WO2023154026A1

WO2023154026A1 - Xeno nucleic acid antisens-oligonucleotide (xna-aso) sequences for the genetic treatment of ush2a induced retinitis pigmentosa disease

Info

Publication number: WO2023154026A1
Application number: PCT/TR2022/051683
Authority: WO
Inventors: Gorkem AKGUL; Ali Dostcan AKYAR; Batuhan YOLVER; Cihan TASTAN
Original assignee: T.C. Uskudar Universitesi
Priority date: 2022-02-09
Filing date: 2022-12-29
Publication date: 2023-08-17
Also published as: TR2022001648A2

Abstract

The invention relates to xenonucleic acid antisense-oligonucleotide (XNA-ASO) sequences that function as exon 13 skipping to bypass pathogenic mutations and transfer of these sequences to the target site for use in the treatment of Retinitis Pigmentosa (RP) disease, which is caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region. With the invention, XNA-ASO sequences with a compact and stable structure are provided for the treatment of Retinitis Pigmentosa (RP) disease, which is caused by pathogenic mutations on the USH2A) gene exon 13 region, which ensures the functional USH2A protein expression and long-term therapeutic effect and ensures that the pathogenic effects are eliminated by skipping the exon 13 region during pre-mRNA processing.

Description

XENO NUCLEIC ACID ANTISENS-OLIGONUCLEOTIDE (XNA-ASO) SEQUENCES FOR THE GENETIC TREATMENT OF USH2A INDUCED RETINITIS PIGMENTOSA DISEASE

Technical Field of the Invention

The invention relates to xenonucleic acid antisense-oligonucleotide (XNA-ASO) sequences that function as exon 13 skipping to bypass pathogenic mutations and transfer of these sequences to the target site for use in the treatment of Retinitis Pigmentosa (RP) disease, which is caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region.

State of the Art

Retinitis Pigmentosa (RP) is a chronic inherited eye disease characterized by black pigmentation and gradual degeneration of light-sensitive retinal cells covering the back of the eye. RP can result from both homozygous and heterozygous mutations. For example, RP can exist in various forms such as autosomal dominant RP (adRP), autosomal recessive RP (arRP), or X-linked RP (X-LRP). Usher syndrome type II (USH2A) is a genetically and phenotypically heterogeneous inherited disorder combining hearing loss and Retinitis Pigmentosa (RP).

Although the treatment options for Retinitis Pigmentosa or Usher syndrome caused by the USH2A gene mutation are limited, there is no approved therapy in the state of the art that can stop or reverse the progression of Usher syndrome and RP. There is only one treatment pending clinical approval in the FDA and EMA. This treatment developed by ProQR Therapeutics is an antisense- oligonucleotide (ASO) treatment named QR-421 a.

Antisense therapy strategy, which is one of the technologies used today to develop treatments for many diseases, including RP, includes the use of oligodeoxyribonucleotides to silence various genes without forming proteins. The antisense mechanism occurs as a result of base pairing of the oligonucleotide sequence to the target pre-mRNA. After binding, two major pathways inhibit protein formation of the gene of interest. The first of these is RNase H-mediated degradation at the binding site of the oligonucleotide. Since translation is not possible from the gene sequence that is cleaved by the RNase enzyme, protein formation is prevented. The second occurs when the oligonucleotide binds and closes the relevant gene region. Thus, the ribosome is sterically prevented from reading the gene sequence, posttranscriptional processes are inhibited, and the gene containing the mutation cannot form a protein.

Various modifications are made to protect these molecules from nucleases. Although antisense oligonucleotides (ASOs) are protected from nucleases with these modifications, their entry into the cell remains limited. In order to overcome this limitation, various methods such as conjugated peptides and lipid-based carrier systems should be used.

QR-421 a ASO treatment, developed by ProQR Therapeutics and awaiting clinical approval, is in phase 1 -2 (NCT 03780257). The most common mutations for RP and Usher syndrome are known to be found in exon 13. For this reason, the region targeted by QR-421 a was chosen as exon 13. The treatment removes exon 13 from the mRNA and then produces a shorter but functional USH2A protein. QR-421 a works with the principle of ASOs closing this place during exon processing and skipping exon 13 region at the end. In order to remove the G deletion (c2299delG) mutation at nucleotide 2299 in exon 13 region of the USH2A gene and G>T wrong base pairing (c2276G>T) mutation at nucleotide 2276, QR- 421 a allows exon 13 to be skipped using ASO during pre-mRNA processing, thus producing a slightly shortened protein the functionality of which is not impaired [1 ]-

QR-421 a performs exon 13 skipping using the classical DNA-ASO system. Classical DNA-ASO systems are therapeutic tools with low hydrogen bonding and low biological stability with a complementary structure. Therefore, the duration of action is quite short. The short duration of action significantly affects drug delivery pathways, especially in ocular diseases such as RP. In ocular disorders, therapeutics given by target tissue-specific routes, such as ASOs, are given by intravitreal and subretinal methods. However, since these transmission routes are given directly into the eye with surgery, they can cause various complications. In conclusion, therapeutics with low biological stability, such as DNA-ASO, increase the frequency of these surgical operations and shorten the duration of action.

