HK1262526B - Antisense oligonucleotides for modulating htra1 expression - Google Patents

Description

Antisense oligonucleotides for regulating HTRA1 expression

Invention Field

This invention relates to antisense oligonucleotides (oligomers) complementary to HTRA1, which lead to the regulation of HTRA1 expression. Regulation of HTRA1 expression is beneficial for a range of medical conditions, such as macular degeneration, including age-related macular degeneration.

background

The human high temperature requirement A (HTRA) family of serine proteases are ubiquitous PDZ-proteases that participate in maintaining protein homeostasis in the extracellular compartment through the dual functions of combinatorial protease and molecular chaperone. HTRA housekeeping proteases are involved in the organization of the extracellular matrix, cell proliferation, and senescence. Regulation of HTRA activity is associated with serious diseases, including Duchenne muscular dystrophy (Bakay et al., 2002, Neuromuscul. Disord. 12: 125-141), arthritis such as osteoarthritis (Grau et al., 2006, JBC 281: 6124-6129), cancer, familial ischemic small vessel disease, age-related macular degeneration, Parkinson's disease, and Alzheimer's disease. Human HTRA1 contains an insulin-like growth factor (IGF) binding domain. HTRA1 has been proposed to regulate IGF availability and cell growth (Zumbrunn and Trueb, 1996, FEES Letters 398: 189-192) and has shown tumor-suppressive properties. HTRA1 expression is downregulated in metastatic melanoma and can therefore indicate the extent of melanoma progression. In xenograft mouse models, overexpression of HTRA1 in metastatic melanoma cell lines reduced in vitro proliferation and invasion, and decreased tumor growth (Baldi et al., 2002, Oncogene 21: 6684-6688). HTRA1 expression is also downregulated in ovarian cancer. In ovarian cancer cell lines, HTRA1 overexpression induces cell death, while antisense HTRA1 expression promotes anchorage-independent growth (Chien et al., 2004, Oncogene 23: 1636-1644).

In addition to its effects on the IGF pathway, HTRA1 also inhibits the signaling of TGFβ family growth factors (Oka et al., 2004, Development 131: 1041-1053). HTRA1 can cleave amyloid precursor protein (APP), and HTRA1 inhibitors cause the accumulation of Aβ peptide in cultured cells. Therefore, HTRA1 is also associated with Alzheimer's disease (Grau et al., 2005, Proc. Nat. Acad. Sci. USA. 102: 6021-6026).

On the other hand, HTRA1 upregulation has been observed and appears to be associated with Dischene muscular dystrophy (Bakay et al. 2002, Neuromuscul. Disord. 12: 125-141), osteoarthritis (Grau et al. 2006, JBC 281: 6124-6129), and AMD (Fritsche et al. Nat Gen 2013 45(4): 433-9.).

A single nucleotide polymorphism (SNP) in the HTRA1 promoter region (rs11200638) was associated with a 10-fold increased risk of developing age-related macular degeneration (AMD). Furthermore, the HTRA1 SNP was in linkage disequilibrium with the ARMS2 SNP (rs10490924), which is also associated with an increased risk of AMD. Risk alleles were associated with a 2-3 fold increase in HTRA1 mRNA and protein expression, and HTRA1 is present in drusen of AMD patients (Dewan et al., 2006, Science 314: 989-992; Yang et al., 2006, Science 314: 992-993). Overexpression of HtrA1 has been demonstrated in various animal models to induce an AMD-like phenotype in mice. hHTRA transgenic mice (Veierkottn, PlosOne 2011) revealed degradation of the elastic layer of the Bruch membrane, identified choroidal vascular abnormalities (Jones, PNAS 2011), and increased polypoidal choroidal vasculopathy (PCV) lesions (Kumar, IOVS 2014). Furthermore, Bruch membrane damage in hHTRA1 Tg mice has been reported, which determined a 3-fold increase in CNV after exposure to cigarette smoke (Nakayama, IOVS 2014).

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in people over 65 years of age. With the onset of AMD, the light-sensitive photoreceptor cells in the posterior part of the eye, the metabolically supporting underlying pigment epithelial cells, and the clear central vision they provide gradually diminish. Age is a major risk factor for AMD: the likelihood of developing AMD increases threefold after age 55. Smoking, light iris color, sex (women are at greater risk), obesity, and repeated exposure to UV radiation also increase the risk of AMD. There are two forms of AMD: dry AMD and wet AMD. In dry AMD, drusen appear in the macula of the eye, cells in the macula die, and vision becomes blurred. Dry AMD can progress in three stages: 1) early, 2) intermediate, and 3) late dry AMD. Dry AMD can also progress to wet AMD at any of these stages. Wet AMD (also known as exudative AMD) is associated with pathological posterior segment angiogenesis. Posterior segment neovascularization (PSNV) found in exudative AMD is characterized by pathological choroidal neovascularization. Leakage from abnormal blood vessels formed during this process can damage the macula and impair vision, ultimately leading to blindness. Treatment strategies for wet AMD are limited, and symptomatic treatment is preferred. Therefore, there is an unmet medical need to provide effective medications for treating macular degeneration conditions such as wet and dry AMD. WO 2008/013893 claims protection for a composition for treating a subject with age-related macular degeneration, comprising a nucleic acid molecule containing an antisense sequence hybridizing to the HTRA1 gene or mRNA: no antisense molecule is disclosed. WO 2009/006460 provides for siRNA targeting HTRA1 and its use in the treatment of AMD.

The purpose of this invention

This invention provides antisense oligonucleotides that regulate HTRA1 in vivo or in vitro. The invention identifies hidden target sequence motifs present in human HTRA1 mRNA (including pre-mRNA) that can be targeted by antisense oligonucleotides to produce potent HTRA1 inhibition. The invention also provides potent antisense oligonucleotide sequences and compounds capable of inhibiting HTRA1, and their use in treating diseases or conditions indicative of HTRA1.

Invention Overview

This invention relates to oligonucleotides that target mammalian HTRA1 nucleic acid, specifically oligonucleotides capable of inhibiting HTRA1 expression and treating or preventing diseases related to HTRA1 function. The HTRA1-targeting oligonucleotides are antisense oligonucleotides, meaning they are complementary to their HTRA1 nucleic acid target.

The oligonucleotides of the present invention may be in the form of pharmaceutically acceptable salts, such as sodium or potassium salts.

Therefore, the present invention provides antisense oligonucleotides comprising a continuous nucleotide sequence of 10-30 nucleotides having at least 90% complementarity (e.g., complete complementarity with mammalian HTRA1 nucleic acids such as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4).

In another aspect, the present invention provides pharmaceutical compositions comprising the oligonucleotides of the present invention and pharmaceutically acceptable diluents, carriers, salts and/or adjuvants.

This invention provides LNA antisense oligonucleotides, such as LNA gapmer oligonucleotides, comprising a continuous nucleotide sequence of 10-30 nucleotides having at least 90% complementarity (e.g., being completely complementary to the sequence of HTRA1 nucleic acid selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4).

This invention provides an antisense oligonucleotide comprising a continuous nucleotide region of 10-22 (e.g., 12-22) nucleotides that are at least 90% (e.g., 100%) complementary to SEQ ID NO 147:

SEQ ID NO 147：5′CCAACAACCAGGTAAATATTTG 3′

The present invention provides an antisense oligonucleotide comprising a continuous nucleotide region of 10-17 nucleotides (e.g., 11, 12, 13, 14, 15, 16, e.g., 12-16 or 12-17) complementary to a sequence selected from the group consisting of SEQ ID NO 148-155.

The present invention provides an antisense oligonucleotide comprising a continuous nucleotide region of 10-17 nucleotides (e.g., 11, 12, 13, 14, 15, 16, e.g., 12-16 or 12-17) complementary to SEQ ID NO 148 or 155.

The present invention provides antisense oligonucleotides of 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide region of 10-22 nucleotides that is at least 90% to 100% complementary to SEQ ID NO 147.

SEQ ID NO 147：5′CCAACAACCAGGTAAATATTTG 3′

The present invention provides antisense oligonucleotides of 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide region of at least 10, such as at least 12, consecutive nucleotides that are complementary to the sequence presented in the sequence selected from SEQ ID NO 148-155.

The present invention provides antisense oligonucleotides with a length of at least 12 nucleotides, wherein the antisense oligonucleotides comprise a sequential sequence of SEQ ID NO 146.

SEQ ID NO 146: 5′TTTACCTGGGTT 3′.

The present invention provides oligonucleotides as described in the embodiments. The present invention provides oligonucleotides, such as antisense oligonucleotides, comprising at least 10, such as at least 12, sequences selected from the group consisting of SEQ ID NO 5-145.

The present invention provides a conjugate comprising an oligonucleotide according to the invention, and at least one conjugate moiety covalently linked to said oligonucleotide.

This invention provides pharmaceutically acceptable salts of the oligonucleotides or conjugates of this invention.

In another aspect, the present invention provides an in vivo or in vitro method for regulating HTRA1 expression in cells by administering an effective amount of the oligonucleotide, conjugate, or composition of the present invention to cells expressing HTRA1.

In another aspect, the present invention provides a method for treating or preventing diseases, conditions, or dysfunctions associated with the in vivo activity of HTRA1, comprising administering a therapeutically or preventively effective amount of the oligonucleotide or conjugate thereof of the present invention to a subject suffering from or susceptible to said disease, condition, or dysfunction.

On the other hand, the oligonucleotides or compositions of the present invention are used to treat or prevent macular degeneration, as well as other conditions involving HTRA1.

The present invention provides oligonucleotides or conjugates thereof for the treatment of diseases or conditions selected from a list including: Dechené muscular dystrophy, arthritis such as osteoarthritis, familial ischemic small vessel disease, Alzheimer's disease, and Parkinson's disease.

This invention provides oligonucleotides or conjugates for the treatment of macular degeneration, such as wet or dry age-related macular degeneration (e.g., wAMD, dAMD, geographic atrophy, intermediate dAMD) or diabetic retinopathy.

This invention provides the use of the oligonucleotides, conjugates, or compositions of the invention in the preparation of medicaments for the treatment of macular degeneration, such as wet or dry age-related macular degeneration (e.g., wAMD, dAMD, geographic atrophy, intermediate dAMD) or diabetic retinopathy.

This invention provides the use of the oligonucleotides, conjugates, or compositions of the invention in the preparation of medicaments for treating diseases or conditions selected from the group consisting of: Dechené muscular dystrophy, arthritis such as osteoarthritis, familial ischemic small vessel disease, Alzheimer's disease, and Parkinson's disease.

The present invention provides a method for treating a subject suffering from a disease or condition selected from the group consisting of: Dechené muscular dystrophy, arthritis such as osteoarthritis, familial ischemic small vessel disease, Alzheimer's disease, and Parkinson's disease, the method comprising the step of administering an effective amount of the oligonucleotide, conjugate, or composition of the present invention to the subject.

The present invention provides a method for treating a subject suffering from an eye disease such as macular degeneration, such as wet or dry age-related macular degeneration (e.g., wAMD, dAMD, geographic atrophy, intermediate dAMD) or diabetic retinopathy, the method comprising the step of administering an effective amount of the oligonucleotide, conjugate or composition of the present invention to the subject.

The present invention provides a method for treating subjects with eye diseases such as macular degeneration, such as wet or dry age-related macular degeneration (e.g., wAMD, dAMD, geographic atrophy, intermediate dAMD) or diabetic retinopathy, the method comprising administering at least two doses of the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof in an intraocular injection at a dose of about 10 μg to 200 μg, wherein the dose interval between consecutive administrations is at least 4 weeks or at least 1 month.

Brief description of the attached diagram

Figure 1. Screening of libraries containing n=129 HTRA1 LNA oligonucleotides at 25 μM in U251 and ARPE19 cell lines. Readout: HTRA1 qPCR. The n=6 oligonucleotides located between positions 33042-33064 were relatively active.

Figure 2. Libraries of n=116 HTRA1 LNA oligonucleotides in the hotspot 33042-33064 were screened at 5 and 25 μM in U251 and ARPE19 cell lines, respectively. Oligonucleotides with n=7 were selected for further analysis. Readout: HTRA1 qPCR.

Figure 3. Dose-response relationship of HTRA1 mRNA levels after treatment of human primary RPE cells with LNA oligonucleotides 139,1 and 143,1.

Figure 4. qPCR quantification of HTRA1 expression relative to the efficacy in rats, 30 μg/eye, after 7 days of treatment via intravitreal (IVT) injection.

Figure 5. In vivo efficacy study in rats, 7-day treatment, IVT administration, dose-response. Retinal samples from rat eyes treated with PBS, 140.1, or 143.1 were analyzed. Htra1 ISH RNAscope was performed; A) shows representative samples and B) shows an overview of the results. GFAP IHC was also performed; GFAP is a marker of reactive gliosis and retinal fibrosis. C) Retinal samples for Htra1 qPCR. RE: Right eye, LE: Left eye. D) Oligonucleotide content bioanalysis was performed, and dose-response curves of the bioanalysis are shown compared to relative mRNA expression. _EC50 assays were performed in GraphPad Prims. For PBS-treated samples, the oligonucleotide content was set at 0.01 μg/g tissue.

Figure 6. Proof-of-concept (Poc) study in albino mice, blue light-induced retinal degeneration. A) Recovery (%) of a- and b-wave amplitudes in electroretinogram 14 days after blue light exposure. Bars represent group means and 95% CI. Each data point indicates the mean values for the right and left eyes of each study animal. B) ISH RNA scope, instances from two different regions of the retina. C) Htra1 qPCR of retinal samples. D) PK PD correlation.

Figure 7. In vivo efficacy kinetics in rats, IVT administration, 50 μg/eye, treatment days 3, 7, and 14. A) HTRA1 mRNA levels measured in the retina by qPCR. B) HTRA1 mRNA levels quantified by ISH. Residual HTRA1 mRNA expression levels are shown as the percentage of control (PBS-treated cells) in A and B, and C) dose-response curves of oligonucleotide content at each time point compared to qPCR data.

Figure 8. Non-human primate (NHP) PK/PD study, IVT administration, 25 μg/eye. A) HTRA1 mRNA level measured in the retina by qPCR. B) HTRA1 mRNA level displayed by ISH. C-D) HTRA1 protein levels in the retina and vitreous humor, respectively, quantified by IP-MS. Dots indicate data for individual animals. Error bars show the standard deviation of technique replicates (n=3).

Figure 9. The compound of the present invention (Compound ID NO 143,1). The compound may be in the form of a pharmaceutical salt, such as a sodium or potassium salt.

Figure 10. Compound of the present invention (Compound ID No. 145,3). The compound may be in the form of a pharmaceutical salt, such as a sodium or potassium salt.

