl-4-triazolopyridine, 3-methylcytosine, 3- (3-amino-3-carboxy-propyl) uracil, 4-acetylcytosine, 4-thiouracil, N4, N4-vinylcytosine (ethanocytosine), 4- (6-aminohexylcytosine), 5-methylcytosine, 5-ethylcytosine, 5- (C3, C6) -alkynylcytosine, 5-bromouracil, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-chlorouracil, 5-ethyluracil, 5-fluorouracil, 5-iodouracil, 5-propyluracil, 5-propynyluracil, thiouracil, 5-carboxymethylaminomethyl uracil, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-hydroxyacetic acid-methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 5-iodo-2' -deoxyuracil, 5-fluorouracil, 5-methyluracil, 5-chlorouracil, 5-ethyluracil, 5-fluorouracil, 5-methylcarbamoyluracil, tricyclic carbazole-based pyrimidine analogs, tricyclic phenoxazine-based pyrimidine analogs, isocytosine (isocytosine), pseudoisocytosine (pseudoisocytosine), dihydrouracil, pseudouracil, and universal nucleotides (univeral nucleotides).

In some embodiments, purines, purine derivatives, and purine analogs include, but are not limited to, azapurine, azaguanine, azaadenine, deazapurine (deazapurine), deazaguanine, deazaadenine, 1-methylguanine, 1-methyladenine, 1-methylinosine, 2-aminopurine, 2-chloro-6-aminopurine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 2-methylthio-N6-isopentenyladenine, 2, 6-diaminopurine, 6-aminopurine, 6-thioguanine, 6-thioadenine, 6-thiopurine, 6-hydroxyaminopurine, N6-methyladenine, N-dimethyladenine, N-methyladenine, N-, N6-isopentenyladenine, N6, N6-ethano-2, 6-diaminopurine, 7-deazaxanthine, 7-deazaguanine, 7-methylguanine, 7-halo-7-deazapurine wherein halo is bromo, fluoro, iodo or chloro, 7-propyne-7-deazapurine, 8-bromoadenine, 8-hydroxyadenine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-hydroxyguanine, 8-methylguanine, 8-thioguanine, 8-oxo-N6-methyladenine, N- ((9-beta-D-ribofuranosylpurin-6-yl) -carbamoyl) threonine, Methylthioadenine, xanthine, hypoxanthine, inosine, wybutosine, isoguanine, Q nucleoside (queuosine), β -D-mannosyl Q nucleoside, β -D-galactosyl Q nucleoside, and universal nucleotides.

The universal base may base pair with more than one type of specific base or any specific base. In other embodiments, the universal base does not specifically form hydrogen bonds with any base but interacts with an adjacent base on the same nucleic acid strand by hydrophobic stacking. Universal bases include, but are not limited to, indoles such as 7-azaindole, 6-methyl-7-azaindole, propynyl-7-aza-indole, dienyl-7-azaindole, isoquinolone (isocarbostyril), propynyl isoquinolone, imidazopyridine (imidizopyradine), and pyrrolpyrizine.

In some embodiments, the modified nucleotides are present in the siRNA in a particular pattern (referred to herein as a "format"), as exemplified by the patterns or formats shown in figures 1A through 1K. In some embodiments, the chemically synthesized passenger (sense) oligonucleotide has a length of 15 to 30 nucleotides and comprises one of a sequence-independent modification (1), a sequence-independent modification (2), and a sequence-independent modification (3):

wherein each m is independently a bicyclic nucleotide or a tricyclic nucleotide; each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide; and when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide; and when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide.

In some embodiments, a chemically synthesized passenger (sense) oligonucleotide is provided, the oligonucleotide consisting essentially of a length of 15 to 30 nucleotides and consisting essentially of one of a sequence-independent modified form (1), a sequence-independent modified form (2), and a sequence-independent modified form (3), as follows:

(2)5′N_p-m-m-N_y-m-N_z-m-N_q-n_r3' wherein p is 0 or1; y is 7, 8 or 9 and z is 1; or y is 7 and z is 2 or 3; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (p + y + z + r + 4);

wherein each m is independently a bicyclic nucleotide or a tricyclic nucleotide; each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide; when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide; and when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide.

In some embodiments, the guide (antisense) oligonucleotide comprises a region of 15 to 30 nucleotides that is continuously complementary to a passenger (sense) oligonucleotide of at least 12 nucleotides and has complementarity to at least a portion of the target mRNA. In some embodiments, the guide (antisense) oligonucleotide comprises a region that is continuously complementary to a passenger (sense) oligonucleotide of at least 13 nucleotides to a number of nucleotides corresponding to the full length of the oligonucleotide. In some embodiments, the guide (antisense) oligonucleotide comprises a region that is continuously complementary to the full length (except for two 3' nucleotides) of the passenger (sense) oligonucleotide.

In some embodiments, the guide (antisense) oligonucleotide has complementarity with at least a portion of the target mRNA by at least 12 contiguous nucleotides. In some embodiments, the guide (antisense) oligonucleotide has complementarity to at least a portion of the target mRNA of at least 13 nucleotides to a number of nucleotides corresponding to the full length of the guide oligonucleotide. In some embodiments, the guide oligonucleotide has complete complementarity (except for 2 3' nucleotides) to a portion of the target mRNA.

In some embodiments, short interfering RNAs are provided comprising a passenger (sense) oligonucleotide having a length of 17 to 30 nucleotides and comprising one of a sequence-independent modification form (4), a form (5) and a form (6), and a guide (antisense) oligonucleotide having a region of 17 to 30 nucleotides that is continuously complementary to the passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand also has complementarity to at least a portion of the target mRNA:

wherein each m is independently a bicyclic nucleotide, a tricyclic nucleotide, or a 2' -modified nucleotide; each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide, and is an overhang nucleotide; when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide, when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide, and when m is a 2 '-modified nucleotide, each N is independently a nucleotide other than a 2' -modified nucleotide.

In embodiments of short interfering RNAs, the passenger (sense) oligonucleotide has a sequence-independent modification (4), p is 2, x is 10, r is 2, each n is an overhang nucleotide, and each n is independently a modified nucleotide, the guide (antisense) oligonucleotide comprises 2 3' overhang modified nucleotides; each modified nucleotide is independently a bicyclic nucleotide, a tricyclic nucleotide, or a 2' modified nucleotide. In one embodiment, at least one of the 3 'penultimate and 3' penultimate positions of the passenger oligonucleotide is a modified nucleotide. The 3' penultimate site is the third site from the last site, i.e., the site preceding the penultimate site (e.g., form DH47 of fig. 1H). The 3' penultimate site is the fourth site from the last site, i.e., the site preceding the penultimate site (e.g., form DH29 of fig. 1H). Such siRNA is particularly stable to nucleases, e.g., in the presence of serum.

In some embodiments of short interfering RNA, when 17 nucleotides in length, at least one of positions 2 and 3 of the guide oligonucleotide is a modified nucleotide; when 18 nucleotides in length, at least one of positions 2, 3 and 4 of the guide oligonucleotide is a modified nucleotide; when 19 nucleotides in length, at least one of positions 2, 3,4 and 5 of the guide oligonucleotide is a modified nucleotide; when 20 nucleotides in length, at least one of positions 2, 3,4, 5 and 6 of the guide oligonucleotide is a modified nucleotide; and when the length is 21 to 30 nucleotides, at least one of positions 2, 3,4, 5, 6 and 7 of the guide oligonucleotide is a modified nucleotide. Form DH1 and form DH39-DH43 of fig. 1K provide examples of siRNA modified forms having a length of 21 nucleotides and at least one of positions 2, 3,4, 5, 6 and 7 of the guide oligonucleotide is a modified nucleotide. Such siRNA is also particularly stable against nucleases, for example in the presence of serum or plasma.

In some embodiments, the passenger (sense) oligonucleotide comprises a sequence-independent modification (1) wherein p is 0, x is 10 and r is 2 (form H, 21 mer with form H is depicted in fig. 1A); or a sequence-independent modified form (1) wherein p is 0, x is 9 or 11, and r is 2 (form H-1 or form H +1, a 21 mer having such a form is depicted in fig. 1B); or a sequence-independent modified form (2) wherein p is 0, y is 9, and r is 2 (form V, 21 mer with form V is depicted in fig. 1C).

In some embodiments, the passenger (sense) oligonucleotide comprises a sequence-independent modification form (1) wherein p is 1, x is 9 or 10, R is 2, (form W or form W +1, a 21 mer having such form is depicted in fig. 1D), and each m has the structure (a) wherein R1 is O, R2 is O, and R3 is CH₂. In some embodiments, the passenger (sense) oligonucleotide comprises a sequence-independent modification form (3), x is 9 or 10, R is 2, (form Y or form Y +1, 21 mer with such form is depicted in fig. 1D), and each m has structure (a), wherein R1 is O, R2 is O, and R3 is CH₂。

In further embodiments, each N of the guide (antisense) strands is a nucleotide other than a bicyclic nucleotide. In some embodiments, the guide (antisense) strand has no modification.

In some embodiments, the siRNA passenger oligonucleotide has a modified form H, wherein modified nucleotide m is at positions 1, 2, 13, and 14 of said form; each m isEach n is a deoxynucleotide; and each N is a ribonucleotide. In some embodiments, the siRNA passenger oligonucleotide has a modified form H, and each m has the structure (a), wherein R1 is O, R2 is O, and R3 is CH₂。

In some embodiments, the siRNA passenger oligonucleotide has a modified form V, wherein modified nucleotide m is at positions 1, 2, 12 and 14 of said form; each m isEach n is a deoxynucleotide; and each N is a ribonucleotide. In some embodiments, the siRNA passenger oligonucleotide has a modified form V, and each m has the structure (a), wherein R1 is O, R2 is O, and R3 is CH₂. In some embodiments, the siRNA passenger oligonucleotide has a modified form V-2, which possesses form (2), wherein p is 0 and y is 7.

In some embodiments, the siRNA passenger oligonucleotide has a modified form Q, wherein modified nucleotide m is at positions 13 and 14 of said form; each m isEach n is a deoxynucleotide; and each N is a ribonucleotide. In some embodiments, the siRNA passenger oligonucleotide has a modified form Q, and each m has the structure (a), wherein R1 is O, R2 is O, and R3 is CH₂。

In some embodiments, the siRNA passenger oligonucleotide has form (4), wherein when p is 0, x is 12 (form Q); when p is 1, x is 11 (form JB 2); when p is 2, x is 10 (form H); when p is 3, x is 9 (form JB5), and when p is 4, x is 8 (form JB 3) and r is 2. In some embodiments, the siRNA passenger oligonucleotide has form (5), wherein x is 11 (form JB1) and r is 2. In a further embodiment, the siRNA passenger oligonucleotide has form (6), wherein p is 3, y is 9 and z is 1 (form JB4), and r is 2.

In some embodiments, the chemically synthesized short interfering RNA comprises a passenger (sense) oligonucleotide and a guide (antisense) oligonucleotide covalently bonded through a nucleotide loop or linker loop, which form a short hairpin oligonucleotide.

In some embodiments, each 3 'end of the siRNA independently has an overhang of 1, 2, or 3 nucleotides, in some embodiments, each 3' end has an overhang of 2 nucleotides, wherein the nucleotides of each overhang comprise deoxyribonucleotides. In some embodiments, at least one overhang nucleotide is a modified nucleotide. In some embodiments, each 3' end of the siRNA has an overhang of 2 nucleotides, and 1, 2, 3, or all 4 overhang nucleotides are modified nucleotides. In some embodiments, at least one internucleoside linkage is not a phosphodiester internucleoside linkage.

Chemical synthesis: interfering RNA oligonucleotide synthesis was performed according to standard methods. Non-limiting examples of synthetic methods include using the diester method, the triester method, the polynucleotide phosphorylase method, and in vitro chemical synthesis by solid phase chemistry. Such methods are discussed in more detail below.

For example as described by Imanishi in U.S. patent nos. 6,770,748 and 6,268,490; as described in Wengel in U.S. Pat. Nos. 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461 and in U.S. published applications 2005/0287566 and 2003/0224377; as described by Kochkine in U.S. patents 6,734,291 and 6,639,059 and U.S. published application 2003/0092905; and oligonucleotides having bicyclic nucleotide structures (including those of structure (a)) were synthesized as described in U.S. published application 2004/0219565 to Kauppinen and as described by Srivastava, P., et al (J.Am.chem.Soc.129, 8362-.

Oligonucleotides having the tricyclic nucleotide structure (b) are synthesized, for example, according to Ittig, D.et al (Nucleic Acids Res.32: 1, 346-353, 2004). Oligonucleotides having the bicyclic nucleotide structure (c) are synthesized, for example, according to Maier, M.A. et al (Nucleic Acids Res.32: 12, 3642-3650, 2004). Oligonucleotides with FANA substituents were synthesized according to Dowler et al (Nucleic Acids Res.2006, 34: 6, p 1669-1675).

The diester method of oligonucleotide synthesis was the first synthesis method developed into a usable state mainly by Khorana et al (Science, 203, 614.1979). The basic step is to join two appropriately protected nucleotides to form a dinucleotide comprising a phosphodiester bond. The diester method is well established and has been used to synthesize DNA molecules.

The main difference between the diester and triester processes is the presence of an additional protecting group on the phosphate atom (phosphate atom) of the reactants and products in the latter (Itakura et al, j. biol. chem., 250: 45921975). The phosphate protecting group is typically chlorophenyl, which allows the dissolution of nucleotide and polynucleotide intermediates in organic solvents. Thus, purification was carried out in chloroform solution. Other improvements to this approach include (i) block coupling of trimers or larger oligomers, (ii) extensive use of high performance liquid chromatography to purify intermediates and end products, and (iii) solid phase synthesis.

Using techniques developed for solid phase synthesis of polypeptides, it has been possible to attach the starting nucleotides to a solid support material and then perform the successive addition of nucleotides. All mixing and washing steps are simplified and the process is easily automated. Such synthesis can now be routinely performed using automated nucleic acid synthesizers.

Phosphoramidite chemistry (Beaucage, S.L. and Iyer, R.P.tetrahedron, 1993(49) 6123; tetrahedron, 1992(48)2223) has hitherto become the most widely used coupling chemistry for oligonucleotide synthesis. As is well known to those skilled in the art, phosphoramidite synthesis of oligonucleotides involves activation of a nucleoside phosphoramidite monomer precursor by reaction with an activator to form an activated intermediate, which is then sequentially added to a growing oligonucleotide chain (typically anchored at one end to a suitable solid support) to form an oligonucleotide product.

siRNA can be purified on reverse phase purification cartridges (reversed phase purification cartridges), ion exchange HPLC, agarose gels, polyacrylamide gels, cesium chloride centrifugation gradients, columns, filters or cartridges containing nucleic acid binding agents (e.g., glass fibers), or by any other method known to those skilled in the art. Gel electrophoresis can be used to determine single-to-double-stranded structure, ion exchange HPLC can be used to determine purity, MALDI-MS can be used to determine identity (identity), UV spectroscopy can be used to quantify siRNA.

The cell containing the endogenous target gene may be derived from or comprised in any organism (e.g., eukaryotes such as plants, animals, fungi; prokaryotes such as bacteria). The plant may be a monocot, a dicot, or a gymnosperm; the animal may be a vertebrate or an invertebrate. Microorganisms may be used in agriculture or industry, or may be pathogenic to plants or animals. Fungi include organisms that exist in the morphological forms of molds and yeasts. Examples of vertebrates include fish and mammals (including cows, goats, pigs, sheep, hamsters, mice, rats, and humans); invertebrates include nematodes, insects, arachnids and other arthropods.