In the prior art, there are xeno nucleic acid antisense-oligonucleotide (XNA-ASO) sequences that differ from conventional ASOs. Xeno nucleic acids (XNA) are synthetic nucleic acid analogues that have a different sugar backbone from the natural nucleic acids DNA and RNA. XNA sequences form a series of artificial genetic polymers that preserve the natural nucleobases and phosphodiester linkages of DNA and RNA sequences. The sugar structure of XNAs are RNA base analogues produced by establishing methylene bridges between the 4'- carbon and 2' oxygen in the furanose structure.

In the design of XNA sequences, HNA, TNA, GNA, CeNA, LNA and PNA are used as nucleotide sequences. HNA (anhydrohexitol nucleic acid), TNA (treofuranosyl nucleic acid) are xeno nucleic acids without ribose instead of ribose sugar found in RNA [2], GNA (glycol nucleic acid), on the other hand, are structures that differ from DNA and RNA in terms of sugar-phosphodiester bond structure. GNAs make the sequence more stable because they increase the melting temperatures of the complementary structures [3]. CeNa (cyclohexene nucleic acid) are nucleic acids that contain cyclohexene structure, unlike classical DNA. Another nucleic acid, LNA (locked nucleic acid), has a structure locked with a methyl bridge, and these nucleotides provide significant resistance, especially against nucleases. PNA (peptide nucleic acid) is nucleic acids that contain N-(2- aminoethyl) repeats linked by peptide bonds in their backbone structure [4],

Advances in nucleic acid chemistry have allowed the development of XNAs, resulting in several FDA-approved nucleic acid therapeutics. Research is rapidly underway to identify new therapies with increased potency, bioavailability, stability, reduced toxicity and minimal off-target effects. However, there are no FDA-approved XNA-based treatments, especially in the treatment of RP, however, studies in this area continue.

The prior art patent application WO2020219981 A2 describes USH2A oligonucleotides, their compositions and methods of use to prevent and/or treat various diseases. The USH2A oligonucleotides described herein include nucleobase modifications, sugar modifications, nucleotidic linkage modifications, and/or patterns thereof. Said oligonucleotides, for example USH2A oligonucleotides, are antisense oligonucleotides (ASOs) having a base sequence that is antisense for the target nucleic acid.

The prior art patent US10745699B2 describes new antisense oligonucleotides that can be used in the treatment, prevention and/or delay of non-syndromic retinal degeneration associated with Usher syndrome type 2A and/or USH2A. In the content of the said invention is the use of classical ASO. However, prior art ASO arrays do not provide long-term therapeutic effects and are insufficient for use in the treatment of Retinitis Pigmentosa due to their low stability.

Since ASO sequences in the prior art designed for use in the treatment of Retinitis Pigmentosa are insufficient to provide a long-term therapeutic effect and to provide the expression of the functional USH2A protein, and since their biological stability are low, there is a need to develop ASO arrays for use in the treatment of Retinitis Pigmentosa, with a compact and stable structure that provides functional USH2A protein expression and long-term therapeutic effect.

Brief Description of the Invention

In the invention, xeno nucleic acid antisense-oligonucleotide (XNA-ASO) sequences that function as exon 13 skipping to bypass pathogenic mutations and transfer of these sequences to the target site for use in the treatment of Retinitis Pigmentosa (RP) disease, which is caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region are described. The aim of the invention is to provide antisense-oligonucleotide-based sequences with a compact and stable structure and long-term therapeutic effect for the treatment of Retinitis Pigmentosa (RP) disease caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region. Within the scope of the invention, xeno nucleic acid antisense-oligonucleotide (XNA-ASO) sequences that function as exon 13 skipping are designed. In the invention, there is the use of ASO obtained by using xeno nucleic acid instead of the classic ASO use, which is very common in the prior art. The sugar structure of XNAs are RNA base analogues produced by establishing methylene bridges between the 4'-carbon and 2' oxygen in the furanose structure. The XNA-ASO polymer structure with this chemical structure creates a more compact and stable structure with the target sequence compared to DNA-ASOs. By means of this structure, XNA-ASOs considerably prolong the therapeutic effect and significantly reduce the frequency of surgical interventions with the possibility of complications.

