Antisense oligonucleotides (ASOs) for efficient and precise RNA editing by endogenous adenosine deaminase acting on RNA (ADAR)
Patent Information
- Application Number
- JP2024532266
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2021-11-30
- Filing Date
- 2022-11-29
- Publication Date
- 2025-12-11
Smart Images
Figure 2023099494000001
Abstract
Description
Detailed Description of the Invention
[0001] The present invention relates to a method and chemically modified nucleic acids for site-specific editing of target RNA. The breakthroughs in molecular biology make it possible to modify the genetic information of a cell. Although DNA editing usually leads to a stable modification of the genetic information of a cell, it may be interesting to modify the genetic information of (m)RNA instead of modifying DNA. The main advantage of (m)RNA editing over DNA is, on the one hand, the dose-dependency of the editing yield, and, on the other hand, the reversibility of the process. By adjusting the concentration of the chemically modified nucleic acid in the cell in which the target RNA is edited, it is possible to make the editing yield, and thus the amount of modified protein after translation of the target RNA, dependent. Moreover, this process is reversible, since the editing of the target RNA stops when the chemically modified nucleic acid is no longer present in the relevant cell, and the edited RNA is replaced by newly transcribed unedited RNA.
[0002] The editing of RNA molecules according to the present invention is mediated by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs). ADARs are members of a family of enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (A-to-I RNA editing). During this enzyme-catalyzed reaction, adenosine is converted to inosine via a hydrated intermediate. Guanosine can form three hydrogen bonds with the complementary base cytidine, whereas inosine can only form two hydrogen bonds with cytidine. The translation machinery reads inosine as guanosine. Thus, ADARs have the effect of introducing a functional adenosine-to-guanosine mutation at the RNA level.
[0003] The requirement for the deaminase belonging to the ADAR family to act on RNA, particularly mRNA, is that a double strand is formed. Therefore, it is necessary to provide a complementary nucleic acid (hereinafter, oligonucleotide or oligoribonucleotide) capable of forming a double-stranded molecule on which ADAR can act.
[0004] The present invention discloses chemically modified nucleic acids that can cause a functional change from adenosine (A) to guanosine (G). Depending on the sequence of the RNA, such a change can have a dramatic effect. The change may correct a point mutation that adversely affects the protein encoded by the mRNA, or other amino acids may be incorporated into the translated protein by substitution. However, strong effects can be observed when stop codons (UAA, UAG, UGA) are edited or at splice sites.
[0005] The substantial advantage of the chemically modified oligonucleotide of the present invention is that such off-target editing is reversible and has less risk of devastating side effects.In addition, treatment can be stopped and reversed if necessary.Due to its better safety profile, the temporary and limited manipulation of human genetic information at RNA level may become widely applicable and may be extended to medical indications where genome editing at DNA level may be dangerous due to unpredictable and irreversible side effects.
[0006] Human tissues express several ADAR enzymes that allow the conversion of adenosine to inosine, which in turn is biochemically read as guanosine during translation. Several ADARs are known in the art. ADARs are found in Xenopus levis and in human and murine cells. All three human ADARs share a common C-terminal deaminase domain, but only ADAR1 and ADAR2 have been shown to have catalytic activity. ADARs share a common functional domain, the double-stranded RNA binding domain (dsRBD). ADAR1 contains three dsRBDs, whereas ADAR2 and ADAR3 share only two dsRBDs. Two isoforms of ADAR1 are known. The constitutively expressed short 110 kDa ADAR1 is the p110 isoform, whereas the long 150 kDa ADAR1 is the p150 isoform, which is expressed from a separate interferon-inducible promoter. Current knowledge suggests that ADAR2 primarily edits coding sites in the brain, whereas ADAR1 is the primary enzyme that edits non-coding sites.
[0007] In order to efficiently edit RNA, it is necessary that ADAR is guided to a specific target site on the mRNA transcript.Previous attempts in the prior art have utilized a specific loop-hairpin structure ADAR recruitment site derived from a natural cis-acting ADAR recruitment sequence to guide the deaminase activity of ADAR to a specific site, thereby bringing the deaminase activity of ADAR to the correct position on the mRNA molecule to be edited.Such an artificial nucleic acid for site-specific RNA editing is disclosed in WO2020 / 001793.The artificial nucleic acid disclosed in the prior art comprises a target sequence, which comprises a nucleic acid sequence that is complementary or at least partially complementary to a target sequence in the target RNA, and a recruitment site for recruiting deaminase.
[0008] The chemically modified nucleic acid according to the present invention is preferably different from the nucleic acid oligonucleotide disclosed in the prior art, in that it does not have a recruiting portion of loop-hairpin structure, particularly for recruiting deaminase.The chemically modified nucleic acid according to the present invention uses a different strategy from the constructs known from the prior art.It is well known that RNA is highly unstable due to the ubiquity of various RNA digestion enzymes, especially RNase A and RNase H.
[0009] RNA editing using the prior art constructs is achieved only by recruiting deaminase with the help of a recruiting moiety of an imperfect hairpin for endogenous ADAR, or other oligonucleotide motif (such as BoxB or MS2 motif). When chemical modifications according to the present invention are used, a separate recruiting moiety motif may no longer be necessary. However, in some embodiments, such a motif may be present to improve efficacy. In general, the recruiting moiety directs deaminase to the desired site of action, i.e., the target adenosine to be converted to inosine, functionally guanosine.
[0010] The chemically modified nucleic acid according to the present invention does not necessarily have a loop-hairpin structure recruitment site for deaminase.Instead, the chemically modified nucleic acid of the present invention forms an RNA duplex to which ADAR enzyme attaches, thereby improving editing efficiency.The latter is achieved by using a chemically modified nucleic acid with a specific optimal chemical modification pattern over its entire length.An important feature of the present invention is that the chemical modification of ASO is not limited to the central triplet, but extends to the flank adjacent to the central triplet.
[0011] The three central bases of the target RNA sequence, including adenosines flanked by one nucleotide on each side, are referred to as the central base triplet. The sequence complementary to the central base triplet in the chemically modified oligonucleotide of the present invention is important with respect to its specific chemical modification. In order to allow functional changes at the translation level of mRNA (editing), it is necessary that the oligonucleotide according to the present invention allows the editing of mRNA. On the one hand, it is essential that the oligonucleotide according to the present invention is sufficiently stabilized against degradation (e.g. caused by RNase), which can be achieved by chemical modification of the oligonucleotide, in particular modification of the sugar moiety of the oligonucleotide, in particular modification of the phosphate backbone, preferably by replacing the phosphate bond with a phosphorothioate bond.
[0012] On the other hand, chemical modification must allow RNA molecule editing.If the chemical modification of oligonucleotide is extensive, the editing efficiency will be reduced to unacceptable levels.Therefore, the modification of oligonucleotide must follow the guidelines described herein to obtain optimal editing efficiency.