The target gene may be a gene derived from a cell, an endogenous gene, a transgene, or an exogenous gene such as a gene of a pathogen (e.g., a virus) present in a cell after infection. The cell having the target gene may be derived from a germline or somatic cell, a totipotent or pluripotent cell, a dividing or non-dividing cell, a parenchymal or epithelial cell, an immortalized cell or a transformed cell, or the like. The cell may be a gamete or an embryo; in the case of an embryo, it may be a single cell embryo or a constituent cell from a multicellular embryo (constitutive cell). The term "embryo" thus includes fetal tissue. The cell having the target gene may be an undifferentiated cell such as a stem cell, or a differentiated cell such as a cell from an organ or tissue including fetal tissue or any other cell present in an organism. Differentiated cell types include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelial cells (endothelium), neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

In some embodiments, the target RNA is a transcribed RNA, including coding RNA and non-coding RNA.

As used herein, the terms "cell," "cell line," and "cell culture" include their progeny, which are any and all of the progeny that form through cell division. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. A host cell may be "transfected" or "transformed," which refers to a process by which an exogenous nucleic acid is transferred or introduced into the host cell. Transformed cells include primary test cells and their progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells are intended to refer to cells into which an exogenous nucleic acid sequence, such as a small interfering RNA or a template construct encoding such RNA, has been introduced. Thus, a recombinant cell is distinguishable from a naturally occurring cell that does not comprise a recombinantly introduced nucleic acid.

In certain embodiments, it is contemplated that RNA or proteinaceous sequences may be co-expressed with other selected RNA or proteinaceous sequences in the same host cell. Co-expression can be achieved by co-transfecting host cells with two or more different recombinant vectors. Alternatively, a single recombinant vector comprising a plurality of different RNA coding regions may be constructed and then expressed in a host cell transfected with the single vector.

In some embodiments, the tissue containing the endogenous target gene is human tissue. In certain embodiments, the tissue comprises blood, such as, but not limited to, red blood cells, white blood cells, platelets, plasma, serum, or whole blood. In certain embodiments, the tissue comprises solid tissue (solid tissue). In certain embodiments, the tissue comprises a virus, a bacterium, or a fungus. In certain embodiments, the tissue comprises ex vivo tissue.

The tissue may comprise a host cell to be transformed or contacted with the nucleic acid delivery composition or additional agent. The tissue may be part of an organism or may be isolated from an organism. In certain embodiments, the tissue and its constituent cells may include, but are not limited to, blood (e.g., hematopoietic cells (e.g., artificial blood progenitor cells, artificial blood stem cells, CD34+ cells, CD4+ cells), lymphocytes and other blood lineage cells), bone marrow, brain, stem cells, blood vessels, liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryo, endometrium, endothelium, epithelium, esophagus, face (facial), fibroblasts, follicular, ganglion cells, glial cells, goblet cells, kidney, lymph node, muscle, neurons, ovary, pancreas, peripheral blood, prostate, skin, small intestine, spleen, stomach, testis.

In some embodiments, modified formatted sirnas that are stable to intracellular and extracellular nucleases are provided. It is demonstrated herein that such siRNA remain in the presence of biological fluids for longer periods of time than previously known stable siRNA. Nuclease-stable sirnas are used in biological fluids and tissues (including, e.g., serum, plasma, and particularly vascularized organs and tissues) containing particularly high concentrations of nucleases.

The siRNA can be conjugated to a ligand that targets the cell. As used herein, a "cell-targeting ligand" is a cell-directing molecule (cell-directing molecule) that has specificity for a site being targeted, e.g., a cell surface receptor. By "specific for the site to be targeted" is meant that specific binding will occur under physiological conditions of ionic strength, temperature, pH, etc., after contacting the cell-targeting ligand with the cell. Cell-ligand interactions may occur as a result of specific electrostatic, hydrophobic, or other interactions of certain residues of the ligand with specific residues of the cell, thereby forming a stable complex under conditions effective to promote the interaction.

Exemplary cell-targeting ligands include, but are not limited to, polynucleotides, oligonucleotides, polyamides, peptides having affinity for cellular receptors, proteins such as antibodies, fatty acids, vitamins, flavonoids, sugars, antigens, receptors, reporter molecules, reporter enzymes, chelators, porphyrins, intercalators, steroids and steroid derivatives, hormones such as progesterone (e.g., progesterone), glucocorticoids (e.g., cortisol), mineralocorticoids (e.g., aldosterone), androgens (e.g., testosterone) and estrogens (e.g., estradiol), histamine, mimic hormones (hormonemics) such as morphine and macrocyclic compounds. Peptides with affinity for cellular receptors may include endorphins, enkephalins, growth factors such as epidermal growth factor, poly-L-lysine, hormones, insulin, ribonuclease, serum albumin binding peptides, peptide regions of proteins capable of being transported across cell membranes either actively or passively, and other molecules.

Suitable methods for siRNA delivery to affect RNAi according to embodiments include any method by which siRNA can be introduced into an organelle, a cell, a tissue, or an organism, as described herein or known to one of skill in the art. Such methods include, but are not limited to, direct delivery of siRNA, e.g., by injection (including microinjection), electroporation, calcium phosphate precipitation, use of DEAE-dextran followed by polyethylene glycol, direct ultrasound loading (direct sonic loading), liposome-mediated transfection, microparticle bombardment, agitation using silicon carbide fibers, agrobacterium-mediated transformation, PEG-mediated transformation, desiccation/inhibition-mediated uptake (desiccation/inhibition-mediated uptake), and the like. Organelles, cells, tissues or organisms can be stably or transiently transformed using techniques such as these. Sirnas that are stable to nuclease degradation (embodiments of which are provided herein) are particularly suitable for direct injection, as shown by the data in example 8, in which serum-stable, modified, formatted sirnas are present in vivo at a level of 400% of unmodified sirnas.

Techniques for visualizing or detecting siRNA include, but are not limited to, microscopy, array, fluorimetry, light cycler or other real-time PCR instrument, FACS analysis, scintillation counter, phosphoimager (phosphor), Geiger counter, MRI, CAT, antibody-based detection methods (Western, immunofluorescence, immunohistochemistry), histochemical techniques, HPLC, spectroscopy, mass spectrometry; radiology techniques, capillary gel electrophoresis and mass balance techniques. Alternatively, the nucleic acids may be labeled or tagged to allow their efficient separation. In other embodiments of the invention, the nucleic acid is biotinylated.

"label" or "reporter" refers to a moiety or property that allows detection of a substance bound thereto. Labels may be attached covalently or non-covalently. Examples of labels include fluorescent labels (including, e.g., quenchers or absorbents), colorimetric labels (colorimetric labels), chemiluminescent labels, bioluminescent labels, radioactive labels, mass-modifying groups, antibodies, antigens, biotin, haptens, enzymes (including, e.g., peroxidases, phosphatases, etc.), and the like. Fluorescent labels may include negatively charged dyes, such as dyes of the fluorescein family, including, for example, FAM, HEX, TET, JOE, NAN, and zo; or dyes that are neutral in charge such as dyes of the rhodamine family, including, for example, Texas Red, ROX, R110, R6G, and TAMRA; or positively charged dyes, such as dyes of the cyanine family, including, for example, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy 7. FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, and TAMRA are available from, for example, Perkin-Elmer, Inc. (Wellesley, MA); texas Red is available, for example, from Molecular Probes, Inc. (Eugene, OR); and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 are available from, for example, amersham biosciences Corp. In certain embodiments, the fluorescer molecule is a fluorescein dye and the quencher molecule is a rhodamine dye.

The label or reporter may comprise a fluorophore and a fluorescence quencher. The fluorescence quencher may be a fluorescent fluorescence quencher such as the fluorophore TAMRA or a non-fluorescent fluorescence quencher (NFQ), e.g., a combined NFQ-Minor Groove Binder (MGB) such as provided by Epoch Biosciences (Bothell, WA) and combined with TAQMAN^TMMGB ECLI PSE with probes (Applied Biosystems, Foster City, Calif.)^TMMinor groove binders. The fluorophore may be any fluorophore that can be attached to a nucleic acid, for example, FAM, HEX, TET, JOE, NAN, zo, Texas Red, ROX, R110, R6G, TAMRA, Cy2, Cy3.5, Cy5, Cy5.5, and Cy7 (as referenced above), as well as VIC, NED, LIZ, ALEXA, Cy9, and dR 6G.

Additional examples of labels include Black Hole Quenchers (BHQ) (Biosearch), Iowa Black (DDT), QSY quenchers (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate quenchers (Epoch). Labels may also include sulfonate derivatives of fluorescein dyes, phosphoramidite forms of fluorescein, phosphoramidite forms of CY5 (available, e.g., from Amersham), intercalating labels such as ethidium bromide, and SYBR^TMGreen I and PICOGREN^TM(Molecular Probes)。

In various embodiments, the detection of fluorescence can be performed using any method known to those skilled in the art, and can be either qualitative or quantitative. Quantitative results can be obtained, for example, with the aid of a fluorimeter. In some embodiments, detection can be performed using microarrays and related software, such as the Applied Biosystems Array System using the Applied Biosystems 1700 chemiluminescent microarray analyzer and other commercially available Array systems (see also Gerry et al, J.Mo.biol.292: 251-62, 1999; De Bellis et al, Minerva Biotec 14: 247-52, 2002; and Stears et al, nat. Med.9: 140-45, including supplementary materials, 2003).

A biologically acceptable carrier refers to a carrier that provides adequate preservation (when needed) and delivers one or more interfering RNAs of the embodiments herein in a uniform dose. The type of carrier may depend on whether the siRNA is used in solid, liquid or aerosol form, and on whether it needs to be sterile for the route of administration, e.g., injection. The siRNA composition can be formulated into the composition as a free base, neutral, or salt form. The salt form of the siRNA has the property of reducing off-target effects and maintaining inhibitory activity (comparable to the siRNA described herein). Examples of salt forms include organic acid addition salts and base addition salts. The siRNA composition may be provided in the form of a prodrug that is metabolized to an active form within the cell; such prodrug forms may include protecting groups such as S-acetylthioethyl (S-acetylthioethyl) or S-pivaloylthioethyl (S-pivaloylthioethyl) groups.

In embodiments where the composition is in liquid form, the carrier may be a solvent or dispersion medium, including, but not limited to, rnase-free water, buffers, salt solutions, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), lipids (e.g., triglycerides, vegetable oils, liposomes), glycine, acceptable preservatives (e.g., antibacterial agents, antifungal agents), co-solvents (co-solvents), surfactants, penetration enhancers (e.g., cremephor and TWEEN), and the like^TM80 (polyoxyethylene sorbitan monolaurate, Sigma Aldrich, st. louis, Mo.)), hyaluronic acid, mannitol, benzalkonium chloride, viscosity building agents (e.g., hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose), coatings, surfactants, antioxidants, isotonicity agents, absorption retarders, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, and combinations thereof. When desired, sterile injectable solutions are prepared by incorporating the siRNA into an appropriate solvent with various of the other ingredients enumerated above, followed by filtered sterilization. The formulation comprises up to 99% by weight of the interfering RNA in the embodiments herein, or a salt thereof, in admixture with a biologically acceptable carrier. The interfering RNA in the embodiments may be used in the form of a solution, suspension or emulsion.

Acceptable carriers for interfering RNA in embodiments include the lipid-based agent, siPORT^TM NeoFX^TMTransfection reagent (Ambion Cat. # AM4510), polyamine-based reagent siPORT^TMAmine transfection reagent (Ambion Cat. # AM4502), cationic and neutral lipid based reagent siPORT^TMLipid transfection reagent (Ambion Cat. # AM4504), cationic Lipid-based transfection reagent-TKO(Mirus Corporation，Madison，Wis.)、Lipofectamine 、Oligofectamine^TM(Invitrogen, Carlsbad, Calif.) or Dharmafect^TM(Dharmacon, Lafayette, Colo.), another polycation such as polyethyleneimine, a cationic peptide such as Tat, polyarginine or penetrating protein (penetratin), or a liposome. For example, liposomes can be formed from standard vesicle-forming lipids and sterols, such as cholesterol, and can include targeting molecules such as monoclonal antibodies having binding affinity for cell surface antigens.In addition, the liposome may be a pegylated liposome.

The interfering RNA can be delivered in solution, suspension, or in a bioerodible or non-bioerodible delivery device. Interfering RNA can be delivered alone or as a component of a defined covalent conjugate, e.g., with a polyethylene glycol moiety, a cholesterol moiety, or with a growth factor (for receptor-mediated endocytosis). Interfering RNAs can also be complexed with cationic lipids, cationic peptides, or cationic polymers; complexed with a protein, fusion protein or protein domain having nucleic acid binding properties (e.g., protamine); or encapsulated in nanoparticles or liposomes. Tissue or cell specific delivery can be achieved by including suitable targeting moieties such as antibodies or antibody fragments. Unless it is incompatible with siRNA, embodiments herein contemplate that any conventional carrier can be used in the composition or as a biological carrier.

The interfering RNA in the embodiments herein may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally (intramural), intracranially, intraarticularly, intraventricularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intrathecally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly (intramuraticularly), transmucosally, intrapericardially, intraumbilically (intramurally), intraocularlly, orally, topically (topically), topically (subcalorly), inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, directly locally perfusing water bath target cells (bathwater cells), by catheter, by lavage, in cream, in lipid compositions (e.g., liposomes), or by other methods known to those skilled in the art, or any combination of the foregoing methods (Remington' pharmaceuticals), 18 th edition, Mack Publishing co.1990).

The dose used ex vivo or in vivo is 0.01 μ g to 1g/kg of body weight, and can be administered once or more times per day, week, month or year. Generally, an effective amount of interfering RNA with modified nucleotides and modified forms in embodiments herein is an amount sufficient to reduce off-target effects while maintaining potency and produce an extracellular concentration on the surface of a target cell of 100pM to 1 μ M, or 1nM to 100nM, or 2nM to about 25nM, or to about 10 nM. The amount required to achieve this local concentration may vary depending on a number of factors including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, etc. The concentration at the delivery site may be much higher than the concentration at the surface of the target cell or tissue. The pH of the formulation is about pH 4-9 or pH 4.5 to pH 7.4.

In some embodiments, the ability of an siRNA to inhibit the expression level of an endogenous target gene is assessed in vitro as follows. Using siPORT^TM NeoFX^TMTransfection reagent (Ambion/Applied biosystems Austin, TX Ca t # AM4510), cells were transfected using the "reverse" transfection protocol described by the manufacturer. Unmodified control or modified formatted siRNA is plated into 96-well tissue culture plates to obtain a final concentration of, for example, 1nM or 5 nM. Will SiPORT^TM NeoFX^TMTransfection reagent and siRNA complex, for at least 10 minutes, then will just be trypsin treated cells (approximately 4.0X 10)³) Overlaid on siRNA/transfection reagent complexes. The plates were incubated at 37 ℃ for 48 hours under tissue culture conditions.

After harvesting the transfected cells, MagMAX was used according to the manufacturer's protocol^TM-96 Total RNA isolation kit (Ambion, Inc. Austin TX Cat. # AM1830) for total RNA isolation. RNA was eluted in 50. mu.l nuclease-free water. cDNA was generated in a 30 μ l reaction volume using the highbeacon cDNA Reverse Transcription Kit (Applied Biosystems, inc., Foster City CA, Cat. #4368814) according to the manufacturer's protocol. Using 2. mu.l cDNA in 10. mu.l Total PCR reaction volumeGene expression Assay (Applied Biosystems, Inc., Cat. # 4331182). Technical repeats were performed and the 18S value was calculated for each sample. The homogeneity of the pre-amplification was checked by calculating ddCT using the following formula:

ddCT ═ (Ct treated with siRNA)_GOICt of siRNA treatment_18S) - (Neg control Ct)_GOI-Neg control Ct_18S)

GOI ═ endogenous gene of interest

18S-18S ribosomal RNA normalizer (normarizer)

siRNA-treated versus siRNA-treated biological samples

Neg control biological samples treated with sirnas with no homology to any known target mRNA

The percentage of residue was calculated using the following formula:

remainder%^-(ddCT)。

Target protein levels can be estimated, for example, by western blotting, at about 72 hours post-transfection (actual time depends on protein turnover). Standard techniques for isolating RNA and/or proteins from cultured cells are well known to those skilled in the art.