By means of the XNA-ASO sequences that are the subject of the invention, exon 13 region is skipped during pre-mRNA processing in RP individuals with the USH2A gene exon 13 mutation, thus eliminating pathogenic effects and expressing functional protein. The ESE-SC35 binding site targeted ASO sequences of the USH2A gene exon 13 region, which is the subject of the invention, form a complementary structure with the pre-mRNA transcript to which they are targeted, thus preventing the binding of splicing factors to the exon 13 region, which is complementary during mRNA processing, thus ensuring that this region is not recognized and bypassed. EGF-lam domains shortened from the mRNA transcript with exon 13 region skipped but non-pathogenic functional Usherin protein expression is provided. Each XNA-ASO sequence disclosed within the scope of the invention is specific for pathogenic mutations at the corresponding exon 13 position and is used in the personalised genetic therapy of RP patients who have one or more of the mutations at the relevant position.

ASO used in the prior art contains 2'-0-(2-methoxyethyl) sugar modification and fully phosphorothioated backbone modification. This is particularly due to the use of conventional ASO. In the invention, there is the use of modified XNA instead of the classical nucleotide in the XNA-ASO technique. In the DNA mixmer structure, there are both classical ASO modifications, 2'-0-(2-methoxyethyl) and phosphorothioated modifications, and at least one of the HNA, TNA, GNA, CeNA, LNA and PNA modifications in XNA. By using XNA, the strength of the A-T double and G-C triple -H bonds in the secondary structure formed by ASO with its target sequence is increased. Thus, a much more stable structure is formed compared to classical DNA-ASO oligomers.

In nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequences containing the DNA mixmer that is the subject of the invention, both the classical modifications and xeno sequences mentioned in the ASO are provided by the fact that the A and T nucleotides or G and C nucleotides are XNA at the same time and the other nucleotides are classical nucleotides. The reason why A-T or G-C nucleotides are XNA or classical nucleotides at the same time is that the A nucleotide forms a complementary structure with T and the G nucleotide with C. Among these structures, the A-T structure contains 2 hydrogen bonds, and the G-C structure contains 3 hydrogen bonds, and these hydrogen bond structures are made stronger with xeno structures.

With the invention, XNA-ASO sequences with a compact and stable structure are provided for the treatment of Retinitis Pigmentosa (RP) disease, which is caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region, which ensures the functional USH2A protein expression and long-term therapeutic effect and ensures that the pathogenic effects are eliminated by skipping the exon 13 region during pre-mRNA processing.

Detailed Description of the Invention

The invention relates to xeno nucleic acid-antisense oligonucleotide (XNA-ASO) sequences that function as exon 13 skipping to bypass pathogenic mutations or nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequences containing DNA mixmer and transfer of these sequences to the target site, for use in the treatment of Retinitis Pigmentosa (RP) disease, which is caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region.

In order to obtain the XNA-ASO or XNA/DNA-ASO sequences, which are the subject of the invention, firstly exon splicing enhancer (ESE) and serine/arginine- rich splicing factor SC35 binding site targeted sequences are determined. Then, ASOs are designed as XNA and XNA/DNA according to the determined sequences. After the XNA-ASO sequences are obtained, in vivo transfer of XNA- ASO and XNA/DNA-ASOs to the target sequence is carried out.

ESE regions were determined in order to ensure that exon 13 region is skipped during pre-mRNA processing, which is the main focus of gene therapy with XNA- ASO or XNA/DNA-ASO sequences, which are the subject of the invention. USH2A gene (Gene ID: 7399) exon 13 region sequence information obtained from the NCBI database and ESE Finder Tool bioinformatics tool were used to determine the sequences that are specific to this region and that can interact (secondary structure) in this region. The SC35 binding site portion of the USH2A gene, which will trigger exon skipping, is targeted in the exon 13 ESE region. ESE-SC35 binding site targeted 54 sequence designs were designed using the ESE-Finder tool bioinformatics. The designed sequences ranged in length from 19 to 21 nucleotides.

74 ASO sequences were designed based on the USH2A exon13 pre-mRNA sequence. The reason why these sequences are different is that the sequence information of the ESE regions on exon 13 is not known exactly. Each of the ASO sequences designed for this scans these ESE regions. The technical difference of these sequences is that they form a duplex structure with different places on exon 13. Therefore, the effect of each sequence on pathogenicity is not the same due to its binding at different sites on the exon. However, since the arrays are all designed to function as exon 13 skipping, it solves the same technical problem.