Figure 11. Example of a medicinal salt of compound 143.1. M+ is a suitable cation, typically a positive metal ion, such as sodium or potassium ions. The stoichiometric ratio of the cation to the oligonucleotide anion will depend on the charge of the cation used. Suitablely, cations with one, two, or three positive charges (M ⁺ , M ⁺⁺ , or M ⁺⁺⁺⁺ ) can be used. For illustrative purposes, twice as many cations with a single + charge (monovalent), such as Na ⁺ or K ⁺ , are required compared to divalent cations such as Ca2 ⁺ .

Figure 12. Example of a medicinal salt of compound 145.3. For an explanation of the cation M ⁺ , please refer to the legend in Figure 11.

definition

Oligonucleotides

As used herein, the term "oligonucleotide" is defined as a molecule commonly understood by those skilled in the art to comprise two or more covalently linked nucleosides. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are typically prepared in the laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleobase portion of the covalently linked nucleotide or nucleoside, or its modifications thereof. The oligonucleotides of this invention are artificial and chemically synthesized, and are generally purified or isolated. The oligonucleotides of this invention may comprise one or more modified nucleosides or nucleotides.

antisense oligonucleotides

As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide capable of regulating the expression of a target gene by hybridization with a target nucleic acid, particularly with a sequential sequence on the target nucleic acid. Antisense oligonucleotides are substantially non-double-stranded and therefore not siRNAs. Preferably, the antisense oligonucleotides of the present invention are single-stranded.

Continuous nucleotide region

The term "continuous nucleotide region" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "continuous nucleotide sequence" or "continuous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of the oligonucleotide are present in the continuous nucleotide region. In some embodiments, the oligonucleotide comprises a continuous nucleotide region and optionally includes other nucleotides, such as nucleotide linker regions that can be used to link functional groups to the continuous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the internucleotide links between nucleotides present in the continuous nucleotide region are all phosphate thioside links. In some embodiments, the continuous nucleotide region comprises one or more sugar-modified nucleosides.

Nucleotides

Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of this invention, nucleotides include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides such as DNA and RNA nucleotides contain a ribose moiety, a nucleobase moiety, and one or more phosphate groups (which are absent in nucleosides). Nucleosides and nucleotides may also be interchangeably referred to as "units" or "monomers".

Modified nucleosides

As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified compared to an equivalent DNA or RNA nucleoside by introducing one or more modifications to a sugar moiety or (nucleo)base moiety. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the terms "nucleoside analogue" or modified "unit" or modified "monomer".

Modified nucleoside interlinking

As is generally understood by those skilled in the art, the term "modified internucleotide link" is defined as a link other than a phosphodiester (PO) link that covalently couples two nucleosides together. Nucleotides having modified internucleotide links are also referred to as "modified nucleotides." In some embodiments, modified internucleotide links increase the nuclease resistance of oligonucleotides compared to phosphodiester links. For naturally occurring oligonucleotides, internucleotide links comprise phosphate groups that form phosphodiester bonds between adjacent nucleosides. Modified internucleotide links are particularly suitable for stabilizing oligonucleotides for in vivo use and can be used to protect against nuclease cleavage of DNA or RNA nucleoside regions in the oligonucleotides of the present invention (e.g., within the nick region of a nick-body oligonucleotide and within the modified nucleoside region).

In one embodiment, the oligonucleotide comprises one or more inter-nucleoside links modified from a natural phosphodiester to, for example, links that are more resistant to nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both of which are well known in the art. Inter-nucleoside links capable of enhancing the nuclease resistance of the oligonucleotide are referred to as nuclease-resistant inter-nucleoside links. In some embodiments, all inter-nucleoside links of the oligonucleotide or their sequential nucleotide sequence are modified. It should be recognized that in some embodiments, the nucleoside linking the oligonucleotide of the present invention to a non-nucleotide functional group (e.g., a conjugate) can be a phosphodiester. In some embodiments, all inter-nucleoside links of the oligonucleotide or their sequential nucleotide sequence are nuclease-resistant inter-nucleoside links.

In some embodiments, the modified nucleoside linker may be a phosphate thioside linker. In some embodiments, the modified nucleoside linker is compatible with the RNase H recruitment of the oligonucleotides of the present invention, such as phosphate thiosides.

In some implementations, the nucleoside linkage includes sulfur (S), such as a thiophosphate nucleoside linkage.

Nucleoside linkages of thiophosphates are particularly useful due to their nuclease resistance, beneficial pharmacokinetics, and ease of manufacture. In some embodiments, all nucleoside linkages of the oligonucleotides, or their continuous nucleotide sequence, are thiophosphates.

base

The term nucleobase includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of this invention, the term nucleobase also includes modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this document, “nucleobase” refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. These variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine (e.g., a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazono-cytosine, 5-propynyl-cytosine, 5-propynyluracil, 5-bromouracil, 5-thiazono-uracil, 2-thiouracil, 2′-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine).

The nucleobase moiety can be represented by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, where each letter may optionally include a modified nucleobase with an equivalent function. For example, in an exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, for the LNA nick body, a 5-methylcytosine LNA nucleoside can be used. In some embodiments, the cytosine nucleobase in the 5′cg3′ motif is 5-methylcytosine.

Modified oligonucleotides

The term "modified oligonucleotide" describes an oligonucleotide containing one or more sugar-modified nucleosides and/or modified nucleoside linkages. The term "chimeric oligonucleotide" is used in the literature to describe oligonucleotides having modified nucleosides.

Complementarity

The term complementarity describes the ability of a nucleoside/nucleotide to form a Watson-Crick base pair. A Watson-Crick base pair is guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It should be understood that oligonucleotides can contain nucleosides with modified nucleosides, such as 5-methylcytosine often used instead of cytosine; therefore, the term complementarity includes Watson-Crick base pairing between unmodified and modified nucleosides (see, for example, Hirao et al. (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

As used herein, the term "%complementarity" refers to the percentage of nucleotides in a continuous nucleotide region or sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is complementary (i.e., forms a Watson-Crick base pair) to a continuous nucleotide sequence at a given position in a separate nucleic acid molecule (e.g., a target nucleic acid). The percentage is calculated by counting the number of aligned bases that form a pair between the two sequences, dividing by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In this comparison, misaligned (non-pairing) nucleobases/nucleotides are referred to as mismatches.

It should be understood that when referring to complementarity between two sequences, the determination of complementarity is measured at the length of the shorter of the two sequences (e.g., the length of a continuous nucleotide region or sequence).

The term "perfectly complementary" means 100% complementarity. In the absence of a % value or mismatch indication, complementarity means perfect complementarity.

identity

As used herein, the term "identity" refers to the percentage of consecutive nucleotide sequences in a nucleic acid molecule (e.g., an oligonucleotide) that are identical at a given position to the consecutive nucleotide sequences at a given position in a separate nucleic acid molecule (e.g., a target nucleic acid) (i.e., in terms of their ability to form Watson-Crick base pairs with complementary nucleosides). The percentage is calculated by dividing the number of identical aligned bases (including gaps) between the two sequences by the total number of nucleotides in the oligonucleotide and multiplying by 100. Identity percentage = (matches x 100) / length of the aligned region (with gaps).

When determining the identity of a continuous nucleotide region of an oligonucleotide, identity is calculated over the length of the continuous nucleotide region. In embodiments where the entire continuous nucleotide sequence of the oligonucleotide is a continuous nucleotide region, identity is therefore calculated over the length of the nucleotide sequence of the oligonucleotide. In this respect, the continuous nucleotide region may be identical to a region of a reference nucleic acid sequence, or in some embodiments, it may be identical to the entire reference nucleic acid. Unless otherwise stated, a sequence having 100% identity with a reference sequence is referred to as identical. For example, the reference sequence may be selected from any of the following groups: SEQ ID NOs 5-146 and 156.

However, if the oligonucleotide contains additional nucleotides flanking a continuous nucleotide region (e.g., region D′ or D″), these additional flanking nucleotides can be ignored when determining identity. In some embodiments, identity can be calculated across the entire oligonucleotide sequence.

In some embodiments, the antisense oligonucleotide of the present invention comprises a continuous nucleotide region of 10-22 consecutive nucleotides, which is identical to SEQ ID NO 156:

SEQ ID NO 156：5′CAAATATTTACCTGGTTGTTGG 3′

In some embodiments, the continuous nucleotide region comprises or consists of at least 10 continuous nucleotides of SEQ ID NO 156, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 continuous nucleotides, such as 12-22, or 14-18 continuous nucleotides. In some embodiments, the entire continuous sequence of the oligonucleotide comprises or consists of at least 10 continuous nucleotides of SEQ ID NO 156, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 continuous nucleotides, such as 12-22, or 14-18 continuous nucleotides.

In some embodiments, the continuous nucleotide region is at least 12 consecutive nucleotides of SEQ ID NO 156. In some embodiments, the continuous nucleotide region is at least 14 consecutive nucleotides of SEQ ID NO 156. In some embodiments, the continuous nucleotide region is at least 16 consecutive nucleotides of SEQ ID NO 156.

In some embodiments, the continuous nucleotide region is at least 10, 12, 14 or 16 consecutive nucleotides identical to SEQ ID NO 143.

In some embodiments, the continuous nucleotide region is at least 10, 12, 14 or 16 consecutive nucleotides identical to SEQ ID NO 145.

In some embodiments, the continuous nucleotide region is at least 10, 11, 12, 13, 14, 15 or 16 consecutive nucleotides of SEQ ID NO 143.

In some embodiments, the continuous nucleotide region is at least 10, 11, 12, 13, 14, 15, 16 or 17 consecutive nucleotides identical to SEQ ID NO 145.

In some embodiments, the continuous nucleotide consists of or contains SEQ ID NO 143.

In some implementations, the continuous nucleotide region consists of or contains SEQ ID NO 145.

In some embodiments, the continuous nucleotide region is at least 10, 12, 14, or 16 consecutive nucleotides identical to the sequence selected from the group consisting of SEQ ID NO 138, 139, 140, 141, 142, 143, 144, and 145. In some embodiments, the continuous nucleotide region comprises or consists of the sequence selected from or consisting of the group consisting of SEQ ID NO 138, 139, 140, 141, 142, 143, 144, and 145.

In some implementations, the continuous nucleotide region contains the sequence SEQ ID NO 146: TTTACCTGGTT.

The present invention provides antisense oligonucleotides with a length of 11-30 nucleotides, such as 12-20 nucleotides, comprising the sequence SEQ ID NO 146: TTTACCTGGTT.

Hybridization

As used herein, the terms “hybridization” or “crossover” should be understood as the formation of hydrogen bonds between base pairs on opposite strands of two nucleic acid chains (e.g., an oligonucleotide and a target nucleic acid), resulting in a doublet. The affinity between the two nucleic acid chains is the strength of the hybridization. It is usually described by the melting temperature (T_{_m} ), defined as the temperature at which half of the oligonucleotide and target nucleic acid are double-stranded. Under physiological conditions, T _{_m} is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13: 515-537). The standard-state Gibbs free energy ΔG is a more accurate representation of the binding affinity and is related to the dissociation constant (K_{_d} ) of the reaction by ΔG = -RTln(K_{_d}), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG for the reaction between the oligonucleotide and the target nucleic acid reflects a strong hybridization between them. ΔG is the energy associated with a reaction at an aqueous solution concentration of 1 M, pH 7, and temperature of 37 °C. Hybridization of oligonucleotides with target nucleic acids is a spontaneous reaction, and for spontaneous reactions, ΔG is less than zero. ΔG can be measured experimentally, for example, by using isothermal titration calorimetry (ITC) as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercial equipment is available for ΔG measurement. ΔG can also be numerically estimated using the nearest neighbor model described in Santa Lucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465, using appropriately derived thermodynamic parameters as described in Sugimoto et al., 1995, Biochemistry 34: 11211-11216 and McTigue et al., 2004, Biochemistry 43: 5388-5405. To enable the possibility of modulating their intended nucleic acid targets through hybridization, the oligonucleotides of the present invention hybridize with the target nucleic acid, and for oligonucleotides of 10-30 nucleotides in length, the estimated ΔG value is less than -10 kcal. In some embodiments, the extent or intensity of hybridization is measured by the standard-state Gibbs free energy ΔG. The oligonucleotides can hybridize with the target nucleic acid, wherein for oligonucleotides of 8-30 nucleotides in length, the estimated ΔG value is less than -10 kcal, for example less than -15 kcal, for example less than -20 kcal, and for example less than -25 kcal. In some embodiments, the oligonucleotides hybridize with the target nucleic acid, and the estimated ΔG value is -10 to -60 kcal, for example -12 to -40 kcal, for example -15 to -30 kcal, or -16 to -27 kcal, for example -18 to -25 kcal.

target sequence

The oligonucleotide comprises a continuous nucleotide region complementary to or hybridized to a subsequence of the target nucleic acid molecule. As used herein, the term "target sequence" refers to a nucleotide sequence present in the target nucleic acid that comprises a nucleobase sequence complementary to a continuous nucleotide region or sequence of the oligonucleotide of the present invention. In some embodiments, the target sequence consists of a region on the target nucleic acid that is complementary to a continuous nucleotide region or sequence of the oligonucleotide of the present invention. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide and may, for example, represent a preferred region of the target nucleic acid that can be targeted by several oligonucleotides of the present invention.

The oligonucleotides of the present invention comprise a continuous nucleotide region complementary to a target nucleic acid, such as a target sequence.

An oligonucleotide comprises a continuous nucleotide region of at least 10 nucleotides that is complementary to or hybridizes to a target sequence present in a target nucleic acid molecule. The continuous nucleotide region (and therefore the target sequence) comprises at least 10 consecutive nucleotides, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides, such as 12-22 or 14-18 consecutive nucleotides.

In some implementations, the target sequence is or is present in SEQ ID NO 147.

In some implementations, the target sequence is selected from the group consisting of: SEQ ID NO 148, 149, 150, 151, 152, 153, 154 and 155:

SEQ ID NO 148：AACAACCAGGTAAATA

SEQ ID NO 149：CAACCAGGTAAATATTTG

SEQ ID NO 150: CCAACAACCAGGTAAA

SEQ ID NO 151：AACCAGGTAAATATTTGG

SEQ ID NO 152：ACAACCAGGTAAATATTTGG

SEQ ID NO 153: CAACAACCAGGTAAATAT

SEQ ID NO 154：ACAACCAGGTAAATAT

SEQ ID NO 155：AACAACCAGGTAAATAT

The present invention provides antisense oligonucleotides of 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide region of at least 10 consecutive nucleotides that are complementary to a sequence selected from the sequences of SEQ ID NO 147 & 148-155.

The present invention provides antisense oligonucleotides of 12-30 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide region of at least 12 consecutive nucleotides that is complementary to a sequence selected from the sequences of SEQ ID NO 147 & 148-155.