Techniques for visualizing or detecting siRNA include those cited above. The nucleic acids may be labeled or tagged to enable their efficient separation. Thus, the silencing ability of any given siRNA can be studied by any of a number of assays in the art.

"kit," as used herein, refers to a combination of at least some items for reducing off-target effects in RNA interference. Embodiments of the kit include, for example, at least one siRNA having modified nucleotides in modified forms of embodiments herein. The at least one siRNA may be tailored. In some embodiments, the kit comprises at least one siRNA having a bicyclic nucleotide in modified form H, Q, V-2, Y, Y +1, JB1, JB2, JB3, JB4, or JB5, and a transfection agent or other suitable delivery vehicle, together with a biologically acceptable carrier, such as a buffer.

Embodiments of the kit may further comprise validated positive control sirnas targeting housekeeping genes in all cell types of human, mouse and rat. Embodiments of the kit can also include a validated negative control siRNA (which is non-targeting). siRNA controls can be pre-plated and labeled with a detectable marker.

Embodiments of the kit may also include reagents for assessing the inhibition of a desired target gene, such as antibodies for monitoring inhibition at the protein level by immunofluorescence or Western analysis, reagents for assessing enzymatic activity or the presence of a reporter protein, or reagents for assessing cell viability. RT-PCR primers and probes may be included to detect mRNA for the target or reporter. Embodiments of the kit may further comprise an rnase inhibitor.

The container means (container means) of the kit will generally comprise at least one vial, test tube, flask, bottle, syringe or other packaging means into which the components may be placed and, in some embodiments, into which the components are suitably aliquoted. Where more than one component is included in a kit (which may be packaged together), the kit will typically also include at least one second, third or other additional container into which the additional components may be separately placed. However, different combinations of components may be packaged in the container means. Kits of the present teachings also generally include means for containing the siRNA with modified nucleotides in the modified form shown herein and any other reagent containers (for commercial sale) that are closed. Such containers may include injection or blow molded plastic containers that retain the desired container tools therein. When the components of the kit are provided in one and/or more liquid solutions, the liquid solutions include aqueous solutions, which may be sterile aqueous solutions.

In certain embodiments, at least one kit component is lyophilized and provided in the form of a dry powder. When the agents and/or components are provided in the form of a dry powder, the powder may be reconstituted by the addition of a suitable solvent. In certain embodiments, the solvent is provided in another container means. The kit may also include additional container means for holding sterile, biologically acceptable buffers and/or other diluents.

The kit may also include instructions for using the kit components as well as using any other reagents not included in the kit. The instructions may include variations that may be implemented.

Embodiments of the present methods and kits may be used for high volume screening (high volume screening). A library of sirnas or candidate sirnas can be constructed using embodiments herein. The library can then be used in high throughput assays, including microarrays. Particularly contemplated are chip-based nucleic acid techniques, which include quantitative methods for rapid and accurate analysis of large numbers of genes. By using an array of immobilized probes, chip technology can be used to isolate target molecules and screen these molecules based on hybridization in a high density array. The term "array," as used herein, refers to a systematic arrangement of nucleic acids. For example, a population of nucleic acids representing a desired source (e.g., human adult brain) is divided into a minimum number of pools (where target genes can be detected or depleted using a desired screening method and can be assigned into a single multi-well plate). The array may be an array of aqueous suspensions of a population of nucleic acids obtainable from a desired mRNA source, including: a multiwell plate comprising a plurality of individual wells, each individual well comprising an aqueous suspension of a different content of a population of nucleic acids. Examples of arrays, their use, and their implementation can be found in U.S. patents 6,329,209, 6,329,140, 6,324,479, 6,322,971, 6,316,193, 6,309,823, 5,412,087, 5,445,934, and 5,744,305, which are incorporated herein by reference.

Microarrays are known in the art and consist of a surface to which probes corresponding in sequence to gene products (e.g., cDNA, mRNA, cRNA, polypeptides, and fragments thereof) specifically hybridize or bind at known locations. In one embodiment, a microarray is an array (i.e., matrix) in which each site represents a separate binding site for a product (e.g., a protein or RNA) encoded by a gene, and in which the binding sites are directed against the products of most or nearly all of the genes in the genome of an organism. In a preferred embodiment, a "binding site" is a nucleic acid or nucleic acid analog to which a particular relevant cDNA can specifically hybridize. The nucleic acid or analog of the binding site can be, for example, a synthetic oligomer, a full-length cDNA, a cDNA that is shorter than full-length, or a fragment of a gene.

The solid support can be made of glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other material. Exemplary methods for attaching nucleic acids to a surface include printing on a glass plate or by masking (masking). In principle, any type of array may be used, e.g. dot blot on nylon hybridization membranes, however, as recognized by the person skilled in the art, very small arrays are preferred, as the hybridization volume will be smaller.

Embodiments of the present teachings can be further understood in light of the following examples, which should not be construed as in any way limiting the scope of the present teachings.

Example 1

siRNA modified forms

Short interfering RNA (siRNA) were chemically synthesized according to Beaucage, S.L. and Iyer, R.P, (Tet rahedron, 1993(49) 6123; Tetrahedron, 1992(48)2223) using standard phosphoramidite-based nucleoside monomers and an established solid phase oligomerization cycle. The synthesis of oligonucleotides was carried out on a BioAutomation MerMade192 synthesizer (BioAutomation Corp, Plano, TX). Use of 8 equivalents of the activator forEach equivalent of phosphoramidite provides a satisfactory stepwise coupling yield of greater than 98% (addition per base). Purification of individual siRNA strands was performed using disposable reverse phase purification cartridges or ion exchange HPLC. Gel electrophoresis is used to determine the structure of single strand versus double strand of an annealed sample, ion exchange HPLC is used to determine single strand purity, MALDI mass spectrometry is used to determine single strand identity, UV spectrometry is used to quantify single strand identityOligonucleotides and siRNA. Each siRNA has a guide strand and passenger strand of 21 nucleotides in length, and each siRNA is designed to have an overhang of two bases on both 3' ends. Site 1 of each guide (antisense) strand is designed as an "a nucleotide" or a "U nucleotide". When a cytosine (C) nucleoside is modified to a bicyclic sugar, the substituted nucleobase is a 5-methylated cytosine residue. When uracil (U) nucleosides are modified to bicyclic sugars, the substituted nucleobases are cytosine (T) residues. The overhang nucleotides are as described in fig. 1A to fig. 1K, and (independently) are: deoxynucleotides, modified nucleotides or ribonucleotides. In the case of the form "O", the guide strand is chemically phosphorylated. All other 3 'and 5' termini were synthesized to include hydroxyl groups.

Modified nucleotides for introduction into siRNA molecules include locked nucleic acids: (Residue), particularly those having the structure (a) above, wherein R1 is O, R2 is O, and R3 is CH₂(ii) a 2' -O-methyl nucleotide derivatives; and 2 '-5' linked nucleotides.amidites are available from Prologo (Boulder, CO) and Exiqon A/S (Vedbaek, Denmark), 2 ' -O-methyl nucleotide derivatives, and 3 ' -and 2 ' -TBDMS RNA amidites are available from Chemmenes corporation (Wilmington, MA).

Modified forms into which sirnas were introduced include those shown in fig. 1A (forms E, F, G, H, J, K and O except unmodified control form a), fig. 1B (form M, H (repeated), H (-1), H (-2), H (+1) and Q), fig. 1C (form M (repeated), H (repeated) V, V (-1), V (-2), V (+1)), fig. 1D (forms W, W +1, W-1, Y, Y-1 and Y +1), fig. 1E (forms JB1, JB2, JB3, JB4 and JB5), and those shown in fig. 1F to fig. 1K further described in example 7.

Modified forms include modifications only on the passenger (sense) strand (forms E, H, K, M, H-1, H-2, H +1, Q, V, V-1, V-2, V +1, W, W +1, W-1, Y, Y-1, Y +1, JB1, JB2, JB3, JB4, JB5, DH3, DH30 and DH28 (for the DH form, see example 7)) or on the lead (antisense) strand and the passenger (sense) strand (form F, G, J, O and all DH forms except DH3, DH30 and DH28 (for the DH form, see example 7)). The modified forms of FIGS. 1A-1E are described further below.

Formal modification sites (numbering starting from the 5' end of each strand)

Passenger sequence guide sequence

A, control none

E1, 20, 21 are

F 1，20，21 20，21

G 1，2，13，14 9，10，19，20

H1, 2, 13, 14 is

J 1，2，10，14 11，12，19，20

K1, 2, 10, 14 is

O1, 22 + 5' phosphoric acid

M1, 2 is absent

H (-1) 1, 2, 12, 13 none

H (-2) 1, 2, 11, 12 none

H (+1) 1, 2, 14, 15 is

Q13, 14 is

V1, 2, 12, 14 is

V (-1) 1, 2, 11, 13 none

V (-2) 1, 2, 10, 12 none

V (+1) 1, 2, 13, 15 is

W2, 3, 13, 14 has

W + 12, 3, 14, 15 none

W-12, 3, 12, 13 none

Y1, 3, 13, 14 is

Y-11, 3, 12, 13 is

Y + 11, 3, 14, 15 is

JB 11, 2, 14 none

JB 21, 13, 14 none

JB 31, 2, 3,4, 13, 14 none

JB 41, 2, 3, 13, 15 none

JB 51, 2, 3, 13, 14 none

Form F was previously reported by Mook et al (Molecular Cancer therapeutics March, 20076: 3, 833-. Forms E and F have been previously reported by E l men et al (Nucleic Acids research January 14, 2005.33: 1, 439-. Jackson et al (RNA May 8, 200612: 71197-.

Example 2

Method for measuring siRNA silencing effect

Cleavage activity of exogenously supplied genes mediated by the leader (antisense) strand and passenger (sense) strand of a given siRNA was determined as follows. A cDNA fragment from a gene of interest (GOI) is cloned into pMIR-REPORT in either forward or reverse orientation (relative to the orientation of the coding strand of the gene DNA) using DNA ligase and standard cloning techniques^TMmiRNA expression reporter vector system (Ambion/Applied Biosystems, Austin TX Cat # AM 5795). For the following GOI, full-length cDNA clones were purchased from Open Biosystems (Huntsville, AL).

Gene ID Symbol Description of the invention

983 CDC2 cell division cycle 2

1017 CDK2 cyclin-dependent kinase 2

701 BUB1B not inhibited by beta benzimidazole 1 homolog

Budding of BUB1

2222 FDFT1 farnesyl diphosphate farnesyltransferase 1

3156 HMGCR 3-hydroxy-2-methylglutaryl-coenzyme A reductase

7456 WEE1 WEE1 homolog (Schizosaccharomyces (S.pombe))

5347 PLK1 Polo-like kinase 1

1213 CLTC lattice protein, heavy chain (Hc)

836 CASP3 caspase 3, apoptosis-related caspase

Amino acid peptidase

595 CCND1 cyclin D1

1595 CYP51A1 cytochrome P450, family 51, subfamily A,

polypeptide 1

The size of the length of the GOI coding sequence ranges from 1.2kb to more than 6 kb. Separately subcloning the cDNA fragments from each GOI into pMIR-REPORT^TMThe HindIII (nucleotide position 463)/SpeI (nucleotide position 525) multiple cloning site of a miRNA expression reporter vector comprising a firefly luciferase reporter protein under the control of a mammalian promoter/terminator system, as shown in FIG. 2A. Standard PCR-based techniques were used to engineer HindIII and SpeI restriction endonuclease sites. Generating a reporter vector that expresses an mRNA transcript comprising luciferase mRNA fused to mRNA of the GOI in the forward or reverse direction (relative to fl ucmm, as shown in fig. 3A and 3B). The ffluc-GOI fusion mRNA is expressed under the control of the CMV promoter located closest to the 5' start site of the ffluc coding sequence. When the reporter construct and siRNA complementary to either the ffluc or the cDNA GOI are co-transfected into the cells, inhibition as a result of RNA interference is monitored by measuring the remaining luciferase protein or by the remaining ffluc-GOI fusion mRNA. The 5 'and 3' end 500 nucleotides of all subcloned fragments from each orientation of each GOI were subjected to nucleotide sequence analysis to confirm the identity and orientation of the cDNA contained within the vector. Thus, these vectors serve as a source of substrate for monitoring siRNA cleavage mediated by either the leader strand (forward cloning) or the passenger strand (reverse cloning).

The pMIR-REPORT described in FIG. 2B was used according to the manufacturer's protocol^TMBeta-galactosidase reporter control vector (Ambion/Applied Biosystems, Austin TX Cat # # AM5795) normalized transfection efficiency. The β -gal coding sequence is expressed under the control of a CMV promoter. The signal obtained from the ffluc reporter gene was divided by the signal obtained from the respective β -gal reporter gene to calculate the normalized luciferase activity for each assay.

Transfection: hela cells (ATCC No. CCL-2, Manassas, Va.) at about 8.0X 10³Density of individual cells/well plated in 96-well tissue culture plates. After 24 hours, standard transfection protocols were used, with siPORT^TMAmine transfection reagent (Ambion/Applied biosystems Austin, TX Cat # AM4502) and siRNA to be investigated, 40ngpMIR-REPORT, at a final concentration of 30nM^TMluciferase-GOI plasmid and 4ng pMIR-REPORT^TMTransfection of control plasmids cells were transfected. Cells were washed with complete medium 24 hours after transfection and harvested 48 hours after transfection for analysis. For each plasmid, 3 biological replicates of each siRNA to be studied were performed. In parallel, to calculate the percentage of mRNA inhibition produced by the passenger and guide strands of the test siRNA, the transfection was performed under the same experimental conditionsNegative control siRNA (Ambion/Applied Biosystems, Austin TX Cat # AM4636) to measure baseline levels of expression using non-targeting siRNA.

Dual-Combination reporter assay system: applied Biosystems Dual-A combination reporter assay system (applied biosystems, Foster City CA Cat # T1003) was used to simultaneously detect luciferase and β -gal in the same sample. Luminescence readings were performed using a BMG LABTECH POLARstar Optima reader (BMG LABTECH, Durham, NC).

In Dual-Luciferase assays were performed in the assay system 2 to 5 seconds after addition of the luciferin substrate. The plate is allowed to stand at least before the addition of substrate for detection and measurement of beta-galactosidaseFor 45 minutes. Wells were normalized using appropriate β -gal readings to obtain the luciferase signal/β -gal signal ratio. The% inhibition or remaining luciferase expression was then calculated by normalizing the biological samples to negative control siRNA transfected cells.

Example 3

silencing Effect of siRNA modified forms can be used for Strand switching (Strand-Switch)

To perform this study, 2 sirnas were synthesized in unmodified form a and each of 8 different test modified forms (E, F, G, H, J, K, M and O) for each of 3 exogenously supplied target mrnas. Thus, each test format provides data for 6 sirnas. For the data of FIGS. 4A-4E, the modified nucleotide "m" at the site designated by the formal letter and referenced to FIGS. 1A and 1B is of structure (a) aboveResidue, wherein R1 is O, R2 is O, and R3 is CH₂. It was experimentally determined that the siRNA sequences selected for use in this study, as illustrated in figure 4A, have strand silencing activity on portions of both strands. Such a collection allows analysis of the impact that a modification has on introducing strand bias (i.e., the ability to attenuate the silencing activity of one strand while maintaining the activity of the other strand). For this siRNA pool, an average of about 70% inhibition of luciferase-GOI mRNA mediated by the unmodified guide strand was shown, and an average of about 58% inhibition of luciferase-GOI mRNA mediated by the unmodified passenger strand was shown (fig. 4A, form a).