[0013] EP3507366 discloses chemically modified single-stranded RNA editing oligonucleotides for target adenosine deamination by ADAR enzymes, whereby a central base triplet of three consecutive nucleotides contains a sugar and / or base modification. The flanking regions are uniformly modified in all embodiments (prior art FIG. 2 above) with blocks of 2'-O-methylation in the ribose units and with a small number of additional terminal phosphorothioate linkages.
[0014] However, it was found that the uniform and blocky 2'-O-methyl modification of nucleic acids used in the above prior art leads to a strong loss of editing activity by native ADAR enzymes at endogenous expression levels. This is in accordance with the negative effect of bulky 2'-modification on the binding of double-stranded RNA binding domains to dsRNA substrates. In general, 2'-F and mixtures of 2'-F and 2'-OMe are particularly well tolerated and have an even better stabilizing effect against nuclease digestion when placed at all pyrimidine bases on the nucleic acid. However, 2'-F, especially 2'-O-methylation of the central base triplet, had a significant negative effect on the editing yield. This is in accordance with the literature. However, it was also found that deoxyribose is well tolerated at all three positions of the central base triplet and provides substantial stabilization against nuclease digestion. In a preferred embodiment of the present invention, the three sugar units of the oligonucleotide complementary to the central triplet are deoxyribose units.
[0015] Chemically modified nucleic acids are suitable for use in site-specific editing of target mRNA. Chemically modified nucleic acids contain a sequence that is completely complementary to the target sequence in the target mRNA, except for the central nucleotide of the central base triplet that faces the target adenosine. The central nucleotide of the central base triplet is typically cytosine or a derivative thereof, but can also be a nucleobase analog, typically constructed on an N-heterocyclic compound, usually replacing the complementary thymidine or uracil, usually improving the recognition of the editing site by ADAR. The action of adenosine deaminase functionally changes the target adenosine to guanosine after transcription. Thus, to improve the recognition of the adenosine targeted by ADAR in the dsRNA formed when the administered oligonucleotide hybridizes with the target RNA, the sequence of nucleotides is always complementary to the target region of the mRNA, with one exception as mentioned above. More important is the modification of the oligonucleotide, particularly the sugar moiety and the pattern of the linkage between them.
[0016] The principle of the present invention is based on the fact that chemically modified nucleic acid must be stable for a sufficient period of time to allow mRNA editing. Usually, RNA molecules are degraded very quickly in cells. Therefore, nucleic acid must be chemically modified, but such modification must be performed so that chemically modified nucleic acid can remain present in cells for a sufficient time, and at the same time, such modification does not prevent recognition by ADAR. Modification of oligonucleotides involves the sugar moiety of nucleotides. RNA base is the unmodified part. The preferred modification that stabilizes oligonucleotides is the RNA base with deoxyribose moiety or 2'-O-methyl or 2'-F modification in ribose moiety. Another important modification is replacing phosphate bond between sugar moieties with phosphorothioate bond, and the proportion and position of phosphorothioate bond in core region play a decisive role.
[0017] There are many chemical modifications of oligonucleotides that affect their properties. Modifications of sugar residues are mainly substitutions at the 2'-position, 2'-F, 2'-OMe and 2'-NH2 are known as well as conformationally locked sugars such as LNA, cEt and / or ENA. Such modifications increase nuclease resistance and maintain compatibility with many biochemical activities and ASOs. A modification that is particularly relevant to the present invention is the phosphodiester bond in which the phosphate residue is modified to phosphorothioate, where the oxygen atom of the phosphate group is replaced by a sulfur atom. Stereochemistry can affect the properties of the oligonucleotide. Such modifications increase resistance to degradation by nucleases, but maintain compatibility with many biochemical activities and ASOs.
[0018] In this respect, not only resistance to nuclease degradation but also editing efficiency is the most important.Therefore, it is desirable to achieve a balance between sufficiently high editing efficiency and sufficient resistance to nuclease degradation.The oligonucleotide according to the present invention has a specific pattern of phosphorothioate bond that provides such advantageous properties.
[0019] In the course of the present invention, it was found that the artificial nucleic acid (oligonucleotide) has a length of 15 to 80 nucleotides, preferably 25 to 65 nucleotides, and more preferably 30 to 60 nucleotides. A nucleic acid of such a length is referred to as an oligonucleotide in the present application.
[0020] The chemically modified nucleic acid (oligonucleotide) according to the present invention has a sequence that is complementary with the corresponding sequence in target mRNA with almost 100% complementarity.In some embodiments, the complementarity of the chemically modified oligonucleotide to the corresponding sequence in target mRNA is at least 85%, preferably 95% complementarity.While perfect complementarity is optimal for hybridization process, natural ADAR substrates often contain a small number of mismatches and / or bulges, which aid in editing by allowing the structural perturbation of double-stranded substrates to improve substrate recognition by double-stranded RNA binding domains or substrate recognition within the active site of deaminase.
[0021] The chemically modified oligoribonucleotide according to the present invention comprises a core sequence of Formula I:
[0022] [ka]
[0023] In this formula I, there is a central base triplet of three nucleotides, the central nucleotide is designated as "0". The nucleotide designated as "0" and the two nucleotides directly adjacent to the nucleotide "0", with numbers -1 and +1, are designated as the central base triplet. The central nucleotide designated as "0" is directly opposite the target adenosine in the target RNA. The nucleotide of formula I is flanked at the 5' end (adjacent to nucleotide -5) and 3' end (adjacent to nucleotide +5) with additional oligonucleotide sequences, which can be either the same length or different lengths.
[0024] In the central base triplet of the chemically modified nucleic acid according to the embodiment of the present invention, there is a nucleoside with an N-heterocyclic base, a pyridine or pyrimidine derivative, more preferably a cytosine nucleoside or its derivative, which faces the target adenosine in the target (m)RNA. In a particularly preferred embodiment, this nucleoside and the 5' and 3'-specific adjacent nucleotides contain at least one modified nucleoside, more preferably two modified nucleosides, even more preferably three modified nucleosides, and have a substituent at the 2' carbon atom, which is either 2'-fluoro or 2'-O-methyl. In the most preferred embodiment, all three bases are 2'-desoxyribose moieties.
[0025] In the target (m)RNA,
[0026] [ka]
[0027] When a codon (targeted A is underlined, N=any nucleobase) is targeted, the central base triplet of the chemically modified nucleic acid according to the invention comprises any nucleotide having a 2'-deoxy-inosine or hypoxanthine nucleobase or derivative thereof, which is paired with a cytosine base 5'-adjacent to the targeted adenosine. Preferably, the 2'-deoxy-inosine is located in a central base triplet that comprises two, more preferably three, 2'-deoxynucleotides.
[0028] Since the chemically modified nucleic acids of the present invention have improved stability against degradation and exhibit optimal chemical modification patterns for binding to ADARs, it is preferred that the nucleic acids of the present invention do not necessarily have a specific loop-hairpin structure recruitment portion that attracts deaminases.