The present invention provides antisense oligonucleotides of 14-30 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide region of at least 14 consecutive nucleotides complementary to a sequence selected from sequences of SEQ ID NO 147 & 148-155.

The present invention provides antisense oligonucleotides comprising or consisting of a continuous nucleotide region complementary to or composed of a sequence selected from SEQ ID NO 148-155.

The target sequence can be a subsequence of the target nucleic acid. In some embodiments, the oligonucleotide or continuous nucleotide region is completely complementary to the HTRA1 subsequence (e.g., a sequence selected from the group consisting of SEQ ID NO 148-154) or contains only one or two mismatches. In some embodiments, the oligonucleotide or continuous nucleotide region is completely complementary to the HTRA1 subsequence SEQ ID NO 147 or contains only one or two mismatches.

target cells

As used herein, the term target cell refers to a cell that expresses a target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell is a mammalian cell, such as rodent cells, such as mouse cells, or rat cells, or a primate cell, such as monkey cells or human cells. In some embodiments, the cell may be a pig cell, dog cell, or rabbit cell. In some embodiments, the target cell may be a retinal cell, such as retinal pigment epithelial (PRE) cells. In some embodiments, the cell is selected from the group consisting of: RPE cells, bipolar cells, amacrine cells, endothelial cells, ganglion cells, and microglia. For in vitro evaluation, the target cell may be a primary cell or an established cell line, such as U251, ARPE19, HEK293, or rat C6 cells.

target nucleic acid

According to the present invention, the target nucleic acid is a nucleic acid encoding mammalian HTRA1, and can be, for example, a gene, RNA, mRNA and premRNA, mature mRNA, or cDNA sequence. Therefore, the target can be referred to as the HTRA1 target nucleic acid.

Suitable, the target nucleic acid encodes the HTRA1 protein, particularly mammalian HTRA1, such as human HTRA1 (see, for example, Tables 1 & 2, which provide mRNA and pre-mRNA sequences of human and rat HTRA1).

In some implementations, the target nucleic acid is selected from the group consisting of: SEQ ID NO: 1, 2, 3 and 4, or naturally occurring variants thereof (e.g., sequences encoding mammalian HTRA1 proteins).

The target cell is a cell that expresses the HTRA1 target nucleic acid. In a preferred embodiment, the target nucleic acid is HTRA1 mRNA, such as HTRA1 pre-mRNA or mature HTRA1 mRNA. For antisense oligonucleotide targeting, the polyA tail of HTRA1 mRNA is typically ignored.

If the oligonucleotides of the present invention are used in research or diagnosis, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

The target sequence can be a subsequence of the target nucleic acid. In some embodiments, the oligonucleotide or continuous nucleotide region is completely complementary to the HTRA1 subsequence (e.g., a sequence selected from the group consisting of SEQ ID NO 148, 149, 150, 151, 152, 153, 154 and 155) or contains only one or two mismatches.

Complementarity with the target or its subsequence is measured along the length of an oligonucleotide or its continuous nucleotide region.

For in vivo or in vitro applications, the oligonucleotides of the present invention are generally capable of inhibiting the expression of HTRA1 target nucleic acids in cells expressing HTRA1 target nucleic acids. As measured over the entire length of the oligonucleotide, the sequential nucleobase sequence of the oligonucleotides of the present invention is generally complementary to the HTRA1 target nucleic acid, optionally except for one or two mismatches, and optionally excluding nucleotide-based linker regions or other non-complementary terminal nucleotides (e.g., region D) that can link the oligonucleotide to optional functional groups such as conjugates. In some embodiments, the target nucleic acid may be RNA or DNA, such as messenger RNA, such as mature mRNA or premRNA. In some embodiments, the target nucleic acid is RNA or DNA encoding a mammalian HTRA1 protein, such as human HTRA1, such as the human HTRA1 mRNA sequence disclosed in, for example, SEQ ID NO 1 (NM_002775.4, GI: 190014575). Further information regarding exemplary target nucleic acids is provided in Tables 1 and 2.

Table 1. Genomic and assembly information of HTRA1 in humans and rats

Fwd = Forward strand. Genomic coordinates provide the pre-mRNA sequence (genomic sequence). NCBI reference provides the mRNA sequence (cDNA sequence).

The National Center for Biotechnology Information Reference Sequence Database is a comprehensive, integrated, non-redundant, and well-annotated set of reference sequences, including genomes, transcripts, and proteins. Its server is located at www.ncbi.nlm.nih.gov/refseq.

Table 2. Detailed sequence information of HTRA1 in humans and rats

Naturally occurring variants

The term "naturally occurring variant" refers to a variant of the HTRA1 gene or transcript that originates from the same genetic locus as the target nucleic acid, but differs for example due to: degeneracy of the genetic code (resulting in diversity of codons encoding the same amino acids), or due to alternative splicing of the pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms and allelic variants. Based on the presence of sufficiently complementary sequences in the oligonucleotides, the oligonucleotides of the present invention can therefore target the target nucleic acid and its naturally occurring variants. In some embodiments, the naturally occurring variants have at least 95%, for example, at least 98% or at least 99% homology to the mammalian HTRA1 target nucleic acid (e.g., a target nucleic acid selected from the group consisting of SEQ ID NO 1, 2, 3, or 4).

Regulation of expression

As used herein, the term "regulation of expression" should be understood as the collective ability of an oligonucleotide to alter the amount of HTRA1 compared to the amount of HTRA1 prior to application of the oligonucleotide. Alternatively, regulation of expression can be determined by referring to control experiments in which the oligonucleotides of the present invention are not applied. One type of regulation is the ability of an oligonucleotide, for example, to inhibit, downregulate, reduce, repress, remove, block, prevent, mitigate, reduce, avoid, or terminate HTRA1 expression by means of mRNA degradation or transcriptional blockade. The antisense oligonucleotides of the present invention are capable of inhibiting, downregulating, reducing, repressing, removing, blocking, preventing, mitigating, reducing, avoiding, or terminating HTRA1 expression.

Nucleosides modified with high affinity

High-affinity modified nucleosides are modified nucleotides that, when incorporated into oligonucleotides, enhance the affinity of the oligonucleotide for its complementary target, as measured by, for example, melting temperature (Tm ). The high-affinity modified nucleosides of the present invention preferably result in an increase in melting temperature of +0.5 to +12 °C, more preferably +1.5 to +10 °C, and most preferably +3 °C to +8 °C for the modified nucleoside. Many high-affinity modified nucleosides are known in the art, including, for example, many 2′-substituted nucleosides and locked nucleic acids (LNAs) (see, for example, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

Sugar modification

The oligomers of the present invention may contain one or more nucleosides having a modified sugar moiety, i.e., a modification of the sugar moiety compared to the ribosomes found in DNA and RNA.

Many nucleosides with ribose modifications have been prepared, primarily to improve certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

These modifications include those in which the ribocyclic structure is modified (e.g., by substitution with a hexose ring (HNA) or a bicyclic ring), which typically has a bibasic bridge between the C2 and C4 carbons on the ribocyclic ring (LNA), or an unconnected ribocyclic ring (e.g., UNA) that typically lacks a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include those in which, for example, in the case of peptide nucleic acids (PNA) or morpholino nucleic acids, the sugar moiety is substituted with a non-sugar moiety.

Sugar modification also includes modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to 2′-OH groups naturally present in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2′, 3′, 4′, or 5′ positions. Nucleosides having modified sugar moieties also include 2′-modified nucleosides, such as 2′-substituted nucleosides. In fact, much attention has been focused on developing 2′-substituted nucleosides, and many 2′-substituted nucleosides have been found to possess beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.

2′ modified nucleosides

Nucleosides modified with 2′ sugars are those that have a substituent other than H or -OH at the 2′ position (2′-substituted nucleosides) or contain a 2′-linked bimolecular group, including 2′-substituted nucleosides and LNA (2′-4′ bimolecular bridging) nucleosides. For example, 2′-modified sugars can provide oligonucleotides with enhanced binding affinity and/or increased nuclease resistance. Examples of 2′-substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-fluoro-RNA, and 2′-F-ANA nucleosides. For more examples, please see, for instance, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2′-substituted nucleosides.

Locking in nucleoside (LNA).

LNA nucleotides are modified nucleotides containing a linking group (called a biradicle or bridge) between the C2′ and C4′ of the ribose ring of the nucleotide. These nucleotides are also referred to in the literature as bridging nucleic acids or bicyclic nucleic acids (BNA).

In some embodiments, the modified nucleosides or LNA nucleosides of the oligomers of the present invention have the general structure of formula I or II:

Where W is selected from -O-, -S-, -N( ^Ra )-, -C( ^{RaRb )} -, for example, in some embodiments it is selected from -O-;

B represents the nucleobase portion;

Z indicates a nucleoside linker to an adjacent nucleoside or a 5′ terminal group;

Z* indicates a nucleoside linker to an adjacent nucleoside or a 3′ terminal group;

X represents a group selected from the list of groups consisting of: -C( ^RaRb )-, ^-C ( ^Ra )＝C(Rb)-, ^-C ( ^Ra )＝N-, -O-, -Si( ^Ra ) _2- , -S-, _-SO2- , -N( ^Ra )-, and >C＝Z.

In some implementations, X is selected from the group consisting of: -O-, -S-, NH-, NR ^{a Rb} , _-CH2- , CR ^{a Rb} , -C(＝ _CH2 )-, and -C(＝CR ^{a Rb} )-

In some implementations, X is -O-

Y represents a group selected from the following groups: -C( ^RaRb )-, -C( ^Ra )＝C( ^Rb )-, ^-C ( ^Ra )＝N-, -O-, -Si( ^Ra ) _2- , -S-, _-SO2- , -N( ^Ra )-, and >C＝Z.

In some implementations, Y is selected from the group consisting of: _-CH₂- , -C( ^RaRb )-, _-CH₂CH₂- , -C ₍ ^RaRb )-C( ^{RaRb )} -, _{-CH₂CH₂CH₂-} , ^-C ( ^RaRb )C( _RaRb )C( ^{RaRb )} -, -C ⁽ _Ra ⁾ ＝C( ^Rb )-, ^and -C( ^{Ra ) ＝} N-

In some implementations, Y is selected from ^the group consisting of: _-CH2- , ^-CHRa- , _-CHCH3- , ^CRaRb-

The prefix -XY- together indicates a divalent linker group (also called a group). Together, they represent a divalent linker group consisting of 1, 2, or 3 groups/atoms, selected from the group consisting of: ^-C ( ^RaRb )-, -C( ^Ra )＝C( ^Rb )-, -C( ^Ra )＝N-, -O-, -Si( ^Ra ) _2- , -S-, -SO2-, -N( ^Ra )-, and _> C＝Z.

In some implementations, -XY- represents a bibase selected from the group consisting of: -X- _CH2- , -X- ^CRaRb- , -X- ^CHRa- , -XC( _HCH3 ) ^- , _-OY- , -O-CH2-, -S- ^CH2- , -NH- _CH2- , _-O - _CHCH3- , -CH2-O-CH2, -O-CH( _CH3CH3 )-, _-O - _CH2 - _CH2- , _OCH2 - _CH2 - _CH2- , -O- _CH2OCH2- , _-O - _NCH2- , -C(＝ _CH2 ) _-CH2- , -NRa _{- CH2-} , _{NO-CH2 ,} -S- ^CRaRb- , ^{and -S} - ^CHRa- .

In some implementations, -XY- represents -O- _CH2- or -O-CH( _CH3 )-.

Where Z is selected from -O-, -S-, and -N( ^Ra )-.

Furthermore, ^Ra and ^Rb , when present, are each independently selected from hydrogen, optionally substituted _C1-6 -alkyl, optionally substituted _C2-6 -alkenyl, optionally substituted _C2-6 -alkynyl, hydroxyl, optionally substituted _C1-6 -alkoxy, _C2-6 -alkoxyalkyl, _C2-6 -alkenyloxy, carboxyl, _C1-6 -alkoxycarbonyl, _C1-6 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di( _C1-6 -alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino- _C1-6 -alkyl-aminocarbonyl, mono- and di( _C1-6 -alkyl)amino _{-C1-6 -} alkyl-aminocarbonyl, _C1-6 -alkyl-carbonylamino, urea, _C1-6 -alkanoyloxy, sulfonyl, _C1-6 -alkylsulfonyloxy, nitro, azide, thioalkyl, _C1-6 -alkylthio, halogen, wherein aryl and heteroaryl groups may optionally be substituted, and wherein two twin substituents ^Ra and ^Rb together may represent an optionally substituted methylene (= _CH2 ), wherein for all chiral centers, an asymmetric group in the R or S orientation may be found.

^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are independently selected from the group consisting of: hydrogen, optionally substituted _C1-6 alkyl, optionally substituted _C2-6 alkenyl, optionally substituted _C2-6 alkynyl, hydroxyl, _C1-6 alkoxy, _C2-6 alkoxyalkyl, _C2-6 alkenyloxy, carboxyl, _C1-6 alkoxycarbonyl, _C1-6 alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di( _C1-6 -alkyl)amino, carbamoyl, mono- and di( _C1-6 -alkyl)amino-carbonyl, amino- _C1-6 -alkyl-aminocarbonyl, mono- and di( _C1-6 -alkyl)amino- _C1-6 -alkyl-aminocarbonyl, _C1-6 -alkyl-carbonylamino, ureo, _C1-6 -alkanoyloxy, sulfonyl, _C1-6 -alkylsulfonyloxy, nitro, azide, thioalkyl, _C1-6 -alkylthio, halogen, wherein the aryl and heteroaryl groups may optionally be substituted, and wherein the two twin substituents together may represent oxo, thio, imino, or optionally substituted methylene.

In some embodiments, ^R1 , ^R2 , ^R3 , ^R5 and R5 ^* are independently selected from _C1-6 alkyl groups, such as methyl and hydrogen.

In some implementations, ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are all hydrogen.

In some embodiments, ^R1 , ^R2 , and ^R3 are all hydrogen, and one of ^R5 and R5 ^* is hydrogen and the other of ^R5 and R5 ^* is not hydrogen, such as a _C1-6 alkyl group, such as methyl.

In some embodiments, ^Ra is hydrogen or methyl. In some embodiments, ^Rb is hydrogen or methyl when present.

In some implementations, one or both of ^Ra and ^Rb are hydrogen.

In some implementations, one of ^Ra and ^Rb is hydrogen, and the other is not hydrogen.

In some implementations, one of ^Ra and ^Rb is a methyl group, and the other is hydrogen.

In some implementations, both ^Ra and ^Rb are methyl groups.

In some embodiments, the di-XY- is -O- _CH2- , W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. This LNA nucleotide is disclosed in WO99/014226, WO00/66604, WO98/039352, and WO2004/046160 (all of which are incorporated herein by reference) and includes substances commonly referred to as β-D-oxyLNA and α-L-oxyLNA nucleotides.