The amount of normalized reporter protein present after RNAi studies of unmodified form a and each of the tested modified forms (E, F, G, H, J, K, M and O) were performed is plotted in fig. 4A. Dark bars indicate the remaining activity% due to silencing by the guide strand, since the data was generated by clones with GOI in the forward orientation. The light bars of fig. 4A represent the remaining activity% due to silencing of the passenger strand, since the data was generated by clones with GOI in reverse orientation. Normalization was performed by parallel transfection of scrambled (nonsense) sirnas, which resulted in a small amount of inhibition of both strands. This reduction in signal (approximately 20%) resulted in a significant increase in reporter protein (for all normalized results) because normalization was based on the negative value of the nonsense siRNA. This significant increase is most pronounced for passenger strand data that modifies targets E, F, G, H, J, K, M and O. The model reporting system is used to detect changes in the activity of the passenger and guide chains.

The data in fig. 4A shows that the guide (antisense) strand of the siRNA with modified forms E, F, H, K, M and O achieved essentially the same silencing activity as the control form a guide strand. In the presence of modified forms G and J, the ability of the guide strand of the siRNA to silence their target is reduced, and in the case of form J, the ability of the guide strand of the siRNA to silence their target is reduced to less than 20% of the average inhibitory activity.

While most of the modified forms depicted in fig. 4A provide for guide (antisense) strand-mediated cleavage similar to the unmodified control guide strand, all tested modified forms severely attenuate the ability of the passenger strand to silence their target, thereby substantially inactivating the passenger strand.

To further examine the effect of the modified form on strand activity, the group of sirnas used for strand type assay was expanded to include another 18 sirnas. The data in FIGS. 4B-4E provide the results of the measurement of a larger pool of siRNAs in boxed line format (also called boxed and whisker plots). For each figure, the dark horizontal bars represent the median values of the data sets for each modification. The boxes represent the distribution of data over the lower quartile and upper quartile of the dataset for each modification, while the dashed lines (whiskers) represent the minimum and maximum values of the dataset for each modification. Circles represent possible outliers in the data set for each modified form.

FIG. 4B provides the passenger strand activity after transfection of siRNA into Hela cells for this larger siRNA group. The activity was calculated using the formula: p (passenger strand activity) ═ (remaining fl uc β -gal activity from the reverse clones treated with test siRNA)/(remaining fl β -gal activity from the reverse clones treated with Neg control siRNA). The data in fig. 4B shows the loss of passenger activity for the modified siRNA form as seen by an upward shift in median passenger inhibitory activity (compared to the inhibitory activity of form a). The data for modified form G, H, J, K, M and O were statistically significant (p < 0.05), demonstrating that the modified form was effective in reducing the activity of the passenger chain.

FIG. 4C provides the activity of the guide strand after transfection of Hela cells with the siRNA group analyzed in FIG. 4B. The activity was calculated using the formula: g (activity of guide strand) ═ (remaining fl uc β -gal activity from forward clones treated with test siRNA)/(remaining fl β -gal activity from forward clones treated with Neg control siRNA). Forms E, F, H, M and O have no negative effect (i.e., "harmless") on the activity of the guide strand. Form E appears to increase the ability of the guide strand to reduce luciferase signal in a statistically significant manner, as indicated by the downward shift in median guide strand activity (compared to unmodified form a). Modified forms G, J and K reduced the activity of the guide strand as evidenced by an upward shift in the median of such forms (p < 0.05) (compared to the unmodified siRNA of form A).

The data in table 4D provide the difference in the fhuc activity between the passenger and leader chains of figures 4B and 4C obtained using the following formula: activity difference P-G (using the definitions of P and G indicated above). Because the fLUC reporter assay measures the amount of firefly luciferase activity in cells following siRNA transfection, the more active siRNA strands inhibit more of the fLUC reporter mRNA and result in a lower relative amount of luciferase activity. Thus, if the passenger strand is less active than the guide strand, the value calculated for this activity difference will be greater than 0. If the passenger strand is more active than the guide strand, the value will be less than 0. Modified form J reduced the difference in activity between the two strands of the test siRNA.

The data of fig. 4E provides fold-changes in activity between the passenger and guide strands of the sirnas of fig. 4B and 4C. Fold change in activity was calculated as log using the definitions of P and G shown above₂(P)-log₂(G) In that respect Value 0siRNAs with equal silencing ability for each strand are described. The greater the fold change in activity, the greater the leader strand bias of the siRNA. Comparison of the boxplots of fig. 4E shows a significant increase in strand bias (p < 0.05) between the passenger and guide strands for modified forms E, H, M and O compared to the strand bias for control unmodified siRNA form a. Thus, sirnas with modified forms E, H, M and O exhibited increased strand pattern compared to unmodified form a. Forms G, J and K show a lower fold difference between the passenger and leader, but only form J significantly reduced the fold change in activity (compared to form a). Form H shows the greatest individual chain bias.

For each modification, studies were conducted to examine the ability of the modification to affect the silencing activity of high potency siRNA strands by strand Switching (SW) modification patterns (from passenger strand to guide strand and from guide strand to passenger strand). That is, for example, for modified form F of FIG. 5A_SWModifications of form F at positions 1, 20 and 21 of the passenger (sense) strand (fig. 1A) were introduced into the guide (antisense) strand, and modifications of form F at positions 20 and 21 of the guide (antisense) strand (fig. 1A) were introduced into the passenger (sense) strand.

The data of fig. 5A provides the results of a strand assay in which the amount of normalized reporter protein present after RNAi studies was performed was plotted for 12 sirnas with unmodified siRNA form a and the same 12 sirnas with converted strand modifications (compared to those of fig. 4A). The group of sirnas studied in the data of fig. 5A differed from the group studied in the data of fig. 4A in that the sirnas in the data of fig. 5A had a strand bias as evidenced by the large difference in the percentage of remaining fLUC signal between the leader and passenger strands of the unmodified form a sirnas. The experimental conditions used to measure inhibition in this "chain switch" assay are the same as those used to generate the data of fig. 4A.

As provided by the data of fig. 5A, the silencing activity of the guide strand is significantly reduced due to the strand switch modification. The guide strand with the modified form of strand switch achieved an average of less than 20% inhibition. For this siRNA pool, modified form H of chain transition_SWIs to guide (reverse)Sense) one of the most effective modified forms of chain inactivation.

Test passenger chains with modified forms of chain conversion provide variable results. However, all tested passenger strands with modified forms of strand switching were more effective in inhibition than passenger strands in unmodified siRNA form a in fig. 5A. While not wishing to be bound by theory, as a result of the diminished potency of the guide strand, the potency of the passenger strand is likely to be enhanced indirectly or secondarily as indicated by the enhanced inhibition obtained by the passenger strand in the chain switch modified form.

Fig. 5B provides the data of fig. 5A in the form of a boxplot, which is as described above for fig. 4D. Thus, if the activity of the passenger strand is lower than the activity of the guide strand, the expected difference will be greater than 0, as evidenced by modification form a for which the median activity difference for the pool of sirnas is about 0.65. The modified form of chain transfer had a differential activity of less than 0, indicating form H_SW、M_SW、F_SW、E_SWAnd O_SWThe strand type is switched so that it favors the passenger strand over the guide strand (as compared to unmodified form a). Modified form M for chain transfer_SWThe greatest chain enhancement was observed (with a median activity difference of about-0.5).

In FIG. 5C, for each modification, the fold change (log) in activity between the passenger and guide strands of the siRNA of FIG. 5B is provided₂(P)-log₂(G) ). These results show that: a modified form that confers a leader chain inhibitory activity and substantially inactivates a passenger chain, when a chain transition is performed, the inhibitory activity of the leader chain can be decreased and the inhibitory activity of the passenger chain can be increased. Studies with FIGS. 4A and 4D were performed using different modification formats (usingModified nucleotides) in which the modified nucleotides are methylated by 2' -O. To perform this study, 2 sirnas were synthesized in unmodified form a and each test form for each of the 3 target mrnas. Due to the fact thatThus, each test format provides data for 6 sirnas. As with the study of fig. 4A, the particular siRNA sequence selected for this study was one in which both strands of the unmodified form of the AsiRNA showed silencing. The guide (antisense) strand of the unmodified siRNA reduced the target mRNA by 70% on average, while the passenger (sense) strand reduced the target mRNA by 55%.

With respect to the activity of the passenger (sense) strand, the data of fig. 6A shows that after 48 hours of incubation with siRNA in culture, substantially all mRNA product is available for luciferase assay. That is, the test siRNA with the modified form in which the modified nucleotide was methylated by 2' -O showed a significant loss of potency of the passenger (sense) strand as indicated by an increase in% expression of the remaining genes in these samples. Inactivation and use of the passenger chain's inhibitory functionThe data for the modified form of the modified nucleotide are in agreement. And also withThe data for modified forms H, K and O are consistent with the substantially unchanged potency of the guide strand having modified forms H, K and O with 2' -O-methyl nucleotides compared to unmodified control form a. Thus, modified forms of nucleotides with 2' -O-methyl modifications appear to be equally effective in these strand assays (for which the target is provided exogenously) for maintaining the activity of the guide (antisense) strand while inactivating the cleavage activity of the passenger (sense) strand.

Figure 6B provides data in the form of a boxplot of the siRNA studied in figure 6A (6siRNA) and another 18 siRNA sequences. The difference in activity of the form a group shows that most siRNA is biased toward the guide strand (indicated by a median value of about 0.5). However, sirnas with 2' OMe in the indicated modified form showed greater differences between the passenger and guide strands compared to control form a. Forms H and H +1 induced the largest interchain differences. Form O with the 2' -OMe modified base did not increase the inter-strand activity differences.

Fold change in activity of passenger versus guide chains is provided in fig. 6C, calculated as described previously. Fold change analysis showed that form H provided the greatest fold change compared to the unmodified siRNA of form a. However, significant fold changes in interchain activity were also observed for forms H-1, H, H +1, V-2, and K but not form O, as determined by the Wilcoxon values quoted in the drawing illustration of FIG. 6C.

The third type of modified nucleotides was studied in different modification formats. FIG. 7A provides the results of a strand assay in which the modified form of the modified nucleotide is a 2 ', 5' -linked nucleotide. For the unmodified form A and the test forms H-1, H, H +1, V-2, K and O, pMIR-REPORT from GOI cloned in forward and reverse orientation will be used^TMDifferences in activity of the fLuc signals studied (thus representing differences in efficacy and inhibitory activity of the lead and passenger chains) are plotted. Comparison of the strand pattern results for unmodified control form a with the results for the test form containing the 2 ', 5' -linked base showed that a statistically significant change in the strand pattern of the siRNA was not obtained by modification with the base comprising the 2 ', 5' -linked in addition to form O. The base modified form O with 2 '-5' -linkage reduced the difference in activity, indicating that the inhibitory activity of the guide strand was lost in a statistically significant manner.

Figure 7B provides fold activity changes between passenger and guide chains (calculated as described previously). Fold change analysis showed that form O containing the 2 '-5' linked nucleic acid affected the strand pattern of the siRNA in the negative direction, as indicated by a decrease in fold change in activity between the passenger and guide strands. Furthermore, data from 2 '-5' linked nucleic acids present in the forms H-1, H +1, V-2 and K are not statistically significant, and thus, these forms are not suitable for increasing the strand pattern of siRNA. Only form H modified with the 2 '-5' base modification showed a statistically significant increase in strand form as shown by the Wilcoxon values quoted in the figure legend of fig. 7B.

Based on the data of FIGS. 4A and 6A, regarding the difference in cleavage activity of the guide strand for the passenger strandThe statistical analysis of (a) shows: the largest change in chain tropism is represented by modification in form HAnd 2' OMe modified nucleotide introduction. The strand-type analysis also showed that form G, form J and form K reduced the activity of the guide strand in a statistically significant manner, so that their use for enhancing siRNA specificity was not considered anymore afterwards. Has the advantages ofForm H of the modified nucleotide induced an average 8-fold difference in cleavage activity of the guide strand over the passenger strand, as demonstrated by the data in fig. 4A. In contrast, unmodified control form a showed an average 4-fold increased activity of the guide strand over the passenger strand. Thus, the form H modification doubles the strand bias that is typically present in optimally designed sirnas.

In addition, these data prove to haveThe form of chain transition of the modified residues is as effective as they inactivate the passenger chain as they inactivate the leader chain.

The data representation of FIGS. 12A-1 and 12A-2 is included in the specified formInhibitory activity of 12 sirnas modifying nucleotides. The strand type of the test group indicated that most of the siRNA was asymmetrically biased toward the guide strand with higher activity (indicated by the difference in median activity of 0.5 for form a of fig. 12A-1). However, produced have modified forms andsiRNA of residues showed greater difference between passenger and guide strand activity compared to unmodified form a. Form H modified forms increased the inter-chain differences in activity in a statistically significant mannerBut fold-activity changes were not significant, as shown by the data in fig. 12A-2.

The data in FIGS. 13A-1 and 13A-2 represent siRNAs containing 2' OMe modified nucleotides in the indicated format. The strand type of the test group indicated that most of the siRNAs were asymmetrically biased toward the guide strand with higher activity, as indicated by the median activity difference of 0.5 in FIG. 13A-1. However, the resulting siRNA with 2' OMe modified nucleotides showed a greater fold change between the passenger and guide strands compared to unmodified control form a, as shown in fig. 13A-2. Forms H, K and V-2 induced the largest and statistically significant interchain fold change (p < 0.05). Data for all forms, except form O, were statistically significant compared to unmodified siRNA.

Example 4

Effect of siRNA modification forms on silencing of endogenous genes

Silencing of the endogenous gene by the modified form of the siRNA is detected to determine whether the modified form alters the efficacy of the siRNA in inhibiting its mRNA target. To perform this study, inhibition of endogenous genes was examined by transfecting modified versions of siRNA into Hela cells (ATCC No. CCL-2, Manassas, VA), U2OS cells (human osteosarcoma cell line, ATCC No. HTB-96), and HUH7 cells (human hepatoma cell line, # JCRB 0403, Japanese Cancer Research resource Bank, Tokyo, Japan) at low concentrations. Data from Hela cells are discussed herein.

Using the manufacturer's described "reverse" transfection scheme, using siPORT^TMNeoFX^TMHeLa cells were transfected with a transfection agent (Ambion/Applied Biosystems Austin, TX Cat # AM 4510). Unmodified control or modified formatted siRNA was plated into 96-well tissue culture plates to obtain final concentrations of 1nM or 5 nM. Will SiPORT^TMNeoFX^TMThe transfection reagent was complexed with siRNA for at least 10 minutes, and then the Hela cells (approximately 4.0X 10) that had just been trypsinized were added³Individual cells) were overlaid on the siRNA/transfection agent complex. Plates were incubated 48 ℃ at 37 ℃ under tissue culture conditionsAnd (4) hours.

Using the manufacturer's described "forward" transfection protocol, use was made ofTransfection reagent (Qiagen, Gaithersberg, Md.) transfected U2OS cells. Unmodified control or modified formatted siRNA was plated into 96-well tissue culture plates to achieve final concentrations of 3nM or 30 nM. Will be provided withTransfection reagent and siRNA complex, for at least 10 minutes, then the complex is added to the culture of U2OS cells (approximately 4.0X 10)³Individual cells). The plates were incubated at 37 ℃ for 48 hours under tissue culture conditions.