[0029] In one embodiment of the invention, the chemically modified nucleic acid is symmetric, meaning that the two nucleotide sequences flanking the central base triplet have the same length. When the oligonucleotide has, for example, 59 nucleotides, there are 28 nucleotides on either side of the central base triplet.
[0030] In another embodiment, the nucleic acid according to the present invention is not symmetrical, which means that the length of the two sequences adjacent to the central base triplet is different. The asymmetric design allows for a more flexible use of the sequence space around the target. Furthermore, it has been found that the asymmetric design can increase the editing yield of short sequences of nucleic acid, for example 45nt, compared to the symmetric design, provided that the nucleic acid is truncated at the correct end. Preferably, the flanking sequence 5' to the central base triplet is longer than the flanking sequence 3' in the asymmetric embodiment. A preferred embodiment comprises at least 4nt, more preferably at least 9nt in the 3' flanking sequence, and at least 19nt, more preferably at least 28nt, most preferably at least 33nt in the 5' flanking sequence.
[0031] The nucleic acid according to the invention, which comprises a core sequence according to formula I, is linked via phosphorothioate bonds at a rate of at least 40%, more preferably more than 50%, particularly preferably 60%. The phosphorothioate pattern in the core sequence of formula I is of utmost importance. Bonds a, d and e are always phosphorothioate bonds, and in addition, up to three bonds selected from the group consisting of bonds b, c, f, g and j may also be phosphorothioate bonds. However, it is excluded that all bonds a to j are phosphorothioate bonds. In a particularly preferred embodiment, bond f is a phosphorothioate bond.
[0032] In a preferred embodiment of the invention, the sequences flanking the core sequence of formula I comprise at least 10, more preferably at least 15, most preferably 20 or more nucleoside bonds which are largely non-discontinuous, more preferably non-discontinuous phosphorothioate bonds, starting from an end (5' or 3') of the nucleic acid. In another embodiment of the invention, said blocks of preferably continuous phosphorothioate bonds are located on both sides of the nucleic acid starting from both ends (5' and 3') of the nucleic acid.
[0033] However, in the core region of the oligonucleotide according to formula I, there must be less than 60%, particularly preferably less than 50%, preferably less than 40% phosphate bonds, so that a specific pattern is observed. Bonds h and i are always phosphate bonds. In a preferred embodiment of the present invention, not only bonds h and i are phosphate ester bonds, but also bonds b and / or c can be phosphate ester bonds.
[0034] In particularly preferred embodiments, bonds a, d and e are phosphorothioate bonds, while bonds h and i are phosphate bonds. In preferred embodiments, the core sequence of formula I preferably contains up to 6 of the 10 phosphorothioate bonds.
[0035] The chemically modified nucleic acid according to the present invention is substantially more stable against the degradation normally caused by RNase, and as a result, can exist longer in the cell in which the (m)RNA is to be edited.Without wishing to be bound by theory, the life span of the chemically modified nucleic acid is increased in the cellular environment, so that the ADAR can act on the mRNA due to the prolonged stability of the double strand, and therefore no recruitment site is required to recruit deaminase.
[0036] Biological responses are often time-dependent. A wide variety of RNA molecules exist in vertebrate cells, which are subject to constant and rapid turnover. RNA molecules are frequently degraded by various RNases. Thus, the use of RNA molecules for therapeutic purposes is often limited by their rapid degradation. Since in vivo conditions usually differ from in vitro conditions using cell culture test systems, the stability of molecules used for therapeutic purposes can be crucial for the success of the treatment.
[0037] The chemically modified nucleic acid molecules according to the invention provide a good balance between editing ability and sufficient stability within the cell, even allowing for intracellular conditions. The chemically modified oligonucleotides according to the invention are furthermore capable of gymnotic uptake and show acceptable editing efficiency.
[0038] The best results with the chemically modified nucleic acids according to the invention can be achieved when preferably several, more preferably at least two, even more preferably at least three of the following features are realized in the oligonucleotide: In the central core sequence of formula I, up to 4-6 of bonds a-j are phosphate linkages, while the remainder are phosphorothioate linkages; At least one DNA sugar nucleoside is present in the central base triplet opposite the adenosine that is to be deaminated; stabilization of pyrimidine bases outside the central base triplet by 2'-F or 2'-OMe modification of the nucleoside ribose moiety with approximately equal stoichiometry, thereby avoiding blocking of 2'-OMe, a pattern that is preferred; Both ends are stabilized by blocks of three nucleotides with double modifications, namely 2'-OMe modifications of the sugar moieties and phosphorothioate linkages.
[0039] The chemically modified nucleic acid molecules (oligonucleotides) of the present invention have the advantage that the molecules are sufficiently stable in the living body of a vertebrate animal to achieve the desired effect. The molecules according to the present invention are stable to degradation by various RNases for a sufficient period of time to achieve the desired effect.
[0040] Another advantage of the chemically modified nucleic acid of the present invention is that it can be directly delivered to the target cell without other auxiliary mechanisms such as a specific vector or a specific transfection method. The chemically modified nucleic acid according to the present invention can act via gymnosis, which means that it can be directly applied to the target cell without auxiliary means such as a vector or other carrier.
[0041] A further advantage of the chemically modified nucleic acids according to the present invention is that they have a high editing efficiency in clinically relevant targets. The modified nucleic acids can be introduced into target cells via gymnosis and can achieve a relatively high effect on the translation level in the target cells.
[0042] Another advantage of the chemically modified nucleic acids according to the present invention is that A to I editing can be achieved not only in targets that are relatively easy to edit, such as 5'UAG, but also in more difficult triplets, such as 5'CAA.
[0043] The present invention relates to a chemically modified oligoribonucleotide for use in site-specific A-to-I editing of a target RNA in a cell by endogenous ADAR, comprising a sequence of 11-100 nucleotides in length capable of binding to a target sequence in the target RNA, and having a central base triplet of three nucleotides, with the central nucleotide facing the target adenosine in the target RNA to be edited to inosine. The oligonucleotide has a core sequence having the following formula I:
[0044] [ka]
[0045] where Nu represents a nucleotide with a sugar moiety that may be modified. The numbers below the nucleotide sequence indicate the position of the nucleotide adjacent to the central nucleotide bearing the number 0, with negative numbers indicating the 5' end of the oligonucleotide and positive numbers indicating the 3' end of the oligonucleotide. Nucleotide (0) and nucleotides (-1) and (+1) form a central base triplet. The letters a to j indicate the linkages between single nucleotides in the core sequence according to formula I. In the examples and in the tables describing the oligonucleotides used, phosphorothioate linkages are represented as " * Each nucleotide Nu can have different meanings, independently of each other, with respect to the base and sugar, as well as their modifications.