In some embodiments, the di-XY- is -S- _CH2- , W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. This thio-LNA nucleoside is disclosed in WO99/014226 and WO2004/046160, which are incorporated herein by reference.

In some embodiments, the di-XY- is -NH- _CH2- , W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. Such amino-LNA nucleosides are disclosed in WO99/014226 and WO2004/046160, which are incorporated herein by reference.

In some embodiments, the di-XY- is -O- _CH2 - _CH2- or -O- _CH2 - _CH2 - _CH2- , W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. This LNA nucleoside is disclosed in WO00/047599 and Morita et al., Bioorganic & Med. Chem. Lett. 1273-76 (which are incorporated herein by reference) and includes a compound commonly referred to as 2′-O-4′C-ethylidene bridging nucleic acid (ENA).

In some embodiments, the di-XY- is -O- _CH2- , W is O, and one of all of ^R1 , ^R2 , ^R3 and ^R5 and R5 ^* is hydrogen, and the other of ^R5 and R5 ^* is not hydrogen, such as a _C1-6 alkyl group, like methyl. Such 5′-substituted LNA nucleosides are disclosed in WO2007/134181 (which is incorporated herein by reference).

In some embodiments, the bis-XY- is -O- ^CRaRb- , wherein one or both of ^Ra and ^Rb are not hydrogen, for ^example , methyl, W is O, and all of ^R1 , ^R2 , ^R3 and one of ^R5 and R5 ^* are hydrogen, and the other of ^R5 and R5 ^* is not hydrogen, for example, _C1-6 alkyl, such as methyl. Such a bis-modified LNA nucleoside is disclosed in WO2010/077578 (which is incorporated herein by reference).

In some embodiments, bis-XY- represents the divalent linker group -O-CH( _CH₂OCH₃ )-(2′O _- methoxyethyl bicyclic nucleic acid -Seth et al., 2010, J.Org.Chem.Vol 75(5)pp.1569-81). In some embodiments, bis-XY- is -O-CHR ^a- , W is O, and all ^R₁ , ^R₂ _, ^R₃ , _R₅ and ^{R₅ *} are hydrogen. This 6′-substituted LNA nucleoside is disclosed in WO10036698 and WO07090071 (both of which are incorporated herein by reference).

In some embodiments, the di-XY- is -O-CH( _CH₂OCH₃ )-, W is O, and all ^R₁ , ^R₂ , ^R₃ , ^R₅ _, and R₅ ^* are hydrogen. This LNA nucleoside is also known in the art as a cyclic MOE (cMOE) and is disclosed in WO07090071.

In some embodiments, the di-XY- represents the divalent linker group -O-CH( _CH3 )-.- in the R- or S- configuration. In some embodiments, the di-XY- together represent the divalent linker group -O- _CH2 -O-CH2- (Seth at al., 2010, J.Org.Chem). In some embodiments, the di-XY- is -O-CH( _CH3 )-, W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. This 6′-methyl-LNA nucleoside is also known in the art as a cET nucleoside and can be a (S)cET or (R)cET stereoisomer, as disclosed in WO07090071 (β-D) and WO2010/036698 (α-L) (both of which are incorporated herein by reference).

In some embodiments, the di ^- XY- is -O- ^CRaRb- , wherein neither ^Ra nor ^Rb is hydrogen, W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. In some embodiments, both ^Ra and ^Rb are methyl groups. This 6′-disubstituted LNA nucleoside is disclosed in WO 2009006478 (which is incorporated herein by reference).

In some embodiments, the di-XY- is -S-CHR ^α- , W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. This 6′-substituted thio-LNA nucleoside is disclosed in WO11156202 (which is incorporated herein by reference). In some embodiments of the 6′-substituted thio-LNA, ^Ra is methyl.

In some embodiments, the di-XY- is -C(＝CH2)-C( ^{RaRb )} -, for example -C(＝ _CH2 ) _-CH2- , or -C(＝ _CH2 )-CH( _CH3 )-, W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. Such vinyl-carbon-supported LNA nucleosides are disclosed in WO08154401 and WO09067647 (both of which are incorporated herein by reference).

In some embodiments, the di-XY- is -N(-OR ^a )-, W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. In some embodiments, ^Ra is a _C1-6 alkyl group, such as methyl. This LNA nucleoside is also known as N-substituted LNA and is disclosed in WO2008/150729 (which is incorporated herein by reference). In some embodiments, the di-XY- together represent the divalent linker group -O-NR ^a - _CH3- (Seth et al., 2010, J.Org.Chem). In some embodiments, the di-XY- is -N( ^Ra )-, W is O, and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. In some embodiments, ^Ra is a _C1-6 alkyl group, such as methyl.

In some embodiments, one or both of ^R5 and R5 ^* are hydrogen, and when substituted, the other of ^R5 and R5 ^* is a _C1-6 alkyl group, such as methyl. In such embodiments, ^R1 , ^R2 , and ^R3 may all be hydrogen, and the di-XY- may be selected from -O-CH2- or -OC( ^HCRa )-, for example -OC(HCH3)-.

In some embodiments, the ^bibase is ^-CRaRb -O- ^CRaRb- , for example _CH2 -O- ^CH2- , where W is O and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. In some embodiments, ^Ra is a _C1-6 alkyl group, such as methyl. This LNA nucleotide is also known as a conformation- _restricted nucleotide (CRN) and is disclosed in WO2013036868 (which is incorporated herein by reference).

In some embodiments, the bibase is -O- ^CRaRb - ^O - ^CRaRb- , for example O- _CH2 -O- _CH2- , ^where W is O and all ^R1 , ^R2 , ^R3 , ^R5 , and R5 ^* are hydrogen. In some embodiments, ^Ra is a _C1-6 alkyl group, such as methyl. This LNA nucleotide is also known as a COC nucleotide and is disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238 (which is incorporated herein by reference).

Unless otherwise stated, it will be recognized that LNA nucleosides can be β-D or α-L stereoisomers.

Examples of LNA nucleosides are given in Scheme 1.

Option 1

As shown in the examples, in some embodiments of the present invention, the LNA nucleoside in the oligonucleotide is β-D-oxy-LNA nucleoside.

nuclease-mediated degradation

Nuclease-mediated degradation refers to oligonucleotides that can mediate the degradation of a complementary nucleotide sequence when forming a double strand with it.

In some embodiments, the oligonucleotides function through nuclease-mediated target nucleic acid degradation, wherein the oligonucleotides of the present invention are capable of recruiting nucleases, particularly endonucleases, preferably RNases, such as RNase H. Examples of oligonucleotide designs that function through a nuclease-mediated mechanism are oligonucleotides that typically contain regions of at least 5 or 6 DNA nucleosides and are side-joined with affinity-enhancing nucleosides on one or both sides, such as nick bodies, head bodies, and tail bodies.

RNase H activity and recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H in the duplex of a complementary RNA molecule. WO01/23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. An oligonucleotide is generally considered capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, the oligonucleotide has an initial rate of at least 5%, for example at least 10%, or more than 20%, as measured in pmol/L/min, using an oligonucleotide (which has the same base sequence as the modified oligonucleotide being tested but contains only DNA monomers, with phosphate thioester bonds between all monomers) and the method provided in Examples 91-95 of WO01/23613 (incorporated herein by reference).

Gapmer

As used herein, the term nick body refers to an antisense oligonucleotide containing an RNase H-recruiting oligonucleotide region (nick), with its 5′ and 3′ flanked by regions containing one or more affinity-enhancing modified nucleosides (flanks or wings). Various nick body designs are described herein. Head bodies and tail bodies are oligonucleotides capable of recruiting RNase H, with one flanked region missing, meaning only one end of the oligonucleotide contains an affinity-enhancing modified nucleoside. For head bodies, the 3′ flanked region is missing (i.e., the 5′ flanked region contains an affinity-enhancing modified nucleoside), and for tail bodies, the 5′ flanked region is missing (i.e., the 3′ flanked region contains an affinity-enhancing modified nucleoside).

LNA notch

The term LNA nick body is a nick body oligonucleotide in which at least one of the affinity-enhancing modified nucleosides is an LNA nucleoside.

Hybrid wing notch

The term "mixed-wing notched body" refers to an LNA notched body in which the flanking regions contain at least one LNA nucleoside and at least one non-LNA-modified nucleoside, such as at least one DNA nucleoside or at least one 2′-substituted modified nucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-fluoro-RNA, and 2′-F-ANA nucleoside. In some embodiments, the mixed-wing notched body has one flanking region containing an LNA nucleoside (e.g., 5′ or 3′) and another flanking region (3′ or 5′, respectively) containing a 2′-substituted modified nucleoside.

Conjugate

As used in this article, a conjugate refers to an oligonucleotide covalently linked to a nonnucleotide portion (conjugate portion or region C or third region).

As used in this article, a conjugate refers to an oligonucleotide covalently linked to a nonnucleotide portion (conjugate portion or region C or third region).

In some embodiments, the nonnucleotide portion is selected from the group consisting of: proteins, such as enzymes, antibodies or antibody fragments or peptides; lipophilic portions, such as lipids, phospholipids, sterols; polymers, such as polyethylene glycol or polypropylene glycol; receptor ligands; small molecules; reporter molecules; and nonnucleoside carbohydrates.

connector

A linkage, or linker, is a connection between two atoms that links one chemical group or target fragment to another via one or more covalent bonds. The conjugate moiety can be linked to an oligonucleotide directly or through a linker (e.g., a linker or connector). Linkers are used for covalently linking a third region, for example, linking the conjugate moiety to the end of an oligonucleotide (e.g., the region A or C).

In some embodiments of the invention, the conjugates or oligonucleotide conjugates of the invention may optionally include a linker region located between the oligonucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and the oligonucleotide is biocleavable.

A biocleavable adapter comprises or is composed of physiologically unstable bonds that are cleavable under conditions commonly encountered or similar to those encountered in mammals. Conditions under which a physiologically unstable adapter undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reducing conditions or reagents, and salt concentrations similar to those encountered in mammalian cells. Intracellular mammalian conditions also include enzymatic activities commonly present in mammalian cells, such as the activities of proteolytic enzymes or hydrolases or nucleases. In one embodiment, the biocleavable adapter is susceptible to cleavage by an S1 nuclease. In a preferred embodiment, the nuclease-sensitive adapter comprises 1 to 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably 2 to 6 nucleosides, and most preferably 2 to 4 linked nucleosides comprising at least two consecutive phosphodiester links, such as at least 3, 4, or 5 consecutive phosphodiester links. Preferably, the nucleosides are DNA or RNA. Phosphodiester containing a bio-cuttable connector is described in more detail in WO2014/076195 (incorporated hereby by reference) and may be referred to herein as Region D.

The conjugate can also be linked to the oligonucleotide via a non-biologically cleavable linker, or in some embodiments, the conjugate may contain an incleavable linker covalently attached to a biologically cleavable linker. The linker does not necessarily have to be biologically cleavable, but is primarily used to covalently link the conjugate portion to the oligonucleotide or a biologically cleavable linker. Such a linker may comprise an oligomer of a chain structure or repeating unit (e.g., ethylene glycol, amino acid unit, or aminoalkyl). In some embodiments, the linker (region Y) is an aminoalkyl, such as a _C2 - _C36 aminoalkyl, including, for example, _C6 to _C12 aminoalkyl. In some embodiments, the linker (region Y) is a _C6 aminoalkyl. The conjugate linker group can be conventionally attached to the oligonucleotide using an amino-modified oligonucleotide and an activated ester group on the conjugate group.

treat

As used herein, the term 'treatment' refers to treating an existing disease (such as the disease or condition described herein) or preventing disease, i.e., prevention. Therefore, it will be appreciated that in some implementations, the treatment mentioned herein can be preventative.

Invention Details

The oligonucleotides of the present invention

This invention relates to oligonucleotides capable of inhibiting HTRA1 expression. Regulation can be achieved by hybridization with a target nucleic acid encoding HTRA1 or involved in the regulation of HTRA1. The target nucleic acid can be a mammalian HTRA1 sequence, such as a sequence selected from the group consisting of SEQ ID 1, 2, 3 or 4.

The oligonucleotides of the present invention are antisense oligonucleotides that target HTRA1 (e.g., mammalian HTRA1).

In some embodiments, the antisense oligonucleotides of the present invention can regulate target expression by inhibiting or downregulating the target. Preferably, this regulation inhibits expression by at least 20%, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the normal expression level of the target. In some embodiments, using ARPE-19 cells, the compounds of the present invention can inhibit the expression level of HTRA1 mRNA by at least 60% or 70% in vitro. In some embodiments, using ARPE-19 cells, the compounds of the present invention can inhibit the expression level of HTRA1 mRNA by at least 60% or 70% in vitro. In some embodiments, using ARPE-19 cells, the compounds of the present invention can inhibit the expression level of HTRA1 protein by at least 50% in vitro. Suitably, examples provide assays that can be used to measure HTRA1 RNA or protein inhibition (e.g., Example 3). Target regulation is triggered by hybridization between the sequential nucleotide sequence of the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotides of the present invention comprise a mismatch between the oligonucleotide and the target nucleic acid. Despite mismatches, hybridization with the target nucleic acid is still sufficient to demonstrate the desired regulation of HTRA1 expression. The reduced binding affinity caused by mismatches can be advantageously compensated by increasing the number of nucleotides in the oligonucleotide and/or increasing the number of modified nucleosides (e.g., 2′-modified nucleosides present in the oligonucleotide sequence, including LNA) that can increase binding affinity to the target.

One aspect of the invention relates to an antisense oligonucleotide comprising a continuous nucleotide region of 10 to 30 nucleotides having at least 90% complementarity to an HTRA1 target sequence (e.g., being completely complementary to an HTRA1 target sequence (e.g., a nucleic acid selected from the group consisting of SEQ ID NO 1, 2, 3 and 4)).

In some embodiments, the oligonucleotide comprises a continuous sequence that is at least 90% complementary to the region of the target nucleic acid, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% complementary.

In some embodiments, the oligonucleotide or its sequential nucleotide sequence of the present invention is completely complementary (100% complementary) to the region of the target nucleic acid, or in some embodiments, one or two mismatches may be included between the oligonucleotide and the target nucleic acid.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary to the region of SEQ ID NO 147, for example, completely (or 100%) complementary.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary to a region of a sequence selected from the group consisting of SEQ ID NO 148, 149, 150, 151, 152, 153, 154 and 155, for example, completely (or 100%) complementary.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 14 nucleotides thereof is completely (or 100%) complementary to the sequence of SEQ ID NO 147 or the group consisting of SEQ ID NO 148, 149, 150, 151, 152, 153, 154 and 155.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 16 nucleotides thereof is completely (or 100%) complementary to the sequence in SEQ ID NO 147 or selected from the group consisting of SEQ ID NO 148, 149, 150, 151, 152, 153, 154 and 155.