After harvesting the transfected cells, MagMAX was used according to the manufacturer's protocol^TM-96 Total RNA isolation kit (Ambion, Inc. Austin TX Cat. # AM1830) for total RNA isolation. RNA was eluted in 50. mu.l nuclease-free water. The cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied biosystems tems, Inc. Fos ter City CA, Cat. #4368814) in a 30. mu.l reaction volume according to the manufacturer's protocol. Using 2. mu.l cDNA in a 10. mu.l total PCR reaction volumeGene Expression Assays (applied Biosystems, Inc., Cat. # 4331182). Technical repeats were performed and the 18S value was calculated for each sample. The uniformity of pre-amplification was checked by calculating ddCT using the above formula.

The remaining% was calculated using the following formula: remainder as 100X 2^-(ddCT)。

The fraction of mRNA inhibition was calculated using the following formula:

fraction of mRNA inhibition 1- (2)^-(ddCT))

And

mRNA inhibition ═ 100 fraction of mRNA inhibition.

To perform this inhibition study, 6 sirnas were synthesized in each of unmodified form a and 8 test modified forms E, F, G, H, J, K, M and O for each of the 8 endogenous target mrnas. Thus, each test format provided data for 48 sirnas. With respect to the data of FIG. 8, the modified nucleotide "m" at the site designated by the formal letter and referred to FIGS. 1A to 1C is of the structure (a) described aboveResidue, wherein R1 is O, R2 is O, and R3 is CH₂。

FIG. 8 provides silencing of endogenous mRNA targets in Hela cells with modified formatted siRNA at a final concentration of 5nM and 48 hours post transfectionData for% inhibition of target determined by GeneExpression Assays. The results of these studies show that inclusion of forms G, J and K in the group compared to the inhibitory activity observed for the unmodified form AModified formatted siRNA of (1) attenuates the inhibitory activity of the siRNA, however inclusion of forms E, F, H, M and OThe modified formatted siRNA of (a) maintains or increases inhibitory activity. Thus, having at different sitesSeveral modifications of the residues do not negatively affect the inhibitory activity of the siRNA, but are sufficient to enhance the strand pattern of the guide strand to the passenger strand. The assay was also performed on human hepatoma cells (HUH7) and human osteosarcoma cells (U2OS)Inhibitory activity of siRNA in the pool of modified sirnas. In these cell lines, the respective modified forms are obtained by taking into account different transfection efficienciesThe modified siRNA exhibited inhibitory activity similar to that observed in HeLa cells (data not shown). Thus, silencing performance is not affected by the type of cell line used.

FIG. 12B provides information regarding useAdditional inhibitory activity data for modified forms of modified nucleotides H, H-2, H-1, H +1, Q, K, V, V-1 and V-2. The data set represents 24 different sirnas targeting 4 different endogenous mRNA targets. Each of 24 sirnas was synthesized in each form, and the inhibitory activity of the sirnas in each group was analyzed similarly. Hela cells were transfected at a final concentration of 5nM and used 48 hours after transfectionThe measurement was performed by GeneExpression Assay. The results show a slight decrease in the inhibitory activity of the sirnas present in forms K and V-2 (this decrease was statistically insignificant compared to the unmodified s RNA). Modified forms H, H-1, H-2, H +1, Q, V, and V-1, maintained or increased inhibitory activity on the mRNA target compared to the inhibitory activity observed for the unmodified siRNA group.

The data in FIG. 13B provide the inhibitory activity of modified forms H, H +1, H-1, K, V-2, and O using 2' OMe modified nucleotides. The data set represents 24 different sirnas targeting 4 different endogenous mRNA targets. Each of 24 sirnas was synthesized in each form, and then the inhibitory activity of the sirnas in each group was analyzed similarly. Hela cells were transfected at a final concentration of 5nM and used 48 hours after transfectionGene Expression Assay. The results show that the modified forms H, H +1, H-1, K, V-2, and O, retained or increased inhibitory activity compared to that observed for the unmodified form Asi RNA.

FIG. 14A provides a use compared to form AAdditional data for modified forms of modified nucleotides H, M, W, W +1, W-1, Y, Y +1 and Y-1. The data set represents 15 different sirnas targeting 5 different endogenous mRNA targets. Each of 15 sirnas was synthesized in the designated form, and then the inhibitory activity of the sirnas in each group was analyzed similarly. U2OS cells were transfected at a final concentration of 30nM and used 48 hours after transfectionThe measurement was performed by GeneExpression Assay. Result display has utilityThe siRNA of the residue modified forms W, W +1, W-1, Y, Y +1, and Y-1 maintained inhibitory activity when compared to the inhibitory activity of unmodified siRNA form A. The siRNA with modified form M, when compared to the inhibitory activity of the siRNA with unmodified form a, had attenuated (in a statistically significant manner) the inhibitory activity, and the siRNA with modified form H, when compared to the inhibitory activity of unmodified form a, had increased (in a statistically significant manner) the inhibitory activity.

Data provision usage of FIG. 14BModified forms of the modified nucleotides H, M, JB1, JB2, JB3, JB4 and JB5 have inhibitory activity compared to form a. The data set represents 15 different sirnas targeting 5 different endogenous mRNA targets. Each of the 15 siRNAs was synthesized in the indicated format and then analyzed identicallyInhibitory activity of siRNA in each group. U2OS cells were transfected at a final concentration of 30nM and used 48 hours after transfectionThe measurement was performed by GeneExpression Assay. The results show that the sirnas with the modified forms JB2, JB3 and JB5 were not statistically significantly different from the unmodified form a in terms of inhibitory activity; the siRNA with form H, JB1 and JB4, improved the inhibitory activity compared to form a, and the siRNA with form M, had lower inhibitory activity when compared to form a.

In the modified forms of siRNA examined in this example, forms G, J, K and M attenuated the inhibitory activity when compared to the unmodified form.

Example 5

Effect of siRNA with modified form H on the Whole Gene expression Profile as a measure of off-target Effect

Form H by measuring Gene profiles Using microarray analysisAnd 2' -O-methyl modified nucleotides compared to unmodified siRNA. Microarray genomic analysis was performed on quadruplicate biological samples transfected with 4 sirnas designed to target the FDFT1 gene. FDFT1 is a gene involved in cholesterol biosynthesis, which is not essential for cells used for detection. This criterion ensures that the differences in gene expression detected by array analysis are entirely the result of siRNA mediated cleavage and not the result of any biological cascade due to loss of functional mRNA target.

Using standard methods as outlined above, in unmodified form A,Modified forms of H and 2' -O-methylModified form H each of 3 siRNA sequences (designated siRNA #183, #184 and #192) was transfected into Hela cells, and the transfected cells were harvested after 24 hours. RNA was isolated for microarray analysis using standard methods. Affymetrix U133V2 microarray chips (Affymetrix, Santa Clara, Calif.) containing human probe sets were used for these studies. Controls included negative control siRNA, "mock" (mock) transfected cells and untreated cells (to record baseline levels of gene expression in the absence of experimental treatment). After normalizing the data, the results are analyzed to determine the identity of genes that vary by a factor of 2 or more in each sample of the sample set compared to the results of the mock (delivery agent only) control sample. One-way analysis of variance (1-way ANOVA) using planned contrasts (planed constrast) was then used to determine the p-value for each simulated treatment for the experimental conditions. Differentially expressed gene probe sets were then determined at 2-fold change and p < 0.001. The number of differentially expressed genes is a measure of off-target effects due to siRNA.

Figures 9A-9C provide venn plots of the results for siRNA #183 (figure 9A), siRNA #184 (figure 9B), and siRNA #192 (figure 9C) (3 different sirnas designed to target the FDFT1 gene). Each figure depicts the use of the indicated unmodified, 2' -O-methyl orResults for each of 3 sirnas in the form of modified sirnas. The venn plot shows the number of specific genes that changed in each experimental condition and the intersection of genes between siRNA conditions. For each figure, the lower left group (red if colored) shows the number of specific genes that changed 2-fold or more in the unmodified siRNA treated samples, the upper group (blue if colored) shows the number of specific genes that changed 2-fold or more in the 2' -O-methyl form H modified siRNA treated samples, and the lower right group (green if colored) showsForm H modified siRNA treated samplesThe number of specific genes that vary by a factor of 2 or more.

For siRNA #183 (fig. 9A), unmodified siRNA induced a 2-fold or greater reduction in mRNA levels of 31 genes (2+19+10+ 0). The 2' -O-methyl modified siRNA induced a 2-fold or greater reduction in mRNA levels of 32 genes (3+0+10+19), 29 of which were also affected by the unmodified control siRNA. The samples treated with 2' -O-methyl modified siRNA differed from the unmodified siRNA-treated samples by only 3 genes in the number of off-target genes.The modified siRNA induced a 2-fold or greater reduction in mRNA levels of 10 genes (0+0+10+0), all of which were also affected by the unmodified control and the 2' -O-methyl modification. Thus, these 10 genes represent off-target effects that are sequence dependent and cannot be attenuated by the teachings provided herein. Thus, compared to the 2' -O-methyl modification (reduction of off-target effect by 0%),the modified form had a more significant effect on the elimination of off-target gene expression changes (68% reduction in off-target effect).

For siRNA #184 (fig. 9B), unmodified siRNA induced a 2-fold or greater reduction in mRNA levels of 70 differentially expressed genes (14+23+33+1) in Hela cells. The 2' -O-methyl modified siRNA induced a 2-fold or greater reduction in mRNA levels of 66 genes (9+1+33+ 23). In contrast, compared to the results for unmodified siRNA,the modified siRNA reduced the number of differentially expressed genes by 50% (35 genes (0+1+33+ 1)). In thatThe vast majority of differentially expressed genes (33 genes) in the modified samples were also modified in unmodified siRNA and 2' -O-methylIs identified as differentially expressed in the siRNA treated sample. Such genes represent off-target effects that are sequence dependent and cannot be attenuated by the teachings provided herein. Thus, compared to the 2' -O-methyl modification (reduction of off-target effect of 6%),the modified form again had a more significant effect on the elimination of off-target gene expression changes (50% reduction in off-target effect).

For siRNA #192 (fig. 9C), unmodified siRNA induced a 2-fold or greater reduction in mRNA levels of 103 differentially expressed genes (28+13+61+ 1). The 2' -O-methyl modified siRNA induced a 2-fold or greater reduction in mRNA levels of 81 genes (7+0+61+ 13).The modified siRNA induced a 2-fold or greater reduction in mRNA levels of 64 genes (2+1+61+ 0). Therefore, compared with the unmodified siRNA,the modification eliminated off-target effects by about 38%. For this siRNA, 61 genes represent off-target effects that are sequence dependent and cannot be attenuated by the teachings provided herein. Compared to the 2' -O-methyl modification (reduction of off-target effect by 21%),the modified form again had a more significant effect on the elimination of off-target gene expression changes (38% reduction in off-target effect).

In summary, microarray analysis showed that, when compared to unmodified form A siRNA,the modified form H siRNA produced a 38% to 68% reduced differentially expressed gene, 2' -O-formazan when compared to the unmodified form A siRNAThe base modified form H siRNA produced genes with reduced differential expression of 0% to 22%. Therefore, the temperature of the molten metal is controlled,the modified form H siRNA provides the least number of off-target effects.

Example 6

Performance of modified formatted siRNA in cell biology studies

Cell biology studies were performed to measure the ability of the modified forms and types of modified nucleotides to affect the on-target and off-target phenotypes caused by siRNA.

To determine whether the modified formatted siRNA maintains an "on-target" or desired phenotype and reduces an "off-target" or undesired phenotype, sirnas are selected that induce a measurable and highly characterized biological response. Thus, in addition to eliminating off-target phenotypes, the maintenance of on-target phenotypes was monitored. Sirnas were synthesized in the test modified form, transfected into appropriate cells for assay as described below.

An assay referred to herein as a "growth assay" was performed to study the difference in cellular levels of modified formatted siRNA compared to unmodified siRNA and negative control siRNA. Growth assays include measurement of cell phenotype-related parameters such as proliferation, apoptosis and morphology in the U2OS osteosarcoma cell line following siRNA transfection at 30nM and at 3 nM. Results from the 30nM assay are discussed herein because more off-target effects are observed at higher siRNA concentrations, thus the barrier to abrogate such off-target effects (barrier) is higher. The growth assay includes immunofluorescence detection of the following antigens that are markers of a designated phenotype:

1. cleaved lamin a (apoptosis),

2. phosphorylated histone H3 (mitosis),

3. tubulin (cytoskeletal morphology, which is a control measure of properties, i.e. background determination for cell structure, and which labels all cells), and

hoescht staining (nuclear morphology, which provides cell number normalization).

Using IMAGEXPRESS^MicroTMAutomated fluorescence microscopy (Molecular Devices, Toronto, Canada) using computer-controlled image acquisition software (Cellinger, Munich, Germany) andimmunofluorescence was collected with a 10X objective lens driven by the image analysis software package (molecular devices, Toronto, Canada). 4 sites in each 384 well sample were collected and the average of the data from 3 biological replicates was calculated and compared to a negative siRNA control sample (spiked non-targeting siRNA) treated in a similar manner. After image analysis, the data were evaluated according to the following protocol:

1) and (3) index calculation: for each site in the well, normalizing the number of mitotic nuclei and apoptotic nuclei against the total number of nuclei, subtracting the area of the non-cellular background from the total image area to calculate the area occupied by the cell, and then normalizing against the number of nuclei;

2) sample well mean calculation: calculate the average of all sites in the wells, in addition, calculate the average of all sites of all negative control wells per plate;

3) and (3) standardization: samples and positive controls from each plate in triplicate were normalized to the average of negative siRNA control wells;

4) mean calculation of triplicate replicates: the average of triplicate plates was calculated for each treatment and reading (readout).

Test genes were selected that were well characterized functionally in different cells (according to published literature). The test genes include those of table 1.

TABLE 1

Identification of strongly induced siRNAs that provide a measurable expected phenotype (e.g., a 300% increase in mitosis after inhibition by Weel or PLK1 (Watanabe, N. et al (2004) PNAS, 101, 4419-. Inhibition of the BUB1B gene is expected to reduce the percentage of mitosis compared to Neg controls (Lampson, m.a. and Kapoor, t.m. (2004) Nature Cell Biology, 7, 93-98).

In addition, sirnas exhibiting off-target phenotypes were identified. For example, sirnas directed against LDLR (a gene that is not expected to induce apoptosis) (see table 1 for other such genes) were shown to increase apoptosis by 500%. Such siRNA sequences are used as a tool to determine whether a modified form can improve the specificity of the siRNA. Generating separate sets of siRNAs with observable on-target and off-target phenotypes to comprise several modifications and 2' -O-methyl modified nucleotides or siRNA having the structure (a) aboveResidue, wherein R1 is O, R2 is O, and R3 is CH₂. Such modified sirnas are tested for on-target and off-target phenotypes to determine modified forms that can eliminate off-target effects while maintaining the on-target phenotype.

The cell-based assay results of the modified formatted sirnas are plotted in the form of box and whisker plots in fig. 10A-10E, 11A-11F, 12C-12D, 13C-13D, 15A-15D, and 16A-16D for comparison to the normalized value (labeled Neg in the plots) of negative control siRNA treated samples analyzed in parallel in cell-based assays.

FIG. 10A provides a block diagram of a computer system withBoxplots of normalized mitotic cells caused by silencing produced by siRNA from modified forms E, F, G, H, J, K, M and O. siRNA targets BUB1B, WEE1, CDC2, CDK2, CASP3 and CCND1 (these genes have widely different effects on the mitotic phenotype). That is, the set of genes includes a subset that increases mitosis when silenced, a subset that decreases mitosis when silenced, and a subset that is not expected to have an effect on mitosis when silenced. As a result of such disparate effects, the subset of genes whose inhibition provided the expected mitotic-loss phenotype was analyzed, and the data is provided in figure 10B.