[0046] The chemically modified oligonucleotides of the invention have a total length ranging from 11 to 100 nucleotides, preferably a length ranging from 20 to 80 nucleotides. In a particularly preferred embodiment, the chemically modified oligonucleotides according to the invention range from 30 to 60 nucleotides including a core sequence of formula I. The sequences flanking the core sequence having formula I may have the same length ranging from 9 nucleotides to 25 nucleotides. In an alternative embodiment, the strands flanking the core sequence may have different lengths.
[0047] In addition to the specific phosphorothioate pattern, further modifications may be used. Such modifications may be at the 2' position of the sugar moiety. The purines and / or pyrimidines may be modified or unmodified.
[0048] According to the present invention, the core sequence has mandatory phosphorothioate bonds at positions a, d and e. Furthermore, the present invention has mandatory orthophosphate bonds at positions h and i. In other words, five of the ten bonds are defined as either PS or orthophosphate. The remaining five bonds b, c, f, g and j can be selected from both PS and orthophosphate, resulting in several preferred embodiments: In a preferred embodiment, the bonds at positions f, g, and j are phosphorothioate, while the bonds at positions b and c are phosphate. The other five bonds a, d, e, and h, i are as defined above. In another preferred embodiment, the bonds at positions b, c, and f are phosphorothioate, while the bonds at positions g and j are phosphate. The other five bonds a, d, e, and h, i are as defined above. In another preferred embodiment, the bond at position f is phosphorothioate, while the bonds at positions b, c, g, and j are phosphate. The other five bonds a, d, e, and h, i are as defined above. In another preferred embodiment, the bonds at positions f and j are phosphorothioate, while the bonds at positions b, c, and g are phosphate. The other five bonds a, d, e, and h, i are as defined above. In another preferred embodiment, the bonds at positions f and j are phosphorothioate, while the bonds at positions b, c, and g are phosphate. The other five bonds a, d, e, and h, i are as defined above. In another preferred embodiment, the bonds at positions f, g are phosphorothioate, while the bonds at positions b, c, j are phosphate. The other five bonds a, d, e and h, i are as defined above. In another preferred embodiment, the bonds at positions b, c, f, g, j are phosphate bonds. The other five bonds a, d, e and h, i are as defined above.
[0049] The chemically modified oligonucleotides of the present invention can be formulated into compositions together with any suitable excipient, in particular a pharma- ceutically acceptable excipient.
[0050] The chemically modified oligonucleotides of the present invention may be for therapeutic or diagnostic use, but preferably for therapeutic use.
[0051] The chemically modified oligonucleotide of the present invention can be used to treat genetic disease or genetic disorder.In particular, genetic disease or genetic disorder can be metabolic disease, cardiovascular disease, autoimmune disease or neurological disease.In this context, the present invention encompasses the method of treating such disease or disorder by administering an effective amount of said chemically modified oligonucleotide to the subject in need thereof.
[0052] The invention and its preferred embodiments are illustrated but are not limited to those illustrated by the examples and figures.
[0053] In the drawings, a particularly preferred embodiment of the invention is shown.
[0054] Figure 1 shows the effect of phosphorothioate optimization on stability and editing efficiency when the central core is modified. Figure 1A shows the sequence of the central core and phosphorothioate modification. In addition, Figure 1A shows the stability of the composition and editing efficiency of each construct. It can be clearly seen that by increasing the number of phosphorothioate bonds, the stability can be greatly increased, but the editing efficiency is decreased (Figure 1A).
[0055] In Figure 1A, we used an oligonucleotide with phosphorothioate bonds only at positions 1 (a) and 10 (j) [v117.26]. The editing efficiency was quite high (59.2% ± 14), but the stability (t 50 (100% FBS)) took only 30 hours.
[0056] Figure 1A also shows an oligonucleotide with phosphorothioate linkages at positions 1 (a), 7 (g), 8 (h), 9 (i), and 10 (j) [v117.27]. Stability to degradation (t 50 ) improved to 40 hours, but the editing efficiency decreased to 33.0%.
[0057] Figure 1A also shows a construct with six phosphorothioate bonds at positions 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), and 10 (j) [v117.28]. The editing efficiency was improved to 50.3%, but the stability against degradation (t 50 ) only decreased by 20 hours.
[0058] Figure 1A shows a further experiment [v117.29] in which all linkages were phosphorothioate linkages. Editing efficiency only decreased to 32.0%.
[0059] In contrast, Figure 1A shows an experiment [v117.30] in which 6 of the 10 linkages were phosphorothioate (i.e., positions a, d, e, f, g, and j). The editing efficiency increased to 52.0%, and the stability (t 50 ) was >7 days.
[0060] The results shown in Figure 1A show that each linkage in the central core is a phosphorothioate linkage [v117.29] and the stability of the construct is >7 days. Unfortunately, however, the editing efficiency only decreased to 32%, indicating that a pattern of phosphorothioate linkages must be observed to achieve reasonable editing efficiency.
[0061] On the other hand, the number of phosphorothioate bonds is not the only important factor; just two phosphorothioate bonds (positions 1 and 10) significantly increased the t 50 (100% FBS) stability achieved [v117.26].
[0062] In Figures 1A and 1B, the positions of phosphorothioate bonds in the relevant samples are shown together with the editing efficiency and stability. Figure 1A shows that the best balance between high editing efficiency and high stability against degradation was obtained in the sample named v117.39. In this sample, the phosphorothioate bonds of the core structure are located at a(1), d(4), e(5), f(6), and j(10). According to the present invention, this pattern of phosphorothioate bonds is particularly preferred.
[0063] Figure 1A shows the exact location of phosphorothioate bonds in the construct targeting the SERPINA1 E342K mutation, further described in Example 1. Editing efficiency is shown from two different model systems (plasmid and piggyBac). The half-life of the construct was measured in 100% FBS (t(50)). n refers to the number of samples.
[0064] Figure 1B shows the orientations of the internucleotide bonds labeled a–j, with the central base triplet highlighted.
[0065] Figure 2 shows the editing yield results of the experiments performed in Example 1. The editing results are shown in Figure 2A and the serum half-life of the constructs of Example 1 is shown in Figure 2B.
[0066] FIG. 3 shows the editing efficiency results of the experiments performed in Example 2, and the corresponding serum half-lives in 100% FBS are shown in FIG. 3B.
[0067] Figure 4A shows the editing efficiency of constructs targeting the disease-causing W104X mutation in mouse MECP2, and Figure 4B shows the serum half-life.
[0068] Figure 5 shows the results of Example 4. The editing efficiency of the constructs is shown in Figure 5A, and the stability is shown in Figure 5B.
[0069] FIG. 6 shows the editing result of the fifth embodiment.
[0070] Figure 7 shows the results of Example 6. Figure 7A shows the editing efficiency, and Figure 7B shows the stability.
[0071] Figure 8 shows the results of Example 7, where the phosphorothioate design optimized according to Example 1 (V117.39) was transferred to an oligonucleotide targeting the T41 site in mouse CTNNB1. This figure shows that the disclosed pattern can also be applied to other targets.