In some embodiments, the oligonucleotide or its continuous nucleotide region is completely (or 100%) complementary to the sequence selected from the group consisting of SEQ ID NO 148, 149, 150, 151, 152, 153, 154 and 155.

In some embodiments, the oligonucleotide or its continuous nucleotide region comprises or consists of a sequence selected from or composed of the group consisting of SEQ ID NO 143, 138, 139, 140, 141, 142, 144 and 145:

SEQ ID NO 143: TATTTACCTGGTTGTT

SEQ ID NO 138：CAAATATTTACCTGGTTG

SEQ ID NO 139: TTTACCTGGTTGTTGG

SEQ ID NO 140：CCAAATATTTAACCTGGTT

SEQ ID NO 141：CCAAATATTTACCTGGGTTGT

SEQ ID NO 142: ATATTTACCTGGGTTGTTG

SEQ ID NO 144: ATATTTACCTGGGTTGT

SEQ ID NO 145: ATATTTACCTGGTTGTT

It should be understood that oligonucleotide motif sequences can be modified, for example, to increase nuclease resistance and/or binding affinity to target nucleic acids. Modifications are described in the definitions and “Oligonucleotide Design” sections.

In some embodiments, the oligonucleotide or a continuous nucleotide region thereof of the present invention is completely complementary (100% complementary) to a region of the target nucleic acid, or in some embodiments, one or two mismatches may be included between the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary to the target nucleic acid sequence, for example, completely (or 100%) complementary.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 12 nucleotides thereof has 100% identity with a sequence selected from the group consisting of SEQ ID NO 5-107 or SEQ ID NO 108-137.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 14 nucleotides thereof has 100% identity with the sequence selected from the group consisting of SEQ ID NO 5-107 or SEQ ID NO 108-137.

In some embodiments, the oligonucleotide or a continuous nucleotide sequence of at least 16 nucleotides thereof has 100% identity with a sequence selected from the group consisting of SEQ ID NO 5-107 or SEQ ID NO 108-137.

In some embodiments, the oligonucleotide or its continuous nucleotide region comprises or consists of sequences selected from or composed of sequences of SEQ ID NO 5-107 or SEQ ID NO 108-137.

In some embodiments, the compounds of the present invention are selected from the group consisting of:

The uppercase letters represent β-D-oxyLNA nucleosides, all LNA cytosines are 5-methylcytosine (as indicated by the superscript ^m ), and the lowercase letters represent DNA nucleosides. All nucleoside linkages are phosphate thioside linkages (as indicated by the subscript _s ).

Oligonucleotide design

Oligonucleotide design refers to the pattern of nucleoside sugar modification in an oligonucleotide sequence. The oligonucleotides of this invention contain sugar-modified nucleosides and may also contain DNA or RNA nucleosides. In some embodiments, the oligonucleotides contain both sugar-modified nucleosides and DNA nucleosides. Incorporating modified nucleosides into the oligonucleotides of this invention can enhance the affinity of the oligonucleotides for target nucleic acids. In that case, the modified nucleosides may be referred to as affinity-enhancing modified nucleotides.

In one embodiment, the oligonucleotide comprises at least one modified nucleoside, such as at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen modified nucleosides. In another embodiment, the oligonucleotide comprises 1-10 modified nucleosides, such as two to nine, three to eight, four to seven, six, or seven modified nucleosides. In one embodiment, the oligonucleotide of the present invention may contain modifications independently selected from these three types of modifications (modified sugars, modified nucleobases, and modified nucleoside linkages) or combinations thereof. Preferably, the oligonucleotide comprises one or more sugar-modified nucleosides, such as 2′ sugar-modified nucleosides. Preferably, the oligonucleotides of the present invention comprise one or more 2′ sugar-modified nucleosides, independently selected from the group consisting of: 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabinonucleotide (ANA), 2′-fluoro-ANA, and LNA nucleosides. Even more preferably, the one or more modified nucleosides are LNA.

In some embodiments, at least one modified nucleoside is a locked nucleic acid (LNA), for example, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight modified nucleosides are LNAs. In yet another embodiment, all modified nucleosides are LNAs.

In a further embodiment, the oligonucleotide includes at least one modified nucleoside linker. In a preferred embodiment, the nucleoside linker within the continuous nucleotide sequence is a phosphate thioate or borane phosphate nucleoside linker. In some embodiments, all nucleoside links within the continuous sequence of the oligonucleotide are phosphate thioate links.

In some embodiments, the oligonucleotides of the present invention comprise at least one modified nucleoside, which is 2′-MOE-RNA, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-MOE-RNA nucleoside units. In some embodiments, at least one of the modified nucleosides is 2′-fluoroDNA, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-fluoro-DNA nucleoside units.

In some embodiments, the oligonucleotides of the present invention comprise at least one LNA unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, for example 2 to 6 LNA units, for example 3 to 7 LNA units, 4 to 8 LNA units, or 3, 4, 5, 6, or 7 LNA units. In some embodiments, all modified nucleosides are LNA nucleosides. In some embodiments, all LNA cytosine units are 5-methylcytosine. In some embodiments, the oligonucleotide or its continuous nucleotide region has at least one LNA unit at the 5′ end of the nucleotide sequence and at least two LNA units at the 3′ end. In some embodiments, all cytosine nucleobases present in the oligonucleotides of the present invention are 5-methylcytosine.

In some embodiments, the oligonucleotides of the present invention comprise at least one LNA unit and at least one 2′-substituted modified nucleoside.

In some embodiments of the present invention, the oligonucleotide comprises a 2′ sugar-modified nucleoside and a DNA unit.

In one embodiment of the present invention, the oligonucleotide of the present invention is capable of recruiting RNase H.

In some embodiments, the oligonucleotides or their continuous nucleotide regions of the present invention are nicked oligonucleotides.

Notch design

In some embodiments, the oligonucleotides of the present invention, or their continuous nucleotide regions, have a nick body design or structure, also referred to herein as "nick bodies". In a nick body structure, the oligonucleotide comprises at least three distinct structural regions in a 5→3′ orientation: a 5′-flank, a nick, and a 3′-flank, F-G-F′. In this design, the flanking regions F and F′ (also referred to as wing regions) contain at least one sugar-modified nucleoside adjacent to region G, and in some embodiments may contain a continuous segment of 2-7 sugar-modified nucleosides, or a continuous segment of sugar-modified and DNA nucleosides (containing a mixed wing of sugar-modified and DNA nucleosides). Thus, the nucleosides of the 5′ flanking and 3′ flanking regions adjacent to the nick region are sugar-modified nucleosides, such as 2′-modified nucleosides. When the oligonucleotide is a duplex with an HTRA1 target nucleic acid, the nick region G contains a continuous nucleotide segment capable of recruiting RNase H. In some embodiments, region G contains a continuous segment of 5-16 DNA nucleosides. The nick body region F-G-F′ is complementary to the HTRA1 target nucleic acid, and therefore can be a continuous nucleotide region of an oligonucleotide.

Regions F and F′ flanking the 5′ and 3′ ends of region G may contain one or more affinity-enhancing modified nucleosides. In some embodiments, the 3′ flanking region contains at least one LNA nucleoside, preferably at least two LNA nucleosides. In some embodiments, the 5′ flanking region contains at least one LNA nucleoside. In some embodiments, both the 5′ and 3′ flanking regions contain LNA nucleosides. In some embodiments, all nucleosides in the flanking regions are LNA nucleosides. In other embodiments, the flanking regions may contain LNA nucleosides and other nucleosides (mixed flanking regions), such as DNA nucleosides and/or non-LNA-modified nucleosides, such as 2′-substituted nucleosides. In this case, the nick is defined as a continuous sequence of at least five nucleosides recruiting RNase H (e.g., 5-16 DNA nucleosides) with affinity-enhancing modified nucleosides, such as LNA, for example, β-D-oxy-LNA, flanking the 5′ and 3′ ends.

Area F

Region F (5′ flank or 5′ wing), attached to the 5′ end of region G, contains or comprises at least one sugar-modified nucleoside, such as at least two, three, four, five, six, or seven modified nucleosides, or is composed of them. In some embodiments, region F contains 1 to seven modified nucleosides, such as two to six modified nucleosides, such as two to five modified nucleosides, such as two to four modified nucleosides, such as one to three modified nucleosides, such as one, two, three, or four modified nucleosides, or is composed of them.

In one embodiment, one or more or all of the modified nucleosides in region F are 2′ modified nucleosides.

In another embodiment, one or more of the 2′ modified nucleosides in region F are selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabinonucleotide (ANA) units, and 2′-fluoro-ANA units.

In one embodiment of the invention, all modified nucleosides in region F are LNA nucleosides. In another embodiment, the LNA nucleosides in region F are independently selected from the group consisting of β-D or α-L configured oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA, or combinations thereof. In a preferred embodiment, region F has at least one β-D-oxy-LNA unit at the 5′ end of a continuous sequence.

Region G

Region G (gap region) may contain or consist of 5-16 consecutive DNA nucleotides capable of recruiting RNase H. In another embodiment, region G contains or consists of 5 to 12, 6 to 10, or 7 to 9, for example, 8 consecutive nucleotide units capable of recruiting RNase H.

In another embodiment, at least one nucleoside unit in region G is a DNA nucleoside unit, such as 4 to 20 or 6 to 18 DNA units, such as 5 to 16. In some embodiments, all nucleosides in region G are DNA units.

In a further embodiment, region G may consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. In some embodiments, at least 50% of the nucleosides in region G are DNA, for example at least 60%, at least 70%, or at least 80%, or at least 90% DNA.

Region F′

Region F′ (3′ flank or 3′ wing) attached to the 3′ end of region G contains or comprises at least one sugar-modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 modified nucleosides, or is composed of them. In some embodiments, region F′ contains 1 to 7 modified nucleosides, such as 2 to 6 modified nucleosides, such as 2 to 5 modified nucleosides, such as 2 to 4 modified nucleosides, such as 1 to 3 modified nucleosides, such as 1, 2, 3, or 4 modified nucleosides, or is composed of them.

In one embodiment, one or more or all of the modified nucleosides in region F′ are 2′ modified nucleosides.

In another embodiment, one or more of the 2′ modified nucleosides in region F′ are selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabinonucleotide (ANA) units, and 2′-fluoro-ANA units.

In one embodiment of the invention, all modified nucleosides in region F′ are LNA nucleosides. In another embodiment, the LNA nucleosides in region F′ are independently selected from the group consisting of β-D or α-L configured oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA, or combinations thereof. In a preferred embodiment, region F′ has at least one β-D-oxy-LNA unit at the 5′ end of the continuous sequence.

Regions D, D′ and D″

The oligonucleotides of the present invention comprise a continuous nucleotide region complementary to a target nucleic acid. In some embodiments, the oligonucleotide may further comprise additional nucleotides located at the 5′ and/or 3′ of the continuous nucleotide region, referred to herein as region D. Regions D′ and D″ may be attached to the 5′ end of region F or the 3′ end of region F′, respectively. In some embodiments, region D (region D′ or D″) may form part of a continuous nucleotide sequence complementary to the target nucleic acid, or in other embodiments, region D may not be complementary to the target nucleic acid.

In some embodiments, the oligonucleotides of the present invention comprise a continuous nucleotide region and optionally 1-5 additional 5′ nucleotides (region D′) or are composed thereof.

In some embodiments, the oligonucleotides of the present invention comprise a continuous nucleotide region and optionally 1-5 additional 3′ nucleotides (region D″) or are composed thereof.

Regions D′ or D″ may independently contain 1, 2, 3, 4, or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. In this regard, the oligonucleotides of the present invention may, in some embodiments, comprise a continuous nucleotide sequence capable of modulating the target by side-attaching additional nucleotides at the 5′ and/or 3′ ends. These additional nucleotides can serve as nuclease-sensitive, bio-cleavable linkers, and thus can be used to attach functional groups, such as conjugate moieties, to the oligonucleotides of the present invention. In some embodiments, the additional 5′ and/or 3′ terminal nucleotides are linked by phosphodiester bonds and may be DNA or RNA. In another embodiment, the additional 5′ and/or 3′ terminal nucleotides are modified nucleotides, which may be included, for example, to enhance nuclease stability or for ease of synthesis. In some embodiments, in addition to the continuous nucleotide regions, the oligonucleotides of the present invention also comprise regions D′ and/or D″.

In some embodiments, the nicked oligonucleotide of the present invention may be represented by the following formula:

FGF′; especially F _1-7 -G _4-12 -F′ _1-7

D′-FGF′, especially D′ _1-3 -F _1-7 -G _4-12 -F′ _1-7

FGF′-D″, especially F _1-7 -G _4-12 -F′ _1-7 -D″ _1-3

D′-FGF′-D″, especially D′ _1-3 -F _1-7 -G _4-12 -F′ _1-7 -D″ _1-3

Preparation method

In another aspect, the present invention provides a method for preparing the oligonucleotides of the present invention, comprising reacting nucleotide units to form covalently linked sequential nucleotide units contained in the oligonucleotide. Preferably, the method uses phosphoramide chemistry (see, for example, Caruthers et al., 1987, Methods in Enzymology vol. 154, pp. 287-313). In another embodiment, the method further comprises reacting the sequential nucleotide sequence with a conjugated moiety (ligand). In another aspect, a method for preparing the compositions of the present invention is provided, comprising mixing the oligonucleotides or conjugated oligonucleotides of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and/or adjuvant.

medicinal salt

For use as a therapeutic agent, the oligonucleotides of the present invention can be provided as suitable pharmaceutical salts, such as sodium or potassium salts. In some embodiments, the oligonucleotides of the present invention are sodium salts.

Pharmaceutical Composition

In another aspect, the present invention provides pharmaceutical compositions comprising any of the above-described oligonucleotides and/or oligonucleotide conjugates and pharmaceutically acceptable diluents, carriers, salts, and/or adjuvants. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS) and pharmaceutically acceptable salts, including but not limited to sodium and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate-buffered saline. In some embodiments, the oligonucleotide is used in a pharmaceutically acceptable diluent at a concentration of 50 μM to 300 μM solution. In some embodiments, the oligonucleotides of the present invention are administered at doses of 10 μg to 1000 μg.

WO 2007/031091 (incorporated hereby by reference) provides suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants. WO 2007/031091 also provides suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations.

The oligonucleotides or oligonucleotide conjugates of the present invention can be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods used to formulate pharmaceutical compositions depend on many criteria, including but not limited to route of administration, disease severity, or dosage.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug. In particular, for oligonucleotide conjugates, once the prodrug is delivered to the site of action, such as a target cell, the conjugate moiety is cleaved from the oligonucleotide.

application

The oligonucleotides of the present invention can be used as research reagents, for example, for diagnosis, treatment and prevention.