FIG. 10B data shows mitotic cells normalized after transfection with siRNA targeting genes (BUB1B, CDC2, CCND1) whose inhibition is expected to reduce mitosis (Lampson, M.A. and Kapoor, T.M. (2004) Nature Cell Biology, 7, 93-98; Harborth, J. et al (2001) Journal of Cell Science 114, 4557-4565; Klier, M. (2008) Leukemia, EPUB). Thus, these data measure the on-target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. Normalized values for the Neg control treated samples showed a median of approximately 1.0. Unmodified form a siRNA provided a median of about 0.55, indicating an approximately 2-fold decrease in mitosis. All modified forms reduced mitosis compared to the Neg control. Sirnas with modified forms H and J provided statistically significant differences (less on-target effects) in their effect on mitosis compared to unmodified form a, as evidenced by the Wilcoxon values and the deviation of the median mitosis from unmodified form a. Thus, consistent with the results of fig. 8 for mRNA inhibition, cell-based assays showed that form J is incompatible with the desired characteristics of highly functional siRNA.

Cell-based growth assays apoptosis was also measured by quantifying fluorescence signals from cells displaying cleaved lamin a compared to Neg control treated samples (Rao, l. et al (1996) J Cell Biology, 135, 1441-1455). Historically, the most common unexpected phenotype observed in genome-wide siRNA screening experiments was cell death. In the siRNA pool used for such studies, siRNA sequences known to induce unexpected cell death are included and such siRNA sequences are used as a tool to determine whether a modified form can abrogate an undesirable cell phenotype.

FIG. 10C provides a quantitative qualitative approach to haveResults of apoptotic fragments generated from treatment of U2OS cells with siRNA modified forms E, F, G, H, J, K, M and O. Negative control siRNA treated samples were used to determine baseline for apoptosis after siRNA transfection, and data was normalized to 1.0. The sirnas in this pool target the following genes: BUB1B, CDC2, CCND1, WEE1, CASP3 and CDK2 (a panel of gene targets, inhibition of which a subset is expected to increase apoptosis, inhibition of which a subset is expected to have no effect on apoptosis). As a result of such widely differing effects, the subset of genes whose inhibition provided the expected increased apoptosis phenotype was analyzed, and the data is provided in fig. 10D.

Fig. 10D provides results of measurements of apoptosis in U2OS cells after transfection with siRNA targeting WEE 1. Inhibition of WEE1 is expected to increase apoptosis (Watanabe, N. et al (2004) PNAS, 101, 4419-31324; Leach, S.D. et al (1998) Cancer Research, 58, 3132-3136). Thus, these data measure the on-target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. The Neg control siRNA treated samples were used to determine a normalized baseline of apoptosis. Unmodified siRNA targeting form a of WEE1 exhibited a median value of approximately 7.5 compared to NEG treated samples, providing the expected phenotypic change. Modified formatted sirnas having forms J and K reduce the on-target phenotype by reducing the amount of apoptosis (when compared to the unmodified form a siRNA) in a statistically significant manner, demonstrating that forms J and K impair siRNA performance. These data are consistent with those of fig. 8 for inhibition by sirnas with modified forms J and K.

The data of fig. 10E provides an analysis of the ability of modified formatted sirnas to eliminate off-target phenotypes of a subset of genes whose inhibition was expected to have no effect on apoptosis. Sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA was demonstrated by a boxplot of unmodified form a (with increased median and larger data distribution compared to Neg control) (fig. 10E). The siRNA studied had forms A, E, F, G, H, J, K, M and O, and the target genes (whose inhibition is expected not to induce apoptosis) included BUB1B, CCND1, CDK2 and CDC 2. The Neg control treated population was used to determine the baseline for apoptosis. Sirnas with forms G, H, J and K showed statistically significant ability to eliminate off-target effects as evidenced by lower apoptotic signals compared to unmodified form a (fig. 10E).

Forms G, J and K appear to be "detrimental" to functional sirnas according to the data of figure 10D, where sirnas with modified forms J and K were shown to impair the on-target efficacy of the sirnas, and according to the data of figure 8, where sirnas with modified forms G, J and K were shown to impair the inhibitory activity.

By using residues containing 2' O-methylation rather thanModified nucleotides of the residues were further characterized in modified form to determine whether other types of modifications could reduce off-target effects of the siRNA. Figure 11A provides box plots of normalized mitotic cells due to silencing by sirnas with 2' -O-methyl modified forms H and K and targeting WEE1, PLK1, FDFT1, LDLR, and SC5DL, which genes have widely different effects on the mitotic phenotype. That is, the set of genes includes a subset that increases mitosis when silenced, and a subset that is not expected to have an effect on mitosis when silenced. Due to these differencesDifferent effects, analysis of the subset of genes whose inhibition provided the expected mitotically increased phenotype, data are provided in figure 11B.

Figure 11B provides the results of mitotic measurements in U2OS cells after transfection of sirnas targeting WEE1 or PLK1 (by sirnas with 2' -O-methyl modified forms H and K). This silencing of WEE1 or PLK1 resulted in increased mitosis in U2OS cells, as suggested by Watanabe, N.et al ((2004) PNAS, 101, 4419-4424), Leach, S.D. et al ((1998) Cancer Research, 58, 3132-3136) and Cogswell, J.P. et al ((2000) Cell Growth and Diff, 11, 615-623), and by data for siRNA with unmodified form A that induced an approximately 4-fold increase in mitosis compared to Neg control-treated samples. Thus, these data measure the on-target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. As shown by the data in fig. 11B, similar levels of mitotic effect were observed with sirnas with 2' OMe modified forms H and K. This study demonstrated that the 2' OMe modification in forms H and K was not detrimental to functional siRNA.

To determine whether sirnas with 2' OMe modified forms H and K could reduce off-target effects, sirnas targeting gene subgroups (FDFT1, SC5DL, and LDLR) whose inhibition was expected to have no effect on mitosis were transfected into U2OS cells and the effect on mitosis was then determined. However, siRNAs that have been empirically demonstrated to have mitotic off-target effects are specifically selected for study to determine whether such siRNAs, when modified to formalize, abrogate or reduce off-target effects. Fig. 11C provides data. Off-target effect of siRNA can be demonstrated by boxplot of unmodified form a (which has a larger data distribution compared to Neg control) (fig. 11C). When comparing siRNA sequences alone, sirnas with 2' OMe residues in form H reduced many off-target phenotypes observed in unmodified siRNA form a. Sirnas with 2' OMe residues in form K increased median mitotic effects compared to unmodified sirnas. However, the data for this 2' -O-methyl modification in forms H and K are not statistically significantly different from the data for the unmodified form a.

Fig. 11D provides results quantifying apoptotic fragments due to treatment of U2OS cells with sirnas having 2' -O-methyl modified forms H and K. The Neg control siRNA treated samples were used to determine the baseline for apoptosis after siRNA transfection and the data was normalized to 1.0. The sirnas in this collection target genes WEE1, PLK1, FDFT1, LDLR, and SC5DL, inhibition of a subset of which is expected to increase apoptosis, and inhibition of a subset of which is expected to have no effect on apoptosis. As a result of these widely differing effects, the subset of genes whose inhibition provided the expected increased apoptosis phenotype was analyzed, and the data is provided in fig. 11E.

Fig. 11E provides results of measurements of apoptosis in U2OS cells after transfection of sirnas targeting WEE1 and PLK1 (by groups of 12 sirnas with 2' -O-methyl modified forms H and K). These data therefore measure the on-target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. The Neg control siRNA treated samples were used to determine a normalized baseline of apoptosis. Unmodified sirnas targeting form a of WEE1 or PLK1 exhibited a median increase in apoptosis greater than about 4-fold compared to NEG-treated samples, providing the expected phenotypic change. Similar levels of apoptosis were observed for 2' OMe modified sirnas in form H and form K. This study showed that the 2' OMe modification in H and K forms did not reduce the efficacy of the siRNA and provided a phenotype similar to that observed in unmodified siRNA form a.

The data of fig. 11F provides an analysis of the ability of 2' -O-methyl modified formatted sirnas to eliminate off-target phenotypes of a panel of genes (FDFT1, LDLR, and SC5DL) whose inhibition was expected to have no effect on apoptosis. Sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. The siRNA studied had forms A, H and K. The Neg control treated population, which had an exceptionally wide range of apoptotic outcomes, was used to determine the baseline for apoptosis. The median value for control form a was similar to that of the Neg control treated samples. When evaluating individual siRNA sequences, sirnas with 2' OMe modified nucleotides in modified forms H and K reduced many off-target phenotypes observed in unmodified siRNA form a, but in the test population, no reduction in off-target effects was observed in a statistically significant manner.

Additional modified forms were detected (as shown in the schematic diagrams of FIGS. 1A-1C providing forms H, H-2, H-1, H +1, Q, V, V-1, V-2, and K).The modified nucleotide has the structure (a), wherein R1 and R2 are O, and R3 is CH₂R4 and R5 are determined as the position of the nucleotides in the sirnas described herein. An on-target phenotype assay for mitosis (i.e., "harmless" assay) was performed in U2OS cells for the siRNA modified form of fig. 12C. As shown by the data in fig. 12C, unmodified siRNA showed an approximately 2-fold increase in mitosis in siRNA treated cells compared to Neg control treated cells. In these formsThe on-target properties of the modified, formatted siRNA did not negatively impact the expected phenotype. Modified form H provided a statistically significant increase in phenotype.

FIG. 12D provides a block diagram with respect to eliminating FIG. 12CData on off-target effect of modified forms of siRNA. Due to having an unmodified form A and havingModified forms H, H +1, H-2, K, Q, V, V-1 and V-2 siRNA silencing normalized apoptosis resulting from a panel of gene targets (inhibition of which would be expected to have no effect on apoptosis) (FDFT1, LDLR or SC5DL)Box plot of death segments. Negative control (Neg) was a scrambled non-targeting siRNA for which quantification of apoptosis was normalized to a value of 1.0. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. The off-target effect of siRNA was demonstrated by the boxplot of unmodified form a (with increased median and larger data distribution compared to Neg control). The most effective modified forms in eliminating the undesirable apoptotic phenotype are forms H, K, Q and V-2, while the modified forms H-2, H-1, H +1, V and V-1 do not alter the measurement of apoptotic fragments by any statistical significance when compared to the unmodified siRNA.

For modified forms H, H-1, K, and V, siRNA modified by 2' OMe nucleotides also did not negatively affect the central target phenotype in U2OS cell-based assays, as shown by the data in fig. 13C. Figure 13C provides a boxplot of normalized mitotic cells resulting from siRNA with unmodified form a and the modified form with nucleotides having 2' -O-methyl modifications silencing a panel of gene targets (WEE1 and PLK1) whose inhibition is expected to increase mitosis. The assay is a "harmless" assay. Unmodified siRNA showed approximately 2-fold median mitosis compared to Neg control treated samples, as expected. Data for modified formatted sirnas show that sirnas with 2' OMe modified nucleotides do not have a negative impact on the expected phenotype in cell-based assays.

Fig. 13D provides a boxplot of normalized apoptotic fragments due to sirnas with unmodified form a and sirnas with modified forms H, H-1, V, and K with 2' -O-methyl modified nucleotides silencing a panel of gene targets (FDFT1, SC5DL, or LDLR) whose inhibition was expected to have no effect on apoptosis. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (which has a larger data distribution compared to Neg control). For each format, the set of siRNAs comprises 13 different siRNAs. None of the sirnas with the 2 'OMe modified form provided statistically significant difference data from unmodified control form a, demonstrating that the 2' OMe modified formatted siRNA maintained the same off-target effect as observed for the unmodified form a siRNA.

Additional modifications similar to those of form H and shown in figure 1D were investigated. FIG. 15A is provided for a compound having the formulae A, H, M, W, W +1, W-1, Y, Y +1 and Y-1Boxplots of normalized mitotic cells of siRNA modified nucleotides. The group of sirnas was selected to inhibit BUB1B, CCND1 and CDC2 mrnas to produce the expected reduction in the U2OS cell mitogenic index. These data therefore measure the target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. As provided by the data in fig. 15A, the median mitosis for the samples treated with unmodified form a siRNA showed a decrease in mitosis to approximately 0.75 compared to the samples treated with Neg control siRNA. Modified forms of siRNA present in forms H, M, W +1, W-1, Y-1 and Y +1 did not significantly alter the effect of such siRNAs on mitosis, thereby demonstrating that such modified forms are "harmless". However, modified forms Y and W reduce the intended on-target effect of sirnas, demonstrating that such modified forms interfere with on-target function.

Fig. 15B provides a box plot for CASP3 as in fig. 15A, inhibition of CASP3 is not expected to affect mitosis. However, siRNAs that have been empirically demonstrated to have mitotic off-target effects are specifically selected for study to determine whether such siRNAs, when modified to formalize, abrogate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (which has a median value of about 0.7 compared to Neg control). siRNAs with modified forms H, W, W +1, W-1, Y and Y +1 reversed the off-target effect exhibited by the siRNA of form A. Modification of siRNAs with forms M and Y-1 did not reverse the off-target phenotype of form A siRNA. For the data of FIG. 15B, none of the Wilcoxon values had p < 0.05, which is likely due to the smaller population of siRNAs in each format.

FIG. 15C provides a boxplot of normalized apoptotic fragments for a panel of siRNAs (forms A, H, M, W, W +1, W-1, Y, Y +1, and Y-1) targeting WEE1 gene, whose inhibition is expected to increase apoptosis. Unmodified form a siRNA, compared to Neg control siRNA treated samples, showed a median apoptosis of greater than 4.0, demonstrating a strong increase in apoptosis due to WEE1 inhibition. The data was therefore examined for target phenotypic effects in measurements, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional unmodified sirnas. As provided by the data in fig. 15C, sirnas with modified forms H, M and W-1 showed similar increases in apoptosis compared to form a, whereas forms W, W +1, Y, Y +1, and Y-1 showed less effect on apoptosis compared to unmodified siRNA form a. For the data of FIG. 15C, none of the Wilcoxon values had p < 0.05, which is likely due to the smaller population of siRNAs in each format. Although the effect on apoptosis was reduced, chemically modified forms W, W +1, Y, Y +1, and Y-1 retained the ability to inhibit target mRNA (fig. 14A) and induce apoptosis (compared to the Neg control siRNA treated samples).

FIG. 15D provides a boxplot of normalized apoptotic fragments for siRNA with modified forms A, H, M, W, W +1, W-1, Y, Y +1, and Y-1 to silence a panel of gene targets whose inhibition is not expected to affect apoptosis. Sirnas in this group target BUB1B, CCND1, CDC2, or CDK 2. However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. Off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (with a median value greater than 3.5 and a larger data distribution compared to Neg control). Silencing by siRNA with forms H, Y and Y +1 showed a statistically significant reversal of off-target effects compared to form a.

Additional siRNA modification formats shown in figure 1E were investigated. siRNA was transfected into U2OS cells and mitotic effects were measured as described herein. FIG. 16A is provided for having an unmodified form A and havingBoxplots of normalized mitotic cells modifying the sirnas for forms H, M, JB1, JB2, JB3, JB4 and JB 5. The group of sirnas (3 sirnas/target mRNA) was selected to inhibit BUB1B, CCND1, and CDC2 mrnas to produce the expected reduction in the U2OS cell mitotic index. Thus, these data measure the on-target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. As provided by the data in fig. 16A, the median mitosis for the samples treated with unmodified form a siRNA showed a decrease in mitosis to approximately 0.75 compared to the samples treated with Neg control siRNA. Modified versions of sirnas presented as forms H, M, JB1, JB2, JB4 and JB5 did not significantly alter the on-target effect of sirnas on mitosis, demonstrating that such versions can be tolerated by sirnas. However, sirnas with the modified form JB3 significantly reduced the on-target impact of the predicted sirnas targeting these genes, demonstrating that form JB3 interferes with on-target function.