[0072] Example 1: Optimization of PS-localization near the central base triplet of the human SERPINA1 gene at the site of the disease-causing E342K mutation.
[0073] Long stretches of PS (phosphorothioate) bonds improve the stability of oligonucleotides and, consequently, their bioavailability. From a therapeutic point of view, constructs with PS bonds are intended to allow lower doses or less frequent treatments to be sufficient to obtain the desired effect compared to constructs with similar phosphodiester (PO) bonds. However, simply replacing all PO bonds with PS bonds is not favorable for editing efficiency. Here, we screened different arrangements of PS bonds within the 10 phosphodiester bonds surrounding the central base triplet, a region that is particularly sensitive to PO / PS substitutions in terms of editing efficiency and stability. The example is based on the E342K mutation in the SERPINA1 gene, a substrate of great therapeutic relevance. This is the underlying cause of the severe Z-phenotype of alpha-1-antitrypsin deficiency and represents an unmet clinical challenge. A list of all oligonucleotide constructs used is shown in Table 1.
[0074] The results of the editing yield of Example 1 are shown in Figure 2A) and the serum half-life of the construct of Example 1 in Figure 2B). First, it has been shown that uniform placement of phosphorothioate (PS) bonds at all 10 positions significantly reduces the editing efficiency (Figures 1 and 2A). Second, there are several positions, especially a, d, and e, where PS bonds are very well accepted. Third, PS bonds can be added at specific positions, further improving the stability and / or editing efficiency. Overall, optimal PS patterns are available that significantly improve the serum half-life of oligonucleotides without significantly compromising the editing yield (Figures 1 and 2A). The best solution combines improved stability with improved editing yield.
[0075] 2.5×10 4HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. After 24 hours, the cells were forward transfected with a plasmid containing human SERPINA1 E342K mutant cDNA or SERPINA1 healthy cDNA ("wild type"). 300 ng of plasmid and 0.9 μL of FuGENE® 6 (Promega) were each diluted in 50 μL of Opti-MEM and incubated for 5 minutes, then combined and incubated for another 20 minutes. The medium was replaced and the transfection mix was evenly distributed into one well. 24 hours after plasmid transfection, the cells were forward transfected with 5 pmol of construct / well and 1.5 μL / well of Lipofectamine RNAiMAX reagent (ThermoFisher Scientific). After 24 hours, the medium was replaced. 48 hours after transfection, the cells were harvested for RNA isolation and sequencing.
[0076] As shown in Figure 2A, the placement of PS bonds at the observed binding positions can have a strong effect on the editing level. V117.26 (SEQ ID NO: 1) contains PS bonds only at a and j positions. There are no stabilizing PS positions near the central base triplet (CBT). As a result, the editing efficiency of the embodiment is over 50%, but the half-life in 100% FBS is only about 30 hours.
[0077] When the PS linkage is placed 3' of the CBT, i.e., v117.27 (SEQ ID NO:2) and v117.29 (SEQ ID NO:4), the editing level is reduced by about 50% compared to v117.26. Thus, placing the PS linkage at positions h and i significantly impairs editing, whereas placing the PS linkage at position g shows only minor effects on editing, as can be seen by comparing v117.39 (SEQ ID NO:9) with v117.40 (SEQ ID NO:10) and v117.30 (SEQ ID NO:5). As seen in v117.29 (SEQ ID NO:4), adding PS linkages at all positions significantly increases the half-life of the ASO in 100% FBS (>7 days), but significantly reduces the editing yield compared to v117.26. Thus, precise placement of the PS linkage is desirable to increase serum half-life without compromising editing efficiency. This includes avoiding placement of PS linkages at positions h and i.
[0078] In contrast, when the PS bond is placed 5'-adjacent or inside the CBT, the editing yield remains the same as that observed for v117.26, for example, as seen in versions v117.28 (SEQ ID NO: 3), v117.30 (SEQ ID NO: 5) and v117.40 (SEQ ID NO: 10). For some embodiments, the editing yield is further improved, as seen for v117.33 (SEQ ID NO: 6), v117.34 (SEQ ID NO: 7), v117.35 (SEQ ID NO: 8) and v117.39 (SEQ ID NO: 9). These embodiments also show that the PS bonds at positions b and c do not impair editing, but do not appear to be as strongly required for ASO stability. Thus, their role on the overall construct performance is somewhat neutral.
[0079] However, especially with regard to the serum half-life of the ASO, the PS bond at CBT (dg position) is essential, as seen for example in embodiment v117.28 (SEQ ID NO: 3) compared to v117.30 (SEQ ID NO: 5). Although both versions have the same amount of bonds (6 PS, 4 PO), the bond at CBT makes the ASO significantly more stable than the bond 5'-adjacent to CBT (>7 days vs. only 20 hours in 100% FBS). This highlights the importance of the correct positioning of the PS bond within the ASO. This can be highlighted by comparing the serum half-life and editing efficiency of ASOs with the same overall number of PS bonds but different arrangements. For example, embodiments v117.28 (SEQ ID NO: 3), v117.30 (SEQ ID NO: 5) and v117.33 (SEQ ID NO: 6) all have 6 PS bonds. However, the editing efficiency of v117.28 and v117.30 is comparable (about 50%), whereas the editing efficiency of v117.33 is higher (about 66%). Moreover, the serum half-life of v117.30 and v117.33 is significantly longer (>7 days) than that of v117.28 (about 20 hours). Overall, this makes v117.33 the most favorably positioned embodiment with six PS bonds in terms of combining high editing efficiency with high serum resistance. Similarly, the embodiments v117.27 (SEQ ID NO: 2), v117.39 (SEQ ID NO: 9) and v117.40 (SEQ ID NO: 10) can be compared for the most favorable positioning of the five PS bonds, with the latter two clearly superior to v117.27 (SEQ ID NO: 2). A summary of the exact PS-linkage configurations of different embodiments, along with the corresponding editing yields and 100% FBS half-life (t50) is shown in FIG.
[0080] As a result, it was found that the PS bonds at positions a, d, and e are the most essential to increase the half-life of the construct in 100% FBS without compromising the editing yield. However, these properties of the construct can be further improved by introducing PS bonds at positions b, c, f, and j. Introducing a PS bond at position g can also increase the serum half-life of the construct, but is likely to slightly affect the editing efficiency. However, PS bonds should not be placed at positions h and i. These have a clear negative effect on the editing efficiency of the construct. The position of the PS bond from embodiment v117.39 (SEQ ID NO: 9) was selected as the most favorable balance between high editing yield and long half-life in 100% FBS and was further tested in other targets (see further examples below). The corresponding positions are a, d, e, f, and j.
[0081] [Table 1]
[0082] Example 2: Incorporation of an optimized PS-binding pattern into an oligonucleotide targeting endogenous human STAT1 Y701.