In this study, this oligonucleotide can be used to specifically regulate the synthesis of the HTRA1 protein in cells (e.g., in vitro cell cultures) and laboratory animals, thereby facilitating functional analysis of the target or assessing its usefulness as a therapeutic intervention target. Typically, target regulation is achieved by degrading or inhibiting the mRNA that produces the protein, thus preventing protein formation or acting as a regulator of the gene or mRNA that produces the protein.

In diagnostics, oligonucleotides can be used to detect and quantify HTRA1 expression in cells and tissues via RNA blotting, in situ hybridization, or similar techniques.

For treatment, animals or humans suspected of having a disease or symptom can be treated by regulating the expression of HTRA1.

The present invention provides a method for treating or preventing a disease, comprising administering to a subject who has or is susceptible to the disease a therapeutically or preventively effective amount of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention.

The present invention also relates to oligonucleotides, compositions or conjugates as defined herein, which are used as pharmaceuticals.

The oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions according to the present invention are typically administered in an effective amount.

The present invention also provides the use of the oligonucleotides or oligonucleotide conjugates of the present invention as described herein in the preparation of medicaments for treating the conditions described herein or in methods for treating the conditions described herein.

The diseases or conditions mentioned herein are associated with HTRA1 expression. In some embodiments, the disease or condition may be associated with mutations in the HTRA1 gene or in genes whose protein products are associated with or interact with HTRA1. Therefore, in some embodiments, the target nucleic acid is a mutant form of the HTRA1 sequence, and in other embodiments, the target nucleic acid is a regulator of the HTRA1 sequence.

The method of the present invention is preferably used for treating or preventing diseases caused by abnormal levels and/or activity of HTRA1.

The present invention also relates to the use of oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions as defined herein in the preparation of medicaments for treating abnormal levels and/or activity of HTRA1.

In one embodiment, the present invention relates to oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions for the treatment of diseases or conditions selected from: eye diseases, such as macular degeneration, including age-related macular degeneration (AMD), such as dry AMD or wet AMD, and diabetic retinopathy. In some embodiments, the oligonucleotide conjugates or pharmaceutical compositions of the present invention can be used to treat geographic atrophy or intermediate dAMD. HTRA1 has also been indicated in Alzheimer's disease and Parkinson's disease, therefore in some embodiments, the oligonucleotide conjugates or pharmaceutical compositions of the present invention can be used to treat Alzheimer's disease or Parkinson's disease. HTRA1 has also been indicated in Dechené muscular dystrophy, arthritis such as osteoarthritis, and familial ischemic small vessel disease, therefore in some embodiments, the oligonucleotide conjugates or pharmaceutical compositions of the present invention can be used to treat Dechené muscular dystrophy, arthritis such as osteoarthritis, or familial ischemic small vessel disease.

application

The oligonucleotide or pharmaceutical compositions of the present invention can be administered topically (e.g., to the skin, inhaled, through the eye or ear), or enterically (e.g., orally or via the gastrointestinal tract), or parenterally (e.g., intravenously, subcutaneously, intramuscularly, intracerebrally, intravenously, or intrathecally).

In some embodiments, the oligonucleotides, conjugates, or pharmaceutical compositions of the present invention are administered via parenteral routes, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, intrathecal, or intracranial administration, such as intracerebral or intraventricular administration. In some embodiments, the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment, the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

Intraocular injections can be used to treat eye diseases such as macular degeneration, such as AMD (wet or dry).

In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof is administered by intraocular injection at a dose of about 10 μg to about 200 μg per eye, for example about 50 μg to about 150 μg per eye, or for example about 100 μg per eye. In some embodiments, the dosing interval, i.e., the time period between consecutive administrations, is at least one month, for example at least every two months or at least every three months.

Combination therapy

In some embodiments, the oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the present invention are used for combination therapy with another therapeutic agent. The therapeutic agent may, for example, be a standard of care for the aforementioned disease or condition.

Embodiments of the present invention

1. An antisense oligonucleotide of 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide region of 10-22 nucleotides that is at least 90% (e.g., 100%) complementary to SEQ ID NO 147:

SEQ ID NO 147：5′CCAACAACCAGGTAAATATTTG 3′

2. The antisense oligonucleotide according to embodiment 1, wherein the antisense oligonucleotide is capable of inhibiting the expression of HTRA1 mRNA.

3. An antisense oligonucleotide according to embodiment 1 or 2, wherein the continuous nucleotide region is identical to the sequence presented in the group consisting of SEQ ID NO 138, 139, 140, 141, 142, 143, 144 and 145:

SEQ ID NO 138：CAAATATTTACCTGGTTG

SEQ ID NO 139: TTTACCTGGTTGTTGG

SEQ ID NO 140：CCAAATATTTAACCTGGTT

SEQ ID NO 141：CCAAATATTTACCTGGGTTGT

SEQ ID NO 142: ATATTTACCTGGGTTGTTG

SEQ ID NO 143: TATTTACCTGGTTGTT

SEQ ID NO 144: ATATTTACCTGGGTTGT

SEQ ID NO 145: ATATTTACCTGGTTGTT

4. An antisense oligonucleotide according to any one of embodiments 1-3, wherein the continuous nucleotide region comprises the sequence SEQ ID NO: 146:

SEQ ID NO 146: TTTACCTGGGTT

5. An antisense oligonucleotide according to any one of embodiments 1-4, wherein the continuous nucleotide region of said oligonucleotide comprises or consists of a sequence selected from or composed of any one of SEQ ID NO 138, 139, 140, 141, 142, 143, 144 and 145.

6. An antisense oligonucleotide according to any one of embodiments 1-5, wherein the continuous nucleotide region of said oligonucleotide comprises one or more 2′ sugar-modified nucleosides.

7. The antisense oligonucleotide according to embodiment 6, wherein the one or more 2′ sugar-modified nucleosides are independently selected from the group consisting of: ′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabinose (ANA), 2′-fluoro-ANA and LNA nucleosides.

8. An antisense oligonucleotide of any one of embodiments 5-7, wherein one or more modified nucleosides are LNA nucleosides.

9. An antisense oligonucleotide according to any one of embodiments 1-8, wherein the continuous nucleotide region of said oligonucleotide comprises at least one modified nucleoside linker, such as one or more phosphate thioside links.

10. The antisense oligonucleotide according to embodiment 9, wherein all nucleoside linkages within the continuous nucleotide region are phosphate thioside linkages.

11. An antisense oligonucleotide according to any one of embodiments 1-10, wherein the oligonucleotide is capable of recruiting RNase H.

12. An antisense oligonucleotide according to any one of embodiments 1-11, wherein the oligonucleotide or its continuous nucleotide sequence is a nick or contains a nick.

13. The antisense oligonucleotide of embodiment 11 or 12, wherein the oligonucleotide or its sequential nucleotide sequence is a nicked form of formula 5′-F-G-F′-3′, wherein regions F and F′ independently contain 1-7 sugar-modified nucleosides, and G is a region of 6-16 nucleosides capable of recruiting RNase H, wherein the nucleosides of regions F and F′ adjacent to region G are sugar-modified nucleosides.

14. An antisense oligonucleotide according to embodiment 13, wherein region G contains or is composed of 6-16 DNA nucleosides.

15. An antisense oligonucleotide according to embodiment 13 or 14, wherein regions F and F′ each contain at least one LNA nucleotide.

16. The antisense oligonucleotide according to any one of the implementation schemes 1-15 is selected from the group consisting of the following:

C _s A _s A _s A _s t _s a _s t _s t _s t _s a _s c _s c _s t _s g _s G _s T _s T _s G (SEQ ID NO 138, compound #138, 1)

T _s T _s t _s a _{s c s} c _s t _s g _s g _s t _s t _s g _s t _s T _s G _s G (SEQ ID NO 139, compound #139, 1)

C _s C _s A _s A _s a _s t _s a _{s t s t s} t _s a _s c _s c _s t _s g _s G _s T _s T (SEQ ID NO 140, compound #140, 1)

C _s C _s A _s a _s a _s t _s a _s t _s t _s t _s a _s c _s c _s t _s g _s g _s t _s t _s G _s T (SEQ ID NO 141, compound #141, 1)

A _s T _s A _s t _s t _s t _s a _s c _s c _s t _s g _s g _s t _s t _s g _s T _s T _s G (SEQ ID NO 142, compound #142, 1)

T _s A _s T _s t _{s t} s a _{s c s} c _s t _s g _s g _s t _s T _s G _s T _s T (SEQ ID NO 143, compound #143, 1)

T _s A _s t _s t _{s t s} a s _{c s} c _s t _s g _s G _s t _s T _s g _s T _s T (SEQ ID NO 143, compound #143, 2)

T _s A _s T _s t _{s t} s a _{s c s} c _s t _s g _s g _s T _s T _s g _s T _s T (SEQ ID NO 143, compound #143, 3)

A _s T _s A _{s T s t s} t _s a _{s c s} c _s t _s g _s g _s T _s T _s G _s T (SEQ ID NO 144, compound #144, 1)

A _s t _s A _s T _s T _s t _s a _{s c s c s} t _s g s g _s t _s T _s G _s T (SEQ ID NO 144, compound #144, 2)

A _s t _s A _{s T s} t s t _s a _s c _s c _s t _s g _s G _s T _s T _s g _s T _s T (SEQ ID NO 145, compound #145, 1)

A _s T _s A _{s t s} t _s t _s a _{s c s} c _s t _s g _s G _s t _s T _s g _s T _s T (SEQ ID NO 145, compound #145, 2)

A _s t _s A _{s T s} t s t _s a _{s c s} c _s t _s g _s g _s t _s T _s G _s T _s T (SEQ ID NO 145, compound #145, 3)

In this context, uppercase letters represent LNA nucleoside units, lowercase letters represent DNA nucleoside units, and the subscript _'s' represents the linkage between phosphate thioside nucleosides. All LNA cytosines are 5-methylcytosine.

17. The antisense oligonucleotide according to embodiment 16, wherein the LNA nucleosides are all β-D-oxyLNA nucleosides.

18. A conjugate comprising an oligonucleotide according to any one of embodiments 1-17 and at least one conjugate portion covalently attached to said oligonucleotide.

19. A pharmaceutical composition comprising an oligonucleotide of embodiments 1-17 or a conjugate of embodiment 18 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

20. An in vivo or in vitro method for regulating HTRA1 expression in target cells expressing HTRA1, the method comprising administering to the cells an effective amount of an oligonucleotide of any one of embodiments 1-17 or a conjugate according to embodiment 18 or a pharmaceutical composition according to embodiment 19.

21. A method of treating or preventing a disease, comprising administering to a subject suffering from or susceptible to a disease a therapeutically or preventively effective amount of any one of embodiments 1-17, or a conjugate according to embodiment 18, or a pharmaceutical composition according to embodiment 19.

22. The method of implementation scheme 21, wherein the disease is selected from the group consisting of: macular degeneration (e.g., wet AMD, dry AMD, geographic atrophy, intermediate dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Dichenne muscular dystrophy, arthritis, such as osteoarthritis, and familial ischemic small vessel disease.

23. An oligonucleotide of any one of embodiments 1-17, or a conjugate according to embodiment 18, or a pharmaceutical composition according to embodiment 19, for use in a medicament.

24. An oligonucleotide of any one of embodiments 1-17 or a conjugate according to embodiment 18 or a pharmaceutical composition according to embodiment 19, for the treatment or prevention of diseases selected from the group consisting of: macular degeneration (e.g., wet AMD, dry AMD, geographic atrophy, intermediate dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Dichenne muscular dystrophy, arthritis, such as osteoarthritis, and familial ischemic small vessel disease.

25. The use of any oligonucleotide of embodiments 1-17 or a conjugate according to embodiment 18 or a pharmaceutical composition according to embodiment 19 for the preparation of a medicament for the treatment or prevention of diseases selected from the group consisting of: macular degeneration (e.g., wet AMD, dry AMD, geographic atrophy, intermediate dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Dichenne muscular dystrophy, arthritis, such as osteoarthritis, and familial ischemic small vessel disease.

Example

Materials and methods

Oligonucleotide synthesis

Oligonucleotide synthesis is generally known in the art. The following are applicable methods. The oligonucleotides of the present invention can be prepared by methods with slight variations in the apparatus, support, and concentration used.

Oligonucleotides were synthesized on a universal uridine support using a 1 μmol-scale Oligomaker 48 solution via the phosphoramidite method. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using ammonia at 60 °C for 5–16 h. The oligonucleotides were purified by reversed-phase HPLC (RP-HPLC) or by solid-phase extraction, characterized by UPLC, and their molecular weights were further confirmed by ESI-MS.

Oligonucleotide elongation:

The coupling of β-cyanoethyl phosphoramides (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), LNA-T) was carried out using a solution of 0.1 M 5′-O-DMT-protected phosphoramide in acetonitrile and a solution of DCI (4,5-dicyanimidazolium) in acetonitrile (0.25 M) as activators. For the final cycle, phosphoramides with the desired modification (e.g., for attaching conjugated groups or C6 linkers of such conjugated groups) could be used. Thiolization for introducing thiophosphate linkages was carried out using xanthan gum hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphodiester linkages could be introduced using 0.02 M iodine in THF/pyridine/water 7:2:1. The remaining reagents were those commonly used in oligonucleotide synthesis.

For post-solid-phase synthetic conjugations, a commercially available C6 amino-linked phosphoramidite can be used in the final cycle of the solid-phase synthesis, and the amino-linked deprotected oligonucleotide is isolated after deprotection and cleavage from the solid support. The conjugation is introduced by activating the functional groups using standard synthetic methods.

Purified by RP-HPLC:

The crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μm 150x10mm column. A buffer of 0.1M ammonium acetate and acetonitrile at pH 8 was used at a flow rate of 5 mL/min. The collected fractions were lyophilized to obtain the purified compound, typically a white solid.

abbreviation:

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4′-Dimethoxytriphenylmethyl

THF: Tetrahydrofuran

Bz: Benzoyl group

Ibu: Isobutyryl

RP-HPLC: Reversed-phase high-performance liquid chromatography

T _m determination:

The oligonucleotide and RNA target (phosphate-linked, PO) duplex were diluted to 3 mM in 500 mL of RNase-free water and mixed with 500 mL of 2x T_{_m}-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM phosphate, pH 7.0). The solution was heated to 95 °C for 3 min and then annealed at room temperature for 30 min. The duplex melting temperature (T_{_m}) was measured using PE Templab software (Perkin Elmer) on a Lambda 40 UV/VIS spectrophotometer equipped with a Peltier temperature programmer PTP6. The temperature was increased from 20 °C to 95 °C and then decreased to 25 °C, with the absorbance recorded at 260 nm. The first derivatives and local maxima of melting and annealing were used to evaluate the duplex T_{_m} .