74 different ASO sequences (28 different XNA-ASO sequences; 56 different XNA/DNA-ASO sequences in 28 of them A and T nucleotides are xeno nucleic acid, while G and C nucleotides are classical DNA nucleic acid, in the other 28, G and C nucleotides are xeno nucleic acids, while A and T nucleotides are classical DNA nucleic acids) that specifically bind to the ESE region on the exon 13 region and affect the binding of splicing factor proteins to this region used as candidate therapeutic tools for gene therapy of RP caused by pathogenic mutations on exon 13 of the USH2A gene. In the pre-mRNA processing during the transcription of the USH2A gene, the produced specific ASO sequences bind to the pre-mRNA and form the secondary structure. The regions targeted by the produced sequences are specific to the USH2A pre-mRNA, since they are specific to the USH2A gene exon 13 region and are regions that can trigger exon skipping here.

ESE-SC35 binding site targeted detected sequences are designed using XNA and XNA-DNA mixmers and produced using at least one of the synthetic HNA, TNA, GNA, CeNA, LNA and PNA nucleotides. By using XNA, the strength of the A-T double and G-C triple -H bonds in the complementary structure formed with the target sequence is increased. Thus, a much more stable structure is formed compared to DNA-ASO oligomers. XNA sequences have a different sugar chemistry than DNA and RNA molecules, in addition to forming a series of artificial genetic polymers that preserve the natural nucleobase (A, C, T, G) and phosphodiester linkage that DNA and RNA sequences have.

The sugar structure of XNA molecules is designed and produced by establishing methylene bridges between 4'-carbon and 2'-oxygen in the furanose structure. Within the scope of the invention, the polymer structure consisting of XNA and XNA/DNA becomes more resistant to endonucleases (such as hnRNPA1/A2) in the cell. In the designed XNA/DNA-ASO mixmer arrays, 2'-0-methoxyethyl modification (MOE) and/or a phosphorothioate (PS) modification is provided by adding a DNA mixmer to the sequences consisting of XNA. The difference of DNA mixmer designs from XNA-ASOs is that they contain classical nucleic acid structures. Since MOE and PS modifications are only found in classical nucleic acid-based ASOs, MOE and PS modifications are present in DNA-mixmer sequences, unlike XNA-ASOs. In the design of XNA sequences, HNA, TNA, GNA, CeNA, LNA and/or PNA are used as nucleotide sequences, in other words, at least one of HNA, TNA, GNA, CeNA, LNA and PNA nucleotides are present in the final versions of XNA-ASO sequences. In this way, XNAs will be able to form a secondary (double helix) structure in the form of XNA:RNA with the target sequences on exon 13 in the USH2A pre-mRNA sequence.

Table 1 shows the sequences designed as a completely xenonucleic acid- antisense oligonucleotide (XNA-ASO) or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) containing a DNA mixmer, and also, the reverse complement sequences of these sequences are presented in Table 2. The sequence XNA- ASO or XNA/DNA-ASO having a nucleotide sequence selected from the sequences of SEQ ID NO:1 -28, respectively, in Table 1 binds to the reverse complementary sequence with a nucleotide sequence selected from the sequences of SEQ ID NO:29-56, respectively, in Table 2.

In one embodiment of the invention, in XNA-ASO sequences with the sequence of SEQ ID NO:1 -28 given in Table 1 , A and T nucleotides are xeno nucleic acid, and G and C nucleotides are classical, natural DNA nucleic acids. In another embodiment of the invention, in XNA-ASO sequences with the sequence of SEQ ID NO:1 -28 given in Table 1 , G and C nucleotides are xeno nucleic acid, and A and T nucleotides are classical, natural DNA nucleic acids.

Similarly, in one embodiment of the invention, in XNA-ASO sequences with the sequence of SEQ ID NO:29-56 given in Table 2, A and T nucleotides are xeno nucleic acid, and G and C nucleotides are classical, natural DNA nucleic acids. In another embodiment of the invention, in XNA-ASO reverse complementary sequences with the sequence of SEQ ID NO:29-56 given in Table 2, G and C nucleotides are xeno nucleic acid, and A and T nucleotides are classical, natural DNA nucleic acids. In the evaluation of the exon 13 position in the tables, the position calculation was made from the 1 ^st nucleotide of the exon 13 region of the USH2A gene. The negative value represents the ASO sequence located in the intron region, away from the 1^st nucleotide at the 5' end. Table 1. Sequences designed entirely as xeno nucleic acid-antisense oligonucleotide (XNA-ASO) or as a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) containing a DNA mixmer (SEQ ID NO:1 -28).