Fig. 16B provides a box plot of the modified form (for the gene target CASP3, whose inhibition is expected not to affect mitosis) as in fig. 16A. However, siRNAs that have been empirically demonstrated to have mitotic off-target effects are specifically selected for study to determine whether such siRNAs, when modified to formalize, abrogate or reduce off-target effects. The off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (which has a median value of approximately 0.65 compared to Neg control, showing a reduction in mitosis). The modified forms H, JB1, JB2, JB4 and JB5 were able to reverse such off-target effects, as demonstrated by the data in fig. 16B. Modified forms M and JB3 did not reduce the off-target phenotype of sirnas targeting CASP3, demonstrating that such forms are ineffective in eliminating the off-target phenotype. For the data of FIG. 16B, none of the Wilcoxon values had p < 0.05, which is likely due to the smaller population of siRNAs in each format.

Fig. 16C provides a boxplot of normalized apoptotic fragments resulting from siRNA silencing gene target WEE1 (which inhibits expected increase apoptosis) with modified versions A, H, M, JB1, JB2, JB3, JB4, and JB 5. These data therefore measure the on-target phenotypic effect, i.e., the assay ensures that the modified formatted siRNA is "harmless" to the group of functional sirnas. As provided by the data in fig. 16C, the median apoptosis of samples treated with unmodified form a siRNA showed an increase in apoptosis greater than 4.0 compared to the Neg control siRNA treated samples, thereby showing a strong increase in apoptosis due to WEE1 inhibition. Sirnas with modified forms induced the expected phenotype to varying degrees, demonstrating that all tested forms were harmless to groups of functional sirnas. For the data of FIG. 15B, none of the Wilcoxon values had p < 0.05, which is likely due to the smaller population of siRNAs in each format.

Fig. 16D provides a box-line plot as in fig. 16C for the modified versions of sirnas with LNA-modified nucleotides in versions A, H, M, JB1, JB2, JB3, JB4, and JB5 (for the set of gene targets including BUB1B, CCND1, CDC2, and CDK2, inhibition of which is expected to have no effect on apoptosis). However, sirnas that have empirically demonstrated apoptotic off-target effects are specifically selected for study to determine whether such sirnas, when modified to formalize, eliminate or reduce off-target effects. The off-target effect of siRNA can be demonstrated by a boxplot of unmodified form a (with an increased median value of greater than 3 and a larger data distribution compared to Neg control treated samples). As shown by the data in fig. 16D, sirnas with modified forms H, JB1, JB2, JB3, JB4, and JB5 were able to reverse the apoptotic off-target effect in a statistically significant manner when compared to the off-target effect of form a. Sirnas with form M were unable to reverse the apoptotic effect to a statistically significant degree.

Taken together, the data of fig. 10E, 11C, 11F, 12D, 13D, 15B, 15D, 16B, and 16D show that modified forms that provide a reduction in the unexpected off-target phenotypic effect include forms G, H, J, K, Q, V-2, Y, Y +1, JB1, JB2, JB3, JB4, and JB 5. Among these modified forms, forms G, J and K are "detrimental" to the ability of functional siRNA to inhibit activity. Highly functional sirnas have the property of reducing off-target phenotypic effects and are "harmless" in terms of inhibitory activity. Modified forms that provide such functionality to functional sirnas include forms H, Q, V-2, Y, Y +1, JB1, JB2, JB3, JB4, and JB 5.

These modified forms were studied for strand effects (fig. 4E, 6C, 7B, 12A, 13A, data of example 3 herein) and for their ability to reduce off-target effects (as provided above) to assess whether it could be predicted from the strand assay what role modified formatted sirnas would play in the elimination of off-target effects. The chain data of example 3 obtained using the classical reporter assay showed that modified forms E, G, H, K, O and M could be expected to perform well in cell phenotyping. In fact, 3 of these do perform well, but 3 of them do not. Thus, the strand-type assay appears to be ineffective in predicting whether a modified, formatted siRNA is functional in obtaining the desired cell phenotype.

Example 7

Stable modified formatted siRNA

This example provides modified formatted sirnas that, in addition to maintaining inhibitory activity and eliminating off-target effects properties described above, are particularly stable to exposure to nuclease-containing biological fluids. Elmen et al (Nucleic Acids Research 33: 1, 439. cndot. 447, 2005) reported a stable siRNA termed siLNA5 with the same modified form as form F herein and used as a reference control for stability studies in this example. The term "stable to exposure to a biological fluid," as used herein, refers to sirnas that retain their full length in the presence of a nuclease-containing biological fluid to the same or greater extent than sirnas having modified form F that were exposed to the same biological fluid and were subjected to the same time.

Although this example provides a stable modification form, the modified formatted siRNA provided in the previous example is suitable for use in an environment where nuclease activity is hardly considered.

With respect to form F, the data from example 6 and fig. 10E show that sirnas with modified form F are essentially ineffective in eliminating off-target cell effects.

8 different siRNAs targeting CLTC and WEE 1mRNA were studied to determine the modified form that confers siRNA stability in 90% of mouse sera. Synthesis of siRNA was performed as described in example 1 for the modified forms described below and shown in fig. 1F to 1K.

Form(s) of Site of modification (numbering from the 5' end of each strand)

Passenger sequence guide sequence

A, control none

F (E lmen et al) 1, 20, 2120, 21

H1, 2, 13, 14 is

DH21 1，2，13，14 20，21

DH20 1，2，13，14 19，20，21

DH31, 2, 13, 14, 20, 21 none

DH 301, 2, 13, 14, 19, 20 none

DH35 1，2，13，14，20 21

DH6 1，13，14，21 21

DH34 1，2，13，14，20，21 21

DH2 1，2，13，14，20，21 20，21

DH19 1，2，13，14，19，20 19，20，21

DH4 1，2，13，14，19，20 19，20

DH31 1，2，13，14，19，20 20，21

DH27 1，2，7，13，14，19，20 20，21

DH25 1，2，6，9，13，14，19，20 20，21

DH47 1，2，13，14，19，20，21 20，21

DH29 1，2，13，14，18，20，21 20，21

DH 281, 2, 13, 14, 18, 20, 21 none

DH18 1，2，13，14，18，20，21 19，20，21

DH36 1，2，20，21 20，21

DH9 1，2，13，14，20 20，21

DH46 1，2，13，14，21 20，21

DH33 1，2，13，14，20，21 20

DH10 1，2，13，14，20 20

DH7 1，2，20，21 2，20，21

DH23 1，2，13，14 2，20，21

DH1 1，2，13，14，20，21 2，20，21

DH48 1，2，13，14，20，21 2，20

DH49 1，2，13，14，20，21 2，21

DH44 1，2，13，14，20 2，20，21

DH45 1，2，13，14，21 2，20，21

DH38 1，2，13，14，20，21 1，20，21

DH39 1，2，13，14，20，21 3，20，21

DH40 1，2，13，14，20，21 4，20，21

DH41 1，2，13，14，20，21 5，20，21

DH42 1，2，13，14，20，21 6，20，21

DH43 1，2，13，14，20，21 7，20，21

The modified nucleotide into which the modified formatted siRNA is introduced is locked nucleic acid: (Residue), particularly those having structure (a) above, wherein R1 is O, R2 is O, and R3 is CH₂。

As used herein, the stability assay provides the amount of gold outgrowth present after incubation of the modified formatted siRNA in 90% of mouse serum (cat #44135, JR Scientific, inc., Woodland, CA) that has not been heat inactivated. All sirnas were treated with the same batch of mouse serum that had previously been verified to have strong nuclease activity. Similar results were obtained using human serum (cat # CC-5500, SeraCare Life Sciences, I nc., Oceanside, Calif.) and fetal bovine serum (cat # SV 30014, HyClone, Logan, Utah) (data not shown).

For each assay, 10 μ M of the modified formatted siRNA was incubated at 50 μ L final volume at 37 ℃ in 90% mouse serum for the time indicated in the time curve (FIG. 17) or for the time of 5 hours (FIG. 18A-23B). In parallel to incubation in serum, siRNA from each group was treated with PBS (cat #9625, Applied Biosystems, Austin, TX) for the same time as serum treated samples to determine a baseline for the amount of full length product used for HPLC studies. After incubation, the siRNA was extracted with phenol (cat #9700, Applied Biosystems, Austin, TX) and then precipitated with ethanol in the presence of 5. mu.g glycogen carrier (1. mu.l/tube, product No. AM9510, Applied Biosystems, Austin, TX) to increase recovery of small RNA. The siRNA and cleaved products were recovered by centrifugation (15 min at 15,000 xg) and then dissolved in PBS buffer (phosphate buffered saline) prior to HPLC analysis.

Using ion exchange High Performance Liquid Chromatography (HPLC) with continuous gradientsSodium perchlorate (NaClO) containing acetonitrile₄) The amount of full length product was determined. The stationary phase is with Waters 2795Analytical HPLC System and EMPOWER^TMDisolution Software v2(Waters Corp, Milford, Mass.) with Dionex200 columns (4 mm. times.250 mm, Dionex Corporation, Sunnyvale, Calif.). The temperature is maintained so that the siRNA duplexes do not denature. Quantification and UV detection were performed at 254nm using a Waters 2998 photodiode array detector (Waters Corp, Milford, Mass.). 10 μ l to 20 μ l injection volumes of siRNA targeting GAPdH mRNA (21-mer duplex with 2 3' overhang nucleotides on each end) (10 μ M) were used to determine column mass and retention time. Analysis of HPLC results was based on the total area of peaks (area count) for untreated siRNA (taken as 100%) and the total area of peaks (providing calculated remaining percentage) for the same retention time (+/-0.1-0.2 min) for serum treated samples.

The same siRNA used for serum stability assays were tested separately for inhibitory activity of the endogenous gene to determine if the modified form altered the efficacy of the siRNA to inhibit its mRNA target. Transfection was performed using Lipofectamine as described in example 4 (except in the "Forward" protocol described by the manufacturer)^TM2000(Invitrogen, Carlsbad, CA), modified versions of siRNA and unmodified control siRNA were transfected into Hela cells at a final concentration of 5 nM. After harvesting the transfected cells, according to the manufacturer's protocol, useGene Expression Cells-to-C_T ^TMKit (Applied Biosystems, inc. cat. # AM1728) total cell lysates were isolated. Preparation of cDNA, gene expression assay and calculation of inhibitory activity were performed as described in example 4.

The data in fig. 17 show that the unmodified forms of the 8 different sirnas of this study degraded in serum, with more than 50% of the full length molecules degraded within 5 to 10 minutes of exposure to serum.

For unmodified form a, stability control form F, and modified forms H, DH21, DH20, DH3, DH30, DH35, DH6, DH34, and DH2, fig. 18A provides a boxplot of the percentage of full-length siRNA that is retained when treated with 90% serum for 5 hours under the described conditions. Under the conditions described, unmodified form a and modified form H were completely degraded in serum. Under the conditions described, stability has a median of approximately 43% of full-length siRNA in serum with reference to control form F. Analysis of the data of figure 18A shows that modification of nucleotides on and/or near only one 3' -end is insufficient to protect modified sirnas in serum (forms DH21, DH20, DH3, and DH30, compared to form F). Modification of two or three overhang nucleotides is also insufficient to protect modified siRNA in serum (forms DH6, DH34, and DH35, compared to form F). The form DH2, in which all 4 overhang nucleotides are modified, provides protection in serum equal to or better than form F. Fig. 18B provides a boxplot of mRNA inhibition (expressed as a percentage of Neg control) of the sirnas studied in fig. 18A. As shown by figure 18B, the unmodified form a siRNA exhibited a median inhibition of approximately 85% compared to the Neg control siRNA treated samples. siRNA modified form DH20, in particular, showed a reduction in inhibitory activity compared to that of form H. Of the modified forms studied in this group, only form DH2 was considered for further analysis due to its stability and inhibitory activity.

Providing two modified nucleotides in the penultimate and penultimate positions of the 3' terminus of the sense strand and providing modified nucleotides in two or three terminal residues of the antisense strand (DH4, DH31, DH19) appears to have an adverse effect on serum stability, as shown in fig. 19A. These data further support the placement of the modified nucleotide as an overhang nucleotide. Furthermore, as shown by the data in fig. 19B, forms DH19, DH27, and DH25 showed a significant reduction in inhibitory activity compared to form a. Form DH31 has greater than 70% inhibitory activity. Protection of internal residues (e.g., DH27 (position 7) and DH25 (positions 6 and 9)) while leaving terminal residues unprotected, produced the opposite effect in terms of inhibitory activity (when comparing the data to that of form DH 2).

The data provided in figure 20A show that the stability of modified forms DH47 and DH29 was increased, and the stability of DH18 was even further increased, compared to control form F. In addition to the modified nucleotides in the overhang site and the sites of form H, modified forms DH47 and DH29 provide modified nucleotides at one of the third to last and fourth to last positions of the sense strand sequence (i.e., for the 21-mer, one of positions 18 and 19). Form DH18 provides the same modified nucleotides as DH29, and in addition, modified nucleotides are provided at the penultimate position of the antisense strand (i.e., position 19 for the 21 mer). The data for form DH28, when compared directly to form DH29, again show that the absence of modified nucleotides at the overhang site of the antisense strand disrupts the stability provided by the protected sense strand. Data for forms DH47 and DH28 have Wilcoxon values p < 0.05 when compared to form F. The data of figure 20B show that forms DH47, DH29, and DH28 have greater than 70% inhibitory activity. Modification of the penultimate site (site 19) of the antisense strand (e.g., form DH18), while providing enhanced stability, attenuates inhibitory activity to a median of about 60%. Based on the above data, both forms DH47 and DH29 provided increased siRNA stability over control form F, form DH47 provided comparable inhibitory activity to unmodified form a, and form DH29 provided greater than 70% inhibitory activity.

None of the sirnas with the modified form depicted in fig. 21A provided increased serum stability over the siRNA with modified form F or form DH 2. The lack of increased serum stability further supports modified forms in which the overhang nucleotide is modified. The data for the inhibitory activity of the sirnas studied in fig. 21A all showed greater than 70% activity, as shown in fig. 21B.

Comparison of the serum stability of the siRNA with modified form F of fig. 22A with the serum stability of the siRNA with modified forms DH23, DH48, DH49, DH44, and DH45 further supports modified nucleotides at the positions of the overhang nucleotides. In addition to having modified nucleotides at the positions of the overhang nucleotides, modifications of the nucleotides at position 2 of the antisense strand (e.g., forms DH1 and DH7) increased the median value of serum stability (fig. 22A). The data in FIG. 22B shows that all the formatted siRNAs of FIG. 22A, except for form DH23, all obtained greater than 70% inhibitory activity. In general, forms DH1 and DH7 with modified nucleotides at position 2 of the guide strand, in addition to the positions of the overhang nucleotides, provide serum stability and inhibitory activity properties. siRNA with modified form DH7 lack modified nucleotides at positions 13 and 14 and in the vicinity thereof and thus may not provide properties that eliminate off-target effects. However, such sirnas can be used for silencing in environments where off-target effects are not expected.

Figure 23A provides data to examine the effect of one nucleotide modification (DH 38, DH1, DH39, DH40, DH41, DH42, DH43, respectively) at position 1, 2, 3,4, 5, 6 or 7 (position #1 is the 5' nucleotide) of the antisense strand in addition to the modified nucleotides on form H and the overhang position. The data show that all the detected formatted sirnas increased the median serum stability, and that the stability tended to decrease as the modified nucleotides were further away from the 5' end of the guide antisense strand.

As provided by the data in figure 23B, all of the formatted sirnas, except the siRNA with form DH38, had greater than 70% inhibitory activity. This observation highlights the incompatibility of the modified nucleotides located at the 5' end of the antisense strand. Addition of a phosphate group to the 5' end portion of the antisense strand rescued biological activity, but at the expense of reduced stability (data not shown). Overall, the data for serum stability and inhibitory activity show that at least one nucleotide modification at positions 2, 3,4, 5, 6 or 7 (reference 5' end) of the guide antisense strand enhances siRNA stability while also providing at least 70% inhibitory activity.