Example 1: In vitro efficacy of antisense oligonucleotides targeting rat Htra1 in C6 cell line was tested at a single dose concentration.

Rat C6 cell lines were purchased from ATCC and maintained in a humidified incubator at 37°C and 5% _CO2 as recommended by the supplier. For assays, 1500 C6 cells/well were seeded in 96-well plates in medium. Cells were incubated for 2 hours, followed by the addition of oligonucleotides dissolved in PBS. Oligonucleotide concentration: 25 μM. Cells were harvested 4 days after oligonucleotide addition. RNA was extracted using the PureLink Pro 96 RNA Purification Kit (Ambion, according to manufacturer's instructions). cDNA was then synthesized using M-MLT reverse transcriptase, random decameric RETROscript, an RNase inhibitor (Ambion, according to manufacturer's instructions) with a 100 mM dNTP group (PCR grade, Invitrogen) and DNase-free/RNase-free water (Gibco). For gene expression analysis, qPCR was performed in a doublex setting using TagMan Fast Advanced Master Mix (2X) (Ambion). The following TaqMan primer assays were used for qPCR: Htra1, Rn00581870_m1 (FAM-MGB) and housekeeping gene, Tbp, Rn01455646_m1 (VIC-MGB). All primer sets were purchased from Life Technologies. Relative Htra1 mRNA expression levels in the table are shown as a percentage of the control (PBS-treated cells).

Oligonucleotides used:

For compounds: uppercase letters represent LNA nucleosides (using β-D-oxyLNA nucleosides), all LNA cytosines are 5-methylcytosine, lowercase letters represent DNA nucleosides, and DNA cytosines preceded by a superscript ^m represent 5-methyl C-DNA nucleosides. All nucleoside linkages are phosphate thioside linkages.

Example 2. The in vitro potency and efficacy of selected oligonucleotides targeting rat Htra1 in the C6 cell line were tested using dose-response curves.

The rat C6 cell line is described in Example 1. Assays were performed as described in Example 1. Oligonucleotide concentrations: starting at 50 μM, semi-logarithmic dilutions, 8 spots. Cells were harvested 4 days after oligonucleotide addition. RNA extraction, cDNA synthesis, and qPCR were performed as described in Example 1. n = 2 biological replicates. _EC50 assays were performed on a GraphPad Prism6. Relative Htra1 mRNA levels treated with 50 μM oligonucleotides are shown in the table as % of control (PBS). Additional primer sets (Htra1, Rn00668987_m1 [FAM-MGB] vs. Ppia, Rn006900933_m1 [VIC-MGB] and Hprt, Rn01527840_m1 [VIC-MGB]) were also tested, and the same trend was observed using these primers (data not shown).

Example 3 tested the in vitro efficacy of oligonucleotides targeting human HTRA1 in the U251 cell line at a single dose concentration.

Human glioblastoma U251 cell lines were purchased from ECACC and maintained in a humidified incubator at 37°C and 5% _CO2 as recommended by the supplier. For assays, 15,000 U251 cells/well were seeded in 96-well plates in starvation medium (supplier-recommended medium, except 1% FBS instead of 10%). Cells were incubated for 24 hours, and then oligonucleotides dissolved in PBS were added. Oligonucleotide concentration: 5 μM. 3–4 days after adding the compound, the medium was removed and fresh medium (oligonucleotide-free) was added. Cells were harvested 6 days after adding the oligonucleotides. RNA was extracted using the PureLink Pro 96 RNA Purification Kit (Ambion, according to manufacturer's instructions). cDNA was then synthesized using M-MLT reverse transcriptase, random decameric RETROscript, an RNase inhibitor (Ambion, according to manufacturer's instructions), a 100 mM dNTP group (PCR grade, Invitrogen), and DNase-free/RNase-free water (Gibco). For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2X) (Ambion) in doublex settings. The following TaqMan primer assays were used for qPCR: HTRA1, Hs01016151_m1 (FAM-MGB) from Life Technologies and housekeeping gene TBP, Hs4326322E (VIC-MGB). The relative HTRA1 mRNA expression levels in the table are shown as a percentage of the control (PBS-treated cells).

For compounds: uppercase letters represent LNA nucleosides (using β-D-oxyLNA nucleosides), all LNA cytosines are 5-methylcytosine, lowercase letters represent DNA nucleosides, and DNA cytosines preceded by a superscript ^m represent 5-methyl C-DNA nucleosides. All nucleoside linkages are phosphate thioside linkages.

Example 4 tested the in vitro efficacy of an oligonucleotide library targeting human HTRA1 mRNA in ARPE19 and U251 cell lines at two concentrations.

Identifying promising "hotspot" regions of HTRA1. A library of n=129 human/cynomolgus monkey/rat HTRA1 LNA oligonucleotides was screened in U251 and ARPE19 cell lines. From this library, we identified a series of active oligonucleotides targeting human HTRA1 pre-mRNA (SEQ ID NO 2) between positions 33042-33064, as shown in Figure 1.

Human retinal pigment epithelium ARPE19 cell line was purchased from ATCC and maintained at 37°C in a humidified incubator with 5% _CO2 in DMEM-F12 (Sigma, D8437), 10% FBS, and 1% pen/strep. The U251 cell line is described in Example 3. For assays, 5000 ARPE19 cells/well were seeded in medium (except for 5% FBS instead of 10%) in 96-well multi-well plates. Cells were incubated for 1 hour, and then oligonucleotides dissolved in PBS were added. Cells were harvested 4 days after the addition of oligonucleotides. Assays of the U251 cell line were performed as described in Example 3. Oligonucleotide concentrations: 25 μM and 2.5 μM. RNA was extracted using the RNeasy96 Biorobot 8000 kit (Qiagen, according to manufacturer's instructions). cDNA was then synthesized using the Retroscript cDNA Synthesis Kit (ThermoFisher, according to manufacturer's instructions). For gene expression analysis, qPCR was performed using the Fluidigm Biomark system. The following TaqMan primer assays were used for qPCR: HTRA1, Hs01016151_m1 and housekeeping gene, TBP, Hs99999910_m1 and PPIA, Hs99999904_m1 from Life Technologies. n = 2 biological replicates. Relative HTRA1 mRNA expression levels are shown in the table as % of control (PBS). An additional HTRA1 primer set (Hs00170197_m1) was also tested and the same trend was observed (data not shown).

For compounds: uppercase letters represent LNA nucleosides (using β-D-oxyLNA nucleosides), all LNA cytosines are 5-methylcytosine, and lowercase letters represent DNA nucleosides. All nucleoside linkages are phosphate thioside linkages.

Example 5. In vitro efficacy of selected LNA oligonucleotides targeting human/rat HTRA1 in rat C6 cell line tested at single-dose concentrations.

The rat C6 cell line is described in Example 1. Assays were performed as described in Example 1. Oligonucleotide concentration: 25 μM. n = 2 biological replicates. Relative Htra1 mRNA expression levels in the table are shown as % of the control (PBS-treated cells).

Example 6. In vitro potency and efficacy of selected human/cynomolgus/rat LNA oligonucleotides in ARPE19, U251 and C6 cell lines were tested on a dose-response basis.

ARPE19, U251, and C6 cell lines are described in Examples 4, 3, and 1, respectively. For assays, 2000 U251 or ARPE19 cells/well were seeded in 96-well plates in the vendor-recommended medium. Cells were incubated for 2 hours, followed by the addition of oligonucleotides dissolved in PBS. C6 cell line assays were performed as described in Examples 1-2. Oligonucleotide concentrations: starting at 50 μM, semi-logarithmic dilutions, 8 spots. Cells were harvested 4 days after oligonucleotide addition. RNA extraction, cDNA synthesis, and qPCR were performed on all cell lines as described in Example 1. For U251 and ARPE19 cells, the following TaqMan primers were used for assays: HTRA1, Hs01016151_m1 (FAM-MGB) and housekeeping gene, TBP, Hs4326322E (VIC-MGB). All primer sets were purchased from Life Technologies. n = 2 biological replicates. EC50 assays were performed on a Graph Pad Prism 6. Relative HTRA1 mRNA levels after treatment with 50 μM oligonucleotides are shown in the table as a percentage of control (PBS).

Example 7. In vitro efficacy of selected oligonucleotides targeting human HTRA1 in ARPE19 and U251 cell lines was tested at single-dose concentrations.

Example 6 describes the ARPE19 and U251 cell lines and assays. RNA extraction was performed as described in Example 1, and cDNA synthesis and qPCR were performed using a qScript XLT one-step RT-qPCR ToughMix Low ROX, 95134-100 (Quanta Biosciences). For U251 and ARPE19 cells, assays were performed in a doubleplex setting using the following TaqMan primers: HTRA1, Hs01016151_m1 (FAM-MGB) and housekeeping gene, GAPDH, Hs4310884E (VIC-MGB). All primer sets were purchased from Life Technologies. n = 1 biological replicate. Relative HTRA1 mRNA expression levels in the table are shown as % of control (PBS-treated cells). Another primer set (HTRA1, Hs00170197_m1 [FAM-MGB] vs. TBP Hs4326322E [VIC-MGB]) was also tested for U251, and the same trend was observed using those primers (data not shown). See Figure 2.

Example 8. Testing in vitro efficacy and potency in human primary RPE cells.

Human primary retinal pigment epithelium (hpRPE) cells were purchased from Sciencell (Cat#6540). For assays, 5000 hpRPE cells/well were seeded in 96-well plates coated with laminin (Laminin 521, BioLamina Cat#LN521-03) in EpiCM (Sciencell Cat#4101). Cells were expanded using this medium for one week and differentiated for two weeks using the following medium: MEM Alpha medium (Sigma Cat#M-4526) supplemented with N1 supplement (Sigma Cat#N-6530), glutamine-penicillin-streptomycin (Sigma Cat#G-1146), non-essential amino acids (NEAA, Sigma Cat#M-7145), taurine (Sigma Cat#T-0625), hydrocortisone (Sigma Cat#H-03966), triiodothyronine (Sigma Cat#T-5516), and bovine serum albumin (BSA, Sigma Cat#A-9647). Cells were cultured in a humidified incubator at 37°C and 5% _CO2 .

On the day of the experiment, cells were incubated with fresh differentiation medium for 1 hour before the addition of oligonucleotides. They were dissolved in PBS and applied to cells on days 0 and 4. On day 7, cells were harvested with 50 μl of RLT buffer (Qiagen Cat#79216) containing β-mercaptoethanol. RNA extraction was performed according to the user manual for the Qiagen RNeasy Mini Kit (Cat#74104; Lot151048073) (including DNase I treatment (Cat#79254; Lot 151042674)). RNA quality control was performed using the Agilent Bioanalyzer Nano Kit (Agilent; Cat#5067-1511; Lot 1446). Total RNA was reverse transcribed into cDNA (cDNA synthesis) using the High Capacity cDNA Reverse Transcription Kit based on random hexameric oligonucleotides, according to the manufacturer's instructions (Thermo Fisher Scientific, Cat#4368814; Lot 00314158). cDNA samples were measured in triplicate in 384-well plates on a 7900HT real-time PCR instrument (Thermo Fisher Scientific). The following TaqMan primer assays were used for qPCR: HTRA1, Hs01016151_m1 and Hs00170197_m1 from Life Technologies, housekeeping gene, GAPDH, Hs99999905_m1 and PPIA, Hs99999904_m1. n = 3 biological replicates. Relative HTRA1 mRNA expression levels are shown in the table as a percentage of control (PBS). See Figure 3.

Example 9. In vivo efficacy study in rats: 30 μg/eye was injected intravitreally (IVT) after 7 days of treatment.

animal

The experiments were conducted on stained male Brown Norway rats. Each study group included 5 animals, for a total of 15 animals.

Compounds and Dosing Procedures

To begin the experiment, animals were anesthetized with isoflurane, their eyes were disinfected and enlarged, and then 30 μg (3 μl) per eye was injected into the vitreous cavity.

euthanasia

At the end of their lifespan (day 7), all rats were euthanized with _CO2 , and their eyes were harvested for dissection. The retina, sclera, and vitreous fluid were collected for further analysis.

Quantitative analysis of HTRA1 RNA expression

Retinal samples were dissected. Rapidly frozen rat retinal tissue was kept frozen and lysed in RLT lysis buffer (Qiagen RNeasy Mini Kit) in the testing device. RNA extraction was continued according to the user manual of the Qiagen RNeasy Mini Kit (Cat#74104; Lot 151039852), including DNase I treatment (Cat#79254; Lot 151048613). RNA quality controls were performed using the Agilent Bioanalyzer Nano Kit (Agilent; Cat#5067-1511; Lot 1446). Total RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit based on random hexameric oligonucleotides (cDNA synthesis) according to the manufacturer's instructions (Thermo Fisher Scientific, Cat#4368814; Lot00314158). cDNA samples were measured in triplicate in 384-well plates using a 7900HT real-time PCR instrument (Thermo Fisher Scientific). The following TaqMan primer assays were used for qPCR: Htra1, Rn00581870_m1 and housekeeping gene, Gapdh, Rn01775763_g1 and Tbp, Rn01455646_m1 from Life Technologies. Rats/groups: 5, n = 10 eyes. Each eye was considered a separate sample. Relative Htra1 mRNA expression levels are shown as a percentage of control (PBS). See Figure 4.

Example 10: In vivo efficacy study in rats. The rats were treated for 7 days and then injected intravitreally (IVT) at the prescribed dosage.

animal

All experiments were conducted on stained Brown Norway rats. Each study group included 17 animals, for a total of 34 animals.

Compounds and Dosing Procedures

Animals were anesthetized by intramuscular injection of a mixture of toluidine and ketamine. On day 1 of the study, test items and negative controls (PBS) were administered intravitreally in both eyes of the anesthetized animals (3 μL each time).

euthanasia

Euthanasia was performed by intraperitoneal injection of an overdose of pentobarbital at the end of life (day 8).

Oligonucleotide content measurement and quantification of Htra1 RNA expression

Both eyes from all animals in the low-dose and medium-dose groups, and the first 5 eyes from the high-dose and PBS groups, were used for bioanalysis. Immediately after euthanasia, the vitreous (V), retina (R), and choroid (CH) were rapidly and carefully sectioned on ice and stored at -80°C until shipment. Retinal samples were lysed in 700 μL MagNa Pure 96 LC RNA separation tissue buffer and homogenized 2 x 1.5 min using a Precellys homogenizer with one stainless steel bead added per 2 ml tube, followed by incubation at room temperature for 30 min. Samples were centrifuged at 13,000 rpm for 5 min. Half of the samples were used for bioanalysis, and the other half were used for direct RNA extraction.