Table 2. Reverse complementary sequences of the sequences given in Table 1 (SEQ ID NO:29-56), constructed entirely from xenonucleic acid (XNA) acids or from xenonucleic acids (XNA/DNA-ASO) containing a DNA mixmer.

A and T nucleotides or G and C nucleotides being XNA at the same time and other nucleotides being classical nucleotides in nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequences containing DNA mixmer in the sequences given in Table 1 and the sequence list, the presence of both classical modifications and xeno sequences in ASO is ensured. The reason why A-T or G- C nucleotides are XNA or classical nucleotides at the same time is that the A nucleotide forms a complementary structure with T and the G nucleotide with C. Among these structures, the A-T structure contains 2 hydrogen bonds, and the G-C structure contains 3 hydrogen bonds, and these hydrogen bond structures are made stronger with xeno structures.

The designed XNA-ASO and XNA/DNA-ASO mixmers are transferred to photoreceptor cells in vivo. XNA-ASO and XNA/DNA-ASO mixmers target the same region, but they also contain classical, natural nucleotide (A, T, G, C) structures within the mixmers as production. Therefore, in the therapeutic strategy, these structures are given to photoreceptor cells as a separate and alternative therapeutic tool. The ESE-SC35 binding site targeted ASO sequences of the USH2A gene exon 13 region form a complementary structure with the pre- mRNA transcript to which they are targeted, thus preventing the binding of splicing factors to the exon 13 region, which is complementary during mRNA processing, thus ensuring that this region is not recognized and bypassed. EGF- lam domains shortened from the mRNA transcript with exon 13 region skipped but non-pathogenic functional Usherin protein expression is provided. References

1. Slijkerman, R., van Diepen, H., Albert, S., Dona, M., Venselaar, H., Zang, J., Neuhauss, S., Peters, T., Broekman, S., Pennings, R., Kremer, H., Adamson, P., de Vrieze, E., & van Wijk, E. (2020). Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by mutations in USH2A exon 13”, bioRxiv.

2. Ekmekgi, A. (2016). Xeno-Nukleik Asitler ve XNA Dunyasi. Gazi Medical Journal, 27(3).

3. Meggers, E., & Zhang, L. (2010). Synthesis and properties of the simplified nucleic acid glycol nucleic acid. Accounts of chemical research, 43(8), 1092- 1 102.

4. Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., ... & Nielsen, P. E. (1993). PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature, 365(6446), 566-568.

Claims

CLAIMS A xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence that functions as exon 13 skipping, or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer for use in the treatment of Retinitis Pigmentosa disease caused by pathogenic mutations on the Usherin (USH2A) gene exon 13 region, comprising a nucleotide sequence selected from the sequences of SEQ ID NO:1 -28. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to Claim 1 , wherein, in a nucleotide sequence selected from said sequences of SEQ ID NOU -28, nucleotides A and T are xeno nucleic acid, nucleotides G and C are classical DNA nucleic acids. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to Claim 1 , wherein, in a nucleotide sequence selected from said sequences of SEQ ID NOU -28, nucleotides G and C are xeno nucleic acid, nucleotides A and T are classical DNA nucleic acids. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to Claim 1 , wherein, XNA-ASO or reverse complementary sequence to which the XNA/DNA-ASO sequence is linked, having a nucleotide sequence selected from said sequences of SEQ ID NOU -28, respectively, is a nucleotide sequence selected from the sequences of SEQ ID NOs:29-56, respectively. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to Claim 4, wherein, in a nucleotide sequence selected from said sequences of SEQ ID NO:29-56, nucleotides A and T are xeno nucleic acid, nucleotides G and C are classical DNA nucleic acids. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to Claim 4, wherein, in a nucleotide sequence selected from said sequences of SEQ ID NO:29-56, nucleotides G and C are xeno nucleic acid, nucleotides A and T are classical DNA nucleic acids. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to any of the Claims 1 -6, wherein, the length of it is between 19 and 21 nucleotides. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to any of the Claims 1 -3, comprising at least one of anhydrohexitol nucleic acid (HNA), treofuranosyl nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexene nucleic acid (CeNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA) nucleotides. The xenonucleic acid-antisense oligonucleotide (XNA-ASO) sequence or a nucleic acid-antisense oligonucleotide (XNA/DNA-ASO) sequence containing a DNA mixmer according to any of the Claims 1 -3, comprising 2'-0-methoxyethyl modification (MOE) and/or a phosphorothioate (PS) modification.