Combined with the data of fig. 18A-23B, modified forms that provide serum stability greater than form F while maintaining at least 70% inhibitory activity are forms DH47, DH29, DH7, DH1, DH39, DH40, DH41, DH42, and DH 43.

Form DH7 lacks modified nucleotides at positions 13 and 14 and positions near it, and thus may not provide properties that eliminate off-target effects.

Forms DH47, DH29, DH1, DH39, DH40, DH41, DH42 and DH43 have modified nucleotides in the passenger sense strand at positions 1, 2, 13, 14, 20, 21 and for DH47 and DH29 at one of positions 18 or 19; and modified nucleotides at positions 20 and 21 in the guide antisense strand and at any of positions 2, 3,4, 5, 6 or 7 for DH39 to DH 43. A double-stranded base pair in which both nucleotides at position 1 of the passenger sense strand are modified was shown to be unfavorable for inhibitory activity (DH20, DH19, DH4, DH 18).

Example 8

Stable in vivo delivery of siRNA

Unmodified form a siRNA and stable siRNA with modified forms DH1 and DH47 were delivered in vivo, and the amount of full length siRNA present in the livers of control and test animals was compared. Will allow reliable and efficient quantification of siRNA and miRNA and the specific detection of full-length molecules only based onStem-loop RT-PCR assay of (a) ((basedstem-loopRT-PCR assay) for analysis (us published patent application 2005/0266418 to Chen et al, filed 9/21/2004; chen, c. et al Nucleic acids research 33, e 179, 2005).

Mice were administered Fas Gene-targeted using hydrodynamic (high pressure) tail vein injection as described by Zhang et al (Human Gene therapy py 10, 1735-1737.1999) and Lewis et al (Nature Genetics 32, 107-108.2002)siRNA (1nmol, diluted in 2.5ml PBS (10% of mouse body weight)). 4 mice were injected for each modified formatted siRNA. The modified nucleotide is of the structure (a)Residue, wherein R1 is O, R2 is O and R3 is CH₂。

At 5 minutes post injection, mice were sacrificed and intact liver harvested and frozen in dry ice. The mirVana was used according to the manufacturer's protocol with the following modifications^TMPARIS^TMThe kit (Applied Biosystems, Foster City, CA) isolated total RNA from intact liver. Cell lysis buffer (20ml) was added and the sample was homogenized. The lysate (400. mu.l) was transferred to a new tube, and a denaturing solution (400. mu.l) was added to each tube, and the tubes were mixed well by vortexing. Phenol extraction was performed by adding 800. mu.l phenol, vortexing for 5 minutes, and centrifuging for 10 minutes. The upper phase (300. mu.l) was transferred into a new tube and mixed with 375. mu.l (1.25 volumes) of 100% ethanol. According to the kit protocol, the mixture was passed through a filter column and the final elution was performed using 100. mu.l of elution buffer. The concentration of RNA was measured and adjusted to 10 ng/. mu.l.

Assays for quantifying siRNA include reverse transcription of the guide strand of (RT) siRNA using RT primers with stem-loop design, as forMicroRNA reverse transcription Assays (Applied Biosystems, Foster City, Calif.) followed by PCR. In a 10. mu.l RT reaction, 1 ng/. mu.l of total RNA and 50nM RT primer were denatured at 85 ℃ for 5 min, then at 60 ℃ for 5 min, and then annealed at 4 ℃. After addition of the enzyme mixture (0.25mM each dNTP, final concentration; 3.33 units/. mu.l of MultiScriptbe)^TMReverse transcriptase (Applied Biosystems), 1 XT buffer, 0.25 units/. mu.l RNase inhibitor), the reaction mixture was incubated at 16 ℃ for 30 minutes, 42 ℃ for 30 minutes, 85 ℃ for 5 minutes, and thenIncubate at 4 ℃. Standard was used on an Applied Biosystems 7900HT sequence detection System (Applied Biosystems)The PCR protocol performs real-time PCR. 10 μ l PCR reaction mixture containing 1 μ l RT product, 1XUniversal PCR MasterMix、0.2μMProbe, 1.5 μ M forward primer and 0.7 μ M reverse primer. The reaction was incubated at 95 ℃ for 10 minutes and then 40 cycles were performed: at 95 ℃ for 15 seconds and at 60 ℃ for 1 minute.

Frozen whole livers from mice from separate control untreated groups were fed with 300, 200, 100 and 50pmol of siRNA (spike) prior to homogenization. Such "dosed" samples serve as controls for the study and provide a standard curve for comparing the cycle threshold (Ct) between injected and control samples. RNA was isolated from control organs as described above. The term "Ct" denotes the number of PCR cycles when a signal is first recorded as statistically significant. Thus, the lower the Ct value, the higher the concentration of nucleic acid target. In thatIn assays, the amount of PCR product is typically nearly doubled for each cycle, so if there is no inhibition of the reaction and the reaction is nearly 100% efficient for purified nucleic acids, the fluorescence signal should be doubled.

Figure 24 provides Ct values for "dosed" control, unmodified form a injected animals, and form DH1 and form DH47siRNA treated animals. Approximately 5% of unmodified form a siRNA was detected in the liver 5 minutes after hydrodynamic injection. In contrast, approximately 20% of modified formatted sirnas with forms DH1 and DH47 were detected, with a 400% increase compared to unmodified sirnas. This in vivo study shows that siRNA modified for stabilization provides significantly increased delivery of full-length siRNA after systemic administration.

The compositions, methods, and kits of the present teachings have been described broadly and generally herein. Each of the narrower species and subgeneric classifications falling within the generic disclosure also form part of the present teachings. This includes the generic description of the present teachings with a conditional or negative limitation (removing any subject matter from the genus, whether or not the removed matter is expressly recited herein).

Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are presented to better illustrate the present teachings and are not intended to limit the scope of the teachings herein. Certain aspects of the present teachings may be further understood with reference to the following claims.

Claims

1. A chemically synthesized passenger (sense) oligonucleotide having a length of 15 to 30 nucleotides and comprising: one of a sequence-independent modification (1), a sequence-independent modification (2), and a sequence-independent modification (3):

(3)5′m-N₁-m-N_x-m-m-N_q-n_r3', wherein x is 8, 9 or 10; r is 0, 1 or 2; and q is an integer representing the length of the passenger chain minus (x + r + 5);

wherein the content of the first and second substances,

each m is independently a bicyclic nucleotide or a tricyclic nucleotide;

each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide;

when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide, and

when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide.

2. A chemically synthesized short interfering RNA comprising the passenger (sense) oligonucleotide of claim 1 and a guide (antisense) oligonucleotide having a region of 15 to 30 nucleotides that is continuously complementary to the passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand also has complementarity to at least a portion of the target mRNA.

3. The chemically synthesized short interfering RNA of claim 2 wherein the passenger (sense) oligonucleotide comprises a sequence independent modification (1), p is 0, x is 10 and r is 2.

4. The chemically synthesized short interfering RNA of claim 2 wherein the passenger (sense) oligonucleotide comprises a sequence independent modification (1), p is 0, x is 9 or 11 and r is 2.

5. The chemically synthesized short interfering RNA of claim 2 wherein the passenger (sense) oligonucleotide comprises a sequence independent modification (2), p is 0, y is 9 and r is 2.

6. The chemically synthesized short interfering RNA of claim 2 wherein at least one m has the structure (a), (b), or (c):

wherein

R1 and R2 are independently O, S, CH₂Or NR, wherein R is hydrogen or C_1-3-an alkyl group;

r3 is CH₂、CH₂-O、CH₂-S、CH₂-CH₂、CH₂-CH₂-CH₂CH-CH or CH₂-NR, wherein R is hydrogen or C_1-3-an alkyl group;

r4 and R5 are independently an internucleoside linkage, a terminal group, or a protecting group;

at least one of R4 and R5 is an internucleoside linkage; and

b is a nucleobase, a nucleobase derivative or a nucleobase analogue.

7. The chemically synthesized short interfering RNA of claim 6 wherein the passenger (sense) oligonucleotide comprises a sequence independent modification (1), p is 0, x is 10, R is 2, and each m has the structure (a), wherein R1 is O, R2 is O, and R3 is CH₂。

8. The chemically synthesized short interfering RNA of claim 6 wherein the passenger (sense) oligonucleotide comprises a sequence independent modification (1), p is 1, x is 9 or 10, R is 2, and each m has the structure (a), wherein R1 is O, R2 is O and R3 is CH₂。

9. The chemically synthesized short interfering RNA of claim 6 wherein the passenger (sense) oligonucleotide comprises a sequence independent modification (3), x is 9 or 10, r is 2, and each m has the structure (a), whereinR1 is O, R2 is O and R3 is CH₂。

10. The chemically synthesized short interfering RNA of claim 2 wherein the passenger (sense) oligonucleotide and the guide (antisense) oligonucleotide are covalently linked through a nucleotide loop or linker loop to form a short hairpin oligonucleotide.

11. The chemically synthesized short interfering RNA of claim 2 wherein each 3' end of the siRNA independently has an overhang of 1, 2, or 3 nucleotides.

12. The chemically synthesized short interfering RNA of claim 2 wherein the at least one internucleoside linkage is not a phosphodiester internucleoside linkage.

13. The chemically synthesized short interfering RNA of claim 2, wherein the chemically synthesized short interfering RNA is further bound to a cell-targeting ligand.

14. The chemically synthesized short interfering RNA of claim 2, wherein phosphate, C, is used_1-12Alkyl radical, C_1-12Alkyl amine, C_1-12-alkenyl, C_1-12-alkynyl, C_1-12-cycloalkyl, C_1-12-aralkyl, aryl, acyl or silyl substituents further derivatize the 5' terminus.

15. A composition comprising the chemically synthesized short interfering RNA of claim 2 and a biologically acceptable carrier.

16. A kit comprising the chemically synthesized short interfering RNA of claim 2 and a transfection agent.

17. A method of minimizing off-target events with respect to inhibition of expression of a target gene by RNA interference, the method comprising: contacting a cell containing the target gene with the chemically synthesized short interfering RNA of claim 2 in an amount sufficient to reduce off-target events while maintaining potency.

18. The method of claim 17, wherein the gene is an endogenous gene.

19. The method of claim 17, wherein the contacting is in vitro contacting of a cell culture containing the cells or a tissue containing the cells with the chemically synthesized short interfering RNA.

20. The method of claim 17, wherein said contacting is ex vivo contacting of a tissue containing said cells, a bodily fluid containing said cells, or an organ containing said cells with said chemically synthesized short interfering RNA.

21. The method of claim 17, wherein said contacting is in vivo contacting of an organ or animal containing said cell with said chemically synthesized short interfering RNA.

22. A method of minimizing cleavage by a passenger strand in the inhibition of expression of an exogenously provided target gene by RNA interference, the method comprising: contacting a cell containing the target gene with a chemically synthesized short interfering RNA in an amount sufficient to minimize cleavage by the passenger strand while maintaining the efficacy of the guide strand, the short interfering RNA comprising:

a passenger (sense) oligonucleotide having a length of 15 to 30 nucleotides and comprising one of a sequence-independent modification (1), a sequence-independent modification (2), and a sequence-independent modification (3) as follows:

wherein

Each m is independently a 2' -modified nucleotide;

each N is independently an unmodified nucleotide;

and

a guide (antisense) oligonucleotide having 15 to 30 nucleotides, a region of continuous complementarity to a passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand also has complementarity to at least a portion of the target mRNA.

23. The method of claim 22, wherein the 2 'modification of the 2' -modified nucleotide comprises H, bromo, chloro, iodo, fluoro in the arabinose conformation (FANA), SH, NH₂CN, azide OR OR, R, SR, NHR OR N (R)₂Wherein R is alkyl, alkenyl, alkynyl, alkoxy, oxyalkyl, alkoxyalkyl or alkylamine, wherein the alkyl moiety is C1-C6.

24. A short interfering RNA comprising:

a passenger (sense) oligonucleotide having a length of 17 to 30 nucleotides and comprising: one of the sequence-independent modifications (4), (5) and (6):

and

a guide (antisense) oligonucleotide having 17 to 30 nucleotides, a region of continuous complementarity to a passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand also has complementarity to at least a portion of the target mRNA;

wherein

Each m is independently a bicyclic nucleotide, a tricyclic nucleotide, or a 2' -modified nucleotide;

each n is independently a deoxynucleotide, a modified nucleotide, or a ribonucleotide, and is an overhang nucleotide;

when m is a bicyclic nucleotide, each N is independently a nucleotide other than a bicyclic nucleotide,

when m is a tricyclic nucleotide, each N is independently a nucleotide other than a tricyclic nucleotide, and

when m is a 2 '-modified nucleotide, each N is independently a nucleotide other than the 2' -modified nucleotide.

25. The short interfering RNA of claim 24, wherein

The passenger (sense) oligonucleotide has a sequence-independent modification (4), p is 2, x is 10, r is 2, each n is an overhang nucleotide, and each n is independently a modified nucleotide,

the guide (antisense) oligonucleotide comprises 2 3' -overhang modified nucleotides, and

each modified nucleotide is independently a bicyclic nucleotide, a tricyclic nucleotide, or a 2' -modified nucleotide.

26. The short interfering RNA of claim 25 wherein at least one of the 3 'penultimate and 3' penultimate positions of the passenger oligonucleotide is a modified nucleotide.

27. The short interfering RNA of claim 25, wherein

When 17 nucleotides in length, at least one of positions 2 and 3 of the guide oligonucleotide is a modified nucleotide;

when 18 nucleotides in length, at least one of positions 2, 3 and 4 of the guide oligonucleotide is a modified nucleotide;

when 19 nucleotides in length, at least one of positions 2, 3,4 and 5 of the guide oligonucleotide is a modified nucleotide;

when 20 nucleotides in length, at least one of positions 2, 3,4, 5 and 6 of the guide oligonucleotide is a modified nucleotide; and

when the length is 21-30 nucleotides, at least one of positions 2, 3,4, 5, 6 and 7 of the guide oligonucleotide is a modified nucleotide.

28. The short interfering RNA of claim 24, 25, 26 or 27 wherein at least one m of the passenger (sense) oligonucleotide has the structure (a), (b) or (c):

wherein

r3 is CH₂、CH₂-O、CH₂-S、CH₂-CH₂、CH₂-CH₂-CH₂CH or CH₂-NR, wherein R is hydrogen or C_1-3-an alkyl group;

at least one of R4 and R5 is an internucleoside linkage; and

b is a nucleobase, a nucleobase derivative or a nucleobase analogue.

29. The short interfering RNA of claim 24, 25, 26, or 27 wherein each modified nucleotide has the structure (a):

wherein

at least one of R4 and R5 is an internucleoside linkage; and

b is a nucleobase, a nucleobase derivative or a nucleobase analogue.

30. A composition comprising the short interfering RNA of any one of claims 24 to 29 and a biologically acceptable carrier.

31. A method of reducing off-target events associated with inhibition of target gene expression by RNA interference in a subject in need thereof, the method comprising: contacting the subject with the short interfering RNA of any one of claims 25 to 30 in an amount sufficient to reduce off-target events while maintaining potency.

32. The method of claim 31, wherein the contacting is administration by intravascular injection.

33. A method, comprising:

obtaining the short interfering RNA of claim 26 or 27, wherein each modified nucleotide has the structure (a):

wherein

at least one of R4 and R5 is an internucleoside linkage; and

b is a nucleobase, a nucleobase derivative or a nucleobase analogue

And

administering the short interfering RNA in vivo to a subject in need thereof;

wherein after administration, a greater amount of the short interfering RNA is present in vivo as compared to the amount of short interfering RNA lacking the modified nucleotide.