[0083] The results of the editing efficiency of Example 2 are shown in Figure 3A, and the serum half-life of the constructs are shown in Figure 3B. The optimal PS-binding pattern surrounding the CBT (found for the SERPINA1 E342K target in embodiment v117.39 (SEQ ID NO: 9)) was transferred to an embodiment targeting the endogenous human STAT1 transcript, in which RNA editing induces an amino acid change Y701C, which removes the functionally important phosphotyrosine of the STAT1 protein. Here, it is shown that the optimized PS-binding pattern from Example 1 can be successfully introduced into different targets. A list of oligonucleotide constructs is shown in Table 2.
[0084] 10 5HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. After 24 hours, they were forward transfected by diluting 25 pmol of construct / well and 1.5 μL / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) in 50 μL of Opti-MEM (ThermoFisher Scientific) respectively and incubated at room temperature for 5 min. After incubation, both solutions were combined to a total of 100 μL / well and further incubated at room temperature for 20 min. After incubation, the transfection mix was slowly dispensed into one well. 24 hours after transfection, cells were harvested for RNA isolation and sequencing.
[0085] As shown in Figure 3B and A, v117.29 (SEQ ID NO: 13), which contains an optimized PS-binding pattern from v117.39 (SEQ ID NO: 9), outperforms the corresponding construct, v117.28 (SEQ ID NO: 12, disclosed in patent EP21177135.7), which lacks PS-binding around CBT, both in terms of stability in 100% FBS (6 days vs. 30 hours) and in terms of editing yield (50.5% vs. 40.5%), respectively. A comparison was also made with v117.19 (SEQ ID NO: 11). v117.19 is minimally modified and shows a higher editing yield compared to v117.28, a more heavily modified construct from the prior art (EP21177135.7). With the optimized PS pattern, v117.29 reaches an editing yield comparable to the much less modified v117.19. Moreover, v117.29 has a 5-fold longer half-life in 100% FBS (6 days) than v117.28 (30 hours). Clearly, the optimized PS-binding sequence showed the same improvement in editing yield and serum half-life in 100% FBS as already seen in the SERPINA1 target (Example 1). Therefore, it can be concluded that the optimized PS-binding pattern can be introduced to other related targets.
[0086] [Table 2]
[0087] Example 3: Incorporation of an optimized PS-binding pattern into oligonucleotides targeting the disease-causing W104X mutation in mouse MECP2.
[0088] The results of Example 3 are shown in Figure 4. The editing efficiency of the constructs is shown in Figure 4A, and the corresponding serum half-life in 100% FBS is shown in Figure 4B. The transfer of the PS pattern of v117.39 (SEQ ID NO: 9) from the SERPINA1 E342K target into endogenous human STAT1 Y701 was proven successful. Therefore, to further test the transferability of the optimized PS design, a construct targeting the W104X mutation in mouse MECP2 (the underlying cause of severe Rett syndrome) was tested. It was shown that the optimized PS-binding patterns of Examples 1 and 2 can be successfully transferred into another target in a clinically relevant sequence context. A list of the oligonucleotide constructs used showing the complete modification patterns is shown in Table 3.
[0089] 5×10 4 HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. After 24 hours, the cells were forward transfected with a plasmid containing mouse MECP2 W104X mutant cDNA. 300 ng of plasmid and 0.9 μL of FuGENE® 6 (Promega) were each diluted in 50 μL of Opti-MEM and incubated for 5 minutes, then combined and incubated for another 20 minutes. The medium was replaced and the transfection mix was evenly distributed into one well. 24 hours after plasmid transfection, the cells were forward transfected with 25 pmol of construct / well and 1.5 μL / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific). After 24 hours, the medium was replaced. 48 hours after transfection, the cells were harvested for RNA isolation and sequencing.
[0090] Figures 4A and 4B show a comparison of the editing yield and half-life in 100% FBS of the three different constructs tested, respectively. V120.17 (SEQ ID NO: 14), a construct with PS-linkages only at positions a and j, performed well in editing (about 50%) but was unstable in 100% FBS (half-life about 48 hours). Construct v120.24 (SEQ ID NO: 16), on the other hand, had PS-linkages at all positions and a significantly improved serum half-life (about 7 days), but also a significantly reduced editing yield, almost half of the yield observed with v120.17 (about 30%). However, when applying the optimized PS-linkage pattern from v117.39 (SEQ ID NO: 9), construct v120.23 (SEQ ID NO: 15) reached the same editing efficiency as v120.17 (about 50%), while at the same time improving its half-life in 100% FBS by 1.5 times (about 72 hours). This highlights the power of precise positioning of the PS-linkages, even across constructs of different design and length (compare constructs in Examples 1 and 2 with those in Example 3), and further highlights the transferability of the patterns to other targets and oligonucleotide sequence designs.
[0091] [Table 3]
[0092] Example 4: Incorporation of an optimized PS-binding pattern into an oligonucleotide targeting the endogenous human L157 GAPDH site.
[0093] The results of Example 4 are shown in Figure 5. The editing efficiency of the constructs is shown in Figure 5A, and the corresponding serum half-life in 100% FBS is shown in Figure 5B). The optimized PS design was transferred to a construct targeting the endogenous GAPDH transcript at the L157 site. The optimized PS pattern improved the half-life stability in 100% FBS by about 2-fold. A list of the constructs showing the position of the PS used is shown in Table 4.
[0094] 10 5HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. After 24 hours, 25 pmol of construct / well and 1.5 μL / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) were each diluted in 50 μL of Opti-MEM (ThermoFisher Scientific) and incubated at room temperature for 5 minutes. After incubation, both solutions were combined to a total of 100 μL / well and further incubated at room temperature for 20 minutes. After incubation, the transfection mix was slowly dispensed into one well. 24 hours after transfection, cells were harvested for RNA isolation and sequencing.
[0095] Figures 5A and 5B show the editing yield and half-life of the constructs in 100% FBS, respectively. Compared to construct v120.21 (SEQ ID NO: 17) with no optimization of PS binding in the CBT region, construct v120.22 (SEQ ID NO: 18) with optimized PS binding pattern shows a two-fold increase in half-life in 100% FBS. Although the editing yield is reduced compared to v120.21, the editing yield of v120.22 is two-fold higher than construct v120.23 (SEQ ID NO: 19) with all PS binding. Due to the already long half-life of v120.21 in 100% FBS (72 hours) and the low initial editing yield (about 25%), the effect of PS optimization is not as pronounced as the targets shown in Examples 1, 2, and 3. Nevertheless, a strong enough effect is obtained to highlight the introductory and flexibility of the present invention.
[0096] [Table 4]
[0097] Example 5: Introduction of an optimized PS-binding design into an ASO targeting the disease-causing G2019S mutation in human LRRK2.
[0098] The results of Example 5 are shown in Figure 6. The optimized PS designs of Example 1 (v117.34 and 117.39 in SERPINA1) were transferred to oligonucleotides targeting transiently overexpressed LRRK2 transcripts carrying the G2019S mutation that causes Parkinson's disease. A list of constructs showing the location of the PS used is shown in Table 5.
[0099] 5×10 4 HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. After 24 hours, cells were forward transfected with a plasmid containing human LRRK2 G2019S mutant cDNA. 300ng of plasmid and 0.9μL of FuGENE®6 (Promega) were each diluted in 50μL of Opti-MEM and incubated for 5 minutes, then combined and incubated for another 20 minutes. The medium was replaced and the transfection mix was evenly distributed into one well. 24 hours after plasmid transfection, cells were forward transfected with 25pmol of construct / well and 1.5μL / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific). 24 hours after transfection, cells were harvested for RNA isolation and sequencing.
[0100] The editing yields are shown in Figure 6A. Both PS patterns v117.34 (SEQ ID NO: 21) and 117.39 (SEQ ID NO: 22) showed editing yields comparable to or higher than the previous oligonucleotide V117.19 (SEQ ID NO: 20), which lacks a PS in the central region.
[0101] [Table 5]
[0102] Example 6: Introduction of an optimized PS-binding design into an ASO targeting the disease-causing C948Y mutation in human CRB1.
[0103] The results of Example 6 are shown in Figure 7. The optimized PS design of Example 1 (V117.39 targeting SERPINA1) was transferred to an oligonucleotide targeting the C948Y mutation site in human CRB1. Mutations in the CRB1 gene are associated with various early-onset retinal dystrophies, including retinitis pigmentosa and Leber's congenital amaurosis. Furthermore, oligonucleotides targeted to the retina would offer significant benefits due to improved stability, fewer administrations, and the possibility of less invasive injections into the patient's eye. A list of the corresponding constructs used is shown in Table 6.
[0104] 5×10 4 HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. After 24 hours, cells were forward transfected with a plasmid containing human CRB1 C948Y mutant cDNA. 300ng of plasmid and 0.9μL of FuGENE®6 (Promega) were each diluted in 50μL of Opti-MEM and incubated for 5 minutes, then combined and incubated for another 20 minutes. The medium was replaced and the transfection mix was evenly distributed into one well. 24 hours after plasmid transfection, cells were forward transfected with 25pmol of construct / well and 1.5μL / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific). After 24 hours, cells were harvested for RNA isolation and sequencing.
[0105] Figure 7A shows the editing yield of different constructs. For the long and symmetrical embodiment V117.20 (SEQ ID NO: 24) with PS optimization in the core region, only a slight decrease in editing yield is observed compared to the unstable construct V117.19 (SEQ ID NO: 23) without PS optimization. For the shorter and asymmetrical embodiment V120.17 (SEQ ID NO: 25) (with PS-optimized core region), the editing yield is even comparable to version V117.19. However, the 100% FBS half-life of V117.20 (>7 days) and V120.17 (10 hours) shown in Figure 7B is significantly increased compared to V117.19 (<1 minute). This highlights the impact of stabilization measures, such as optimal placement of PS bonds in the core region of the construct, regardless of the design of the construct.
[0106] [Table 6]
[0107] Example 7: Introduction of an optimized PS-binding design into an ASO targeting the endogenous T41 site on mouse CTNNB1.
[0108] The results of Example 7 are shown in Figure 8. The optimized PS design of Example 1 (V117.39 targeting SERPINA1) was transferred to an oligonucleotide targeting the T41 site in mouse CTNNB1. The encoded protein, β-catenin, is a key component in cell proliferation and tissue homeostasis and is degraded by phosphorylation at the T41 site. Mutation at the T41 site extends the time that β-catenin resides in the cell, thus effectively promoting tissue regeneration. A list of the corresponding constructs is shown in Table 7.
[0109] 10 5Mouse embryonic fibroblasts (MEFs) were seeded in 24-well plates. After 24 hours, 25 pmol of construct / well and 1.5 μL / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) were each diluted in 50 μL of Opti-MEM (ThermoFisher Scientific) and incubated at room temperature for 5 minutes. After incubation, both solutions were combined for a total of 100 μL / well and further incubated at room temperature for 20 minutes. After incubation, the transfection mix was slowly dispensed into one well. 24 hours after transfection, cells were harvested for RNA isolation and sequencing.
[0110] Figure 8A shows the editing yield of v117.20 (SEQ ID NO: 26), which reaches about 20%, while Figure 8B shows the half-life of the construct in 100% FBS (>7 days). Thus, the mouse CTNNB1 T41 site represents another example where optimized PS binding configurations can be applied.
[0111] [Table 7]
[0112] The sequences disclosed herein are also shown in the attached sequence listing. However, the sequence listing only shows the sequence of the nucleotides, and the modifications of the nucleotides and the modifications of the bonds between the nucleotides are not shown in the sequence listing. The relevant sequences are disclosed in the above table. [Brief description of the drawings]
[0113] [Figure 1-1]Figure 1 shows the effect of phosphorothioate optimization on stability and editing efficiency when the central core is modified. Figure 1A shows the sequence of the central core and phosphorothioate modification. In addition, Figure 1A shows the stability of the composition and editing efficiency of each construct. It can be clearly seen that by increasing the number of phosphorothioate bonds, the stability can be greatly increased, but the editing efficiency is decreased (Figure 1A).
[0114] In Figure 1A, we used an oligonucleotide with phosphorothioate bonds only at positions 1 (a) and 10 (j) [v117.26]. The editing efficiency was quite high (59.2% ± 14), but the stability (t 50 (100% FBS)) took only 30 hours.
[0115] Figure 1A also shows an oligonucleotide with phosphorothioate linkages at positions 1 (a), 7 (g), 8 (h), 9 (i), and 10 (j) [v117.27]. Stability to degradation (t 50 ) improved to 40 hours, but the editing efficiency decreased to 33.0%.
[0116] Figure 1A also shows a construct with six phosphorothioate bonds at positions 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), and 10 (j) [v117.28]. The editing efficiency was improved to 50.3%, but the stability against degradation (t 50 ) only decreased by 20 hours.
[0117] Figure 1A shows a further experiment [v117.29] in which all linkages were phosphorothioate linkages. Editing efficiency only decreased to 32.0%.
[0118] In contrast, Figure 1A shows an experiment [v117.30] in which 6 of the 10 linkages were phosphorothioate (i.e., positions a, d, e, f, g, and j). The editing efficiency increased to 52.0%, and the stability (t 50 ) was >7 days.
[0119] The results shown in Figure 1A show that each linkage in the central core is a phosphorothioate linkage [v117.29] and the stability of the construct is >7 days. Unfortunately, however, the editing efficiency only decreased to 32%, indicating that a pattern of phosphorothioate linkages must be observed to achieve reasonable editing efficiency.
[0120] On the other hand, the number of phosphorothioate bonds is not the only important factor; just two phosphorothioate bonds (positions 1 and 10) significantly increased the t 50 (100% FBS) stability achieved [v117.26].
[0121] In Figures 1A and 1B, the positions of phosphorothioate bonds in the relevant samples are shown together with the editing efficiency and stability. Figure 1A shows that the best balance between high editing efficiency and high stability against degradation was obtained in the sample named v117.39. In this sample, the phosphorothioate bonds of the core structure are located at a(1), d(4), e(5), f(6), and j(10). According to the present invention, this pattern of phosphorothioate bonds is particularly preferred.
[0122] Figure 1A shows the exact location of phosphorothioate bonds in the construct targeting the SERPINA1 E342K mutation, further described in Example 1. Editing efficiency is shown from two different model systems (plasmid and piggyBac). The half-life of the construct was measured in 100% FBS (t(50)). n refers to the number of samples.
[0123] Figure 1B shows the orientations of the internucleotide bonds labeled a–j, with the central base triplet highlighted. [Figure 1-2] Continued from Figure 1. [Diagram 2]Figure 2 shows the editing yield results of the experiments performed in Example 1. The editing results are shown in Figure 2A and the serum half-life of the constructs of Example 1 is shown in Figure 2B. [Diagram 3] FIG. 3 shows the editing efficiency results of the experiments performed in Example 2, and the corresponding serum half-lives in 100% FBS are shown in FIG. 3B. [Figure 4] Figure 4A shows the editing efficiency of constructs targeting the disease-causing W104X mutation in mouse MECP2, and Figure 4B shows the serum half-life. [Diagram 5] Figure 5 shows the results of Example 4. The editing efficiency of the constructs is shown in Figure 5A, and the stability is shown in Figure 5B. [Figure 6] FIG. 6 shows the editing result of the fifth embodiment. [Figure 7] Figure 7 shows the results of Example 6. Figure 7A shows the editing efficiency, and Figure 7B shows the stability. [Figure 8] Figure 8 shows the results of Example 7, where the phosphorothioate design optimized according to Example 1 (V117.39) was transferred to an oligonucleotide targeting the T41 site in mouse CTNNB1. This figure shows that the disclosed pattern can also be applied to other targets.
Claims
1. 1. A chemically modified oligonucleotide for use in site-specific A-to-I editing of a target RNA in a cell by an endogenous ADAR, comprising: a sequence of 11 to 100 nucleotides in length capable of binding to a target sequence in the target RNA; a central base triplet (CBT) of three nucleotides, with the central nucleotide opposite the target adenosine in the target RNA to be edited to inosine, and having a core sequence shown in formula (I) below: 【Chemistry 1】 where Nu represents a nucleotide having a sugar moiety that can be modified; The numbers below the nucleotide sequence indicate the position of the nucleotide adjacent to the central nucleotide of the CBT having the number 0; Negative numbers indicate the 5' end of an oligonucleotide and positive numbers indicate the 3' end of said oligonucleotide; a to j indicate the nature of the bond between single nucleotides; A chemically modified oligonucleotide, wherein at least bonds a, d, and e are phosphorothioate bonds and at least two bonds are phosphate bonds.
2. The chemically modified oligonucleotide of claim 1, characterized in that bonds h and i are phosphate bonds, and / or bonds f, j and / or g are phosphorothioate bonds, and / or bonds b and / or c are phosphorothioate bonds.
3. The chemically modified oligonucleotide according to claim 2, wherein the bond b and / or c is a phosphate bond.
4. 2. The chemically modified oligonucleotide of claim 1, wherein at least one bond is a stereochemically pure phosphorothioate bond.
5. a) at least 90% of the pyrimidine nucleosides outside the CBT are chemically modified at the 2'-position of the sugar moiety; b) no more than six consecutive nucleosides are chemically modified with 2'-O-methyl at the 2'-position of the sugar moiety; and / or c) The chemically modified oligonucleotide according to claim 1, wherein at least two of the three nucleosides of the CBT are deoxyribonucleosides.
6. 2. The chemically modified oligonucleotide of claim 1, wherein at least 50% of the nucleotides Nu-5 to Nu+5 are independently modified at the 2' position of the ribose with one of the following modifications: 2'-fluoro, or 2'-O-methyl, or 2'-H (deoxy).
7. 2. The chemically modified oligonucleotide of claim 1, wherein at least two of the three nucleotides Nu-1, Nu0, and Nu+1 have an H residue at the 2'-position of the ribose.
8. The chemically modified oligonucleotide according to claim 1, wherein the nucleotide Nu+1 has an N-heterocyclic base.
9. The chemically modified oligonucleotide described in Claim 8, wherein the N-heterocyclic base is a purine derivative.
10. The chemically modified oligonucleotide described in Claim 8, wherein the nucleotide Nu+1 has a hypoxanthine base or a derivative thereof.
11. The chemically modified oligonucleotide according to claim 1, wherein the nucleotide Nu0 has a nucleic acid base based on an N-heterocycle.
12. A chemically modified oligonucleotide as described in claim 11, wherein the nucleotide Nu0 has cytosine or a derivative thereof.
13. The chemically modified oligonucleotide according to claim 1, characterized in that it has a length of 20 to 80 nucleotides or a length of 30 to 60 nucleotides.
14. 2. The chemically modified oligonucleotide of claim 1, wherein within the core sequence of formula I, only a, d, and e are phosphorothioate linkages.
15. The chemically modified oligonucleotide of claim 1, wherein within the core sequence of formula I, less than 40% of the bonds are phosphate bonds.
16. The chemically modified oligonucleotide of claim 1, wherein within the core sequence of formula I, up to 60% of the linkages are phosphorothioate linkages.
17. The chemically modified oligonucleotide described in claim 1, wherein the 5' flanking sequence to the CBT comprises at least 19 nucleotides (nt), at least 28 nt, or at least 33 nt, and the 3' flanking sequence to the CBT comprises at least 4 nt or at least 9 nt.
18. The chemically modified oligonucleotide of claim 1, wherein the target RNA is human SERPINA1 mRNA encoding the disease-causing E342K mutation in the alpha-1-antitrypsin (A1AT) protein.
19. The chemically modified oligonucleotide of claim 1, wherein the oligonucleotide is suitable for A-to-I editing of a target adenosine in human SERPINA1 mRNA transcripts that causes the disease-causing E342K mutation in the A1AT protein.
20. A chemically modified oligonucleotide described in claim 1, which does not contain an ADAR recruitment portion having a hairpin loop structure.
21. A composition comprising the chemically modified oligonucleotide of claim 1 and a pharmaceutically acceptable excipient.
22. 22. The chemically modified oligonucleotide of claim 1 or the composition of claim 21 for use in site-specific A-to-I editing of a target RNA in a cell.
23. 22. A chemically modified oligonucleotide according to claim 1 or a composition according to claim 21 for use in the treatment or prevention of a genetic disease or disorder.
24. 24. The chemically modified oligonucleotide or composition for use according to claim 23, wherein the genetic disease or disorder is a metabolic disease, an autoimmune disease, a cardiovascular disease or a neurological disease.