For bioanalysis, samples are diluted 10–50-fold using a hybridization ELISA method to measure oligonucleotide content. Biotinylated LNA-capture probes and digoxigenin-conjugated LNA-detection probes (35 nM in 5xSSCT, each complementary to one end of the LNA oligonucleotide to be detected) are mixed with diluted homogenate or relevant standards and incubated at room temperature for 30 min. The mixture is then added to a streptavidin-coated ELISA plate (Nunc catalog number 436014).

The plate was incubated at room temperature for 1 hour and washed in 2xSSCT (300 mM sodium chloride, 30 mM sodium citrate, and 0.05% v/v Tween-20, pH 7.0). The captured LNA duplexes were detected using an anti-DIG antibody conjugated with alkaline phosphatase (Roche Applied Sciencecat. No. 11093274910) and the alkaline phosphatase substrate system (Blue Phos substrate, KPL product code 50-88-00). The amount of oligomeric complex was measured as absorbance at 615 nm on a Biotek reader.

For RNA extraction, a large-volume RNA kit (05467535001, Roche) was used with the MagNA Pure 96 system, following the procedure: Tissue FF standard LV3.1 was prepared according to the manufacturer's instructions, including DNase treatment. RNA quality controls and concentrations were measured using an Eon reader (Biotek). RNA concentrations were normalized between samples, followed by cDNA synthesis and qPCR in a one-step reaction using the qScript XLT one-step RT-qPCR ToughMix Low ROX, 95134-100 (Quanta Biosciences). The following TaqMan primer assays were used for the duplex reaction: Htra1, Rn00581870_m1 and Rn00668987_m1 from Life Technologies, and housekeeping gene, HPRT, Rn01527840_m1 and Tbp, Rn01455646_m1. qPCR analysis was performed on a ViiA7 machine (Life Technologies). Rats/group: 5, n = 10 eyes. Each eye was considered a separate sample. Relative Htra1 mRNA expression levels are shown as % of control (PBS).

Histology

The eyes of the remaining two high-dose animals and the PBS animal were removed, fixed in 10% neutral buffered formalin for 24 hours, trimmed, and embedded in paraffin.

For ISH analysis, 4µm thick formalin-fixed, paraffin-embedded rat retinal tissue sections were treated using RNAscope 2.5VS Probe-Rn-HTRA1 (Cat No. 440959, Advanced Cell Diagnostic) with the fully automated Ventana Discovery ULTRA staining module (program: mRNA Discovery Ultra Red 4.0-v0.00.0152). Fastrided hematoxylin II counterstaining agent was used.

Example 11

PoC study: Blue light-induced retinal degeneration in albino rats

animal

All experiments were conducted on albino Sprague-Dawley rats. Each study group included 16 animals, for a total of 42 animals.

Compounds and Dosing Procedures

Animals were anesthetized by intramuscular injection of a mixture of toluidine and ketamine. On day 3 of the study, test items and negative controls (PBS) were administered intravitreally in both eyes of the anesthetized animals (3 μL each time).

The positive control (PBN) was administered intraperitoneally on day 0 at a dose volume of 2.5 mL/kg in the dark using a 25-gauge needle mounted on a 1 mL plastic syringe, four times (0.5 hours before the start of light exposure, 2 hours and 4 hours after the start of light exposure, and after the end of light exposure).

Light exposure

Rats were dark-acclimatized for 36 hours, then exposed to continuous blue fluorescence (400-540 nm) for 6 hours in a transparent plastic cage. After exposure, the rats were placed in a dark room for 24 hours, and then returned to standard circulating light conditions.

Electroretinography (ERG)

After overnight dark adaptation, electroretinograms (ERGs) were recorded in both eyes at baseline and on day 14. The amplitudes of the A and B waves were measured in each ERG recording.

euthanasia

At the end of their lifespan (day 14), the animals were anesthetized and euthanized by intraperitoneal injection of an overdose of pentobarbital.

Outer core layer (ONL) thickness measurement

Both eyes were removed from 10 primary animals in each group, fixed in Bouin-Hollande solution, and embedded in paraffin. Thin sections (5 to 7 μm thick) were cut along the vertical meridian and stained with Trichrome-Masson. The ONL thickness was measured at seven points (every 250 μm) from the optic nerve to the peripheral retina in each segment of the retina (superior and inferior). The thickness of the outer nuclear layer was measured at each point, and the area under the curve (AUC) was calculated.

Oligonucleotide content measurement and quantification of Htra1 RNA expression

Eyes from four satellite animals in the test and PBS groups were used for bioanalysis. Immediately after euthanasia, the vitreous (V), retina (R), and choroid (CH) were rapidly and carefully sectioned on ice and stored at -80°C until shipment. Retinal samples were lysed in 700 μL MagNa Pure 96 LC RNA separation tissue buffer and homogenized 2 x 1.5 min using a Precellys homogenizer with one stainless steel bead added per 2 ml tube, followed by incubation at room temperature for 30 min. Samples were centrifuged at 13,000 rpm for 5 min. Half of the samples were used for bioanalysis, and the other half were used directly for RNA extraction.

For bioanalysis, samples are diluted 10–50-fold using a hybridization ELISA method to measure oligonucleotide content. Biotinylated LNA-capture probes and digoxigenin-conjugated LNA-detection probes (35 nM in 5xSSCT, each complementary to one end of the LNA oligonucleotide to be detected) are mixed with diluted homogenate or relevant standards and incubated at room temperature for 30 min. The mixture is then added to a streptavidin-coated ELISA plate (Nunc catalog number 436014).

The plate was incubated at room temperature for 1 hour and washed in 2xSSCT (300 mM sodium chloride, 30 mM sodium citrate, and 0.05% v/v Tween-20, pH 7.0). The captured LNA duplexes were detected using an anti-DIG antibody conjugated with alkaline phosphatase (Roche Applied Sciencecat. No. 11093274910) and the alkaline phosphatase substrate system (Blue Phos substrate, KPL product code 50-88-00). The amount of oligomeric complex was measured as absorbance at 615 nm on a Biotek reader.

For RNA extraction, a large-volume RNA kit (05467535001, Roche) was used with the MagNA Pure 96 system, following the procedure: Tissue FF standard LV3.1 was prepared according to the manufacturer's instructions, including DNase treatment. RNA quality controls and concentrations were measured using an Eon reader (Biotek). RNA concentrations were normalized between samples, followed by cDNA synthesis and qPCR in a one-step reaction using the qScript XLT one-step RT-qPCR ToughMix Low ROX, 95134-100 (Quanta Biosciences). The following TaqMan primer assays were used for the duplex reaction: Htra1, Rn00581870_m1 and Rn00668987_m1 from Life Technologies, and housekeeping gene, HPRT, Rn01527840_m1 and Tbp, Rn01455646_m1. qPCR analysis was performed on a ViiA7 machine (Life Technologies). Rats/group: 5, n = 10 eyes. Each eye was considered a separate sample. Relative Htra1 mRNA expression levels are shown as % of control (PBS).

Histology

The eyes of the remaining two companion animals from the test items and PBS group were removed, fixed in 10% neutral buffered formalin for 24 hours, trimmed, and embedded in paraffin. ISH RNAscope was then performed as described in Example 10.

Example 12. In vivo efficacy and pharmacokinetic study in rats, treated for 3, 7, and 14 days, intravitreal (IVT) injection, single dose.

Knockdown (KD) of mRNA levels of two selected HTRA1 LNA oligonucleotides targeting a “hotspot” between locations 33042-33064 (SEQ ID NO 147) of human HTRA1 pre-mRNA was observed in the retina. This was observed by both qPCR and ISH readings (see Figures 7A and/or B and the table below).

The knockdown variation was relatively large; see the standard deviations listed in the table. When the dose-response curves of oligonucleotide content versus residual HTRA1 mRNA levels were plotted, it can be seen that this variation was at the administration level (see Figure 7C).

animal

All experiments were conducted on albino Sprague-Dawley rats.

Compounds and Dosing Procedures

Animals were anesthetized with isoflurane. On day 1 of the study, test items and negative controls (PBS) were administered intravitreally in both eyes of the anesthetized animals (3 μL each time).

euthanasia

At the end of their lifespan (days 4, 8, or 15 of the study), the rats were anesthetized and euthanized by decapitation.

Oligonucleotide content measurement and quantification of Htra1 RNA expression

Oligonucleotide content measurement and Htra1 mRNA expression quantification were performed as described in Example 10.

The relative residual Htra1 mRNA expression level was shown as % of the control (PBS).

Histology

Histology was performed as described in Example 10.

Example 13. In vivo pharmacokinetic and pharmacodynamic (PK/PD) study of cynomolgus monkeys (non-human primates, NHP), treated for 21 days, intravitreal injection (IVT), single dose.

For one selected HTRA1 LNA oligonucleotide 145.3 (targeting the “hotspot” at positions 33042-33064 in human HTRA1 pre-mRNA), knockdown of mRNA in the retina and protein levels in the retina and vitreous were observed (see Figure 8).

animal

All experiments were conducted on cynomolgus monkeys (Macaca fascicularis).

Compounds and Dosing Procedures

Buprenorphine was administered for analgesia two days before and after the injection of the test compound. Animals were anesthetized with ketamine and xylazine via intramuscular injection. On day 1 of the study, after local administration of tetracaine, 50 μL of the test item and negative control (PBS) were administered intravitreally in both eyes of the anesthetized animals.

euthanasia

At the end of their lifespan (day 22), all monkeys were euthanized by intraperitoneal injection of an overdose of pentobarbital.

Oligonucleotide content measurement and Htra1 RNA expression quantification were performed using qPCR.

Immediately after euthanasia, eye tissue was rapidly and carefully sectioned on ice and stored at -80°C until shipment. Retinal samples were lysed in 700 μL MagNa Pure 96 LC RNA separation tissue buffer and homogenized 2 x 1.5 min using a Precellys homogenizer with one stainless steel bead added per 2 ml tube, followed by incubation at room temperature for 30 min. The samples were centrifuged at 13,000 rpm for 5 min. Half of the samples were used for bioanalysis, and the other half were used for direct RNA extraction.

For bioanalysis, samples are diluted 10–50-fold using a hybridization ELISA method to measure oligonucleotide content. Biotinylated LNA-capture probes and digoxigenin-conjugated LNA-detection probes (35 nM in 5xSSCT, each complementary to one end of the LNA oligonucleotide to be detected) are mixed with diluted homogenate or relevant standards and incubated at room temperature for 30 min. The mixture is then added to a streptavidin-coated ELISA plate (Nunc catalog number 436014).

The plate was incubated at room temperature for 1 hour and washed in 2xSSCT (300 mM sodium chloride, 30 mM sodium citrate, and 0.05% v/v Tween-20, pH 7.0). The captured LNA duplexes were detected using an anti-DIG antibody conjugated with alkaline phosphatase (Roche Applied Sciencecat. No. 11093274910) and the alkaline phosphatase substrate system (Blue Phos substrate, KPL product code 50-88-00). The amount of oligomeric complex was measured as absorbance at 615 nm on a Biotek reader.

For RNA extraction, a large-volume RNA kit for cells (05467535001, Roche) was used with the MagNA Pure 96 system, following the procedure: Tissue FF standard LV3.1 was prepared according to the manufacturer's instructions, including DNase treatment. RNA quality controls and concentrations were measured using an Eon reader (Biotek). RNA concentrations were normalized between samples, followed by cDNA synthesis and qPCR in a one-step reaction using the qScript XLT one-step RT-qPCR ToughMix Low ROX, 95134-100 (Quanta Biosciences). The following TaqMan primer assays were used for single-stranded body reactions: Htra1, Mf01016150_, Mf01016152_m1, and Rh02799527_m1 from Life Technologies, as well as housekeeping genes, ARFGAP2, Mf01058488_g1, and Rh01058485_m1, and ARL1, Mf02795431_m1. qPCR analysis was run on a ViiA7 machine (Life Technologies). Eye/group: n = 3 eyes. Each eye was considered a separate sample. Relative Htra1 mRNA expression levels are shown as % of control (PBS).

Histology

The eyeball was removed and fixed in 10% neutral buffered formalin for 24 hours, then trimmed and embedded in paraffin.

For ISH analysis, 4 μm thick formalin-fixed, paraffin-embedded retinal tissue sections were treated using RNAscope 2.5 VS Probe-Mmu-HTRA1 (REF 486979, Advanced Cell Diagnostics, Inc.) with the fully automated Ventana Discovery ULTRA staining module (program: mRNADiscovery Ultra Red 4.0-v0.00.0152). Fastred hematoxylin II counterstaining agent was used.

HTRA1 protein was quantified using plate-based immunoprecipitation mass spectrometry (IP-MS).

Sample preparation, retina

The retina was homogenized in 4 volumes (w/v) of RIPA buffer containing protease inhibitors (completely EDTA-free, Roche) (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, Millipore) using Precellys 24 (5500, 15 s, 2 cycles). The homogenate was centrifuged (13,000 rpm, 3 min) and the protein content of the supernatant was determined (Pierce BCA protein assay).

Sample preparation, vitreous body

The vitreous humor (300 μl) was diluted with 5x RIPA buffer (final concentration: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) containing protease inhibitors (completely EDTA-free, Roche) and homogenized using Precellys 24 (5500, 15 s, 2 cycles). The homogenate was centrifuged (13,000 rpm, 3 min) and the protein content of the supernatant was determined (Pierce BCA protein assay).

Plate-based HTRA1 immunoprecipitation and trypsin digestion

96-well plates (Nunc MaxiSorp) were coated with anti-HTRA1 mouse monoclonal antibody (R&D MAB2916, 500 ng/well in 50 μl PBS) and incubated overnight at 4 °C. The plates were washed twice with PBS (200 μl) and blocked with 3% (w/v) BSA in PBS at 20 °C for 30 min, followed by two PBS washes. Samples (75 μg retina, 100 μg vitreous humor, in 50 μl PBS) were randomized and added to the plates, then incubated overnight at 4 °C on a shaker (150 rpm). The plates were then washed twice with PBS and once with water. 10 mM DTT (30 μl) in 50 mM TEAB was added to each well, and the plates were incubated at 20 °C for 1 h to reduce cysteine thiol groups. Then, 5 μl of 150 mM iodoacetamide in 50 mM TEAB was added to each well, and the mixture was incubated in the dark at 20 °C for 30 min to block cysteine sulfhydryl groups. 10 μl of digestion solution (final concentration: 1.24 ng/μl trypsin, 20 fmol/μl BSA peptide, 26 fmol/μl isotopically labeled HTRA1 peptide, 1 fmol/μl iRT peptide, Biognosys) was added to each well, and the mixture was incubated overnight at 20 °C.

Quantification of HTRA1 peptide by targeted mass spectrometry (selective reaction monitoring, SRM)

Mass spectrometry analysis was performed on an Ultimate RSLCnano LC coupled to a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Scientific). Samples (20 μL) were injected directly from a 96-well plate used for IP and loaded at 5 μL/min onto an Acclaim Pepmap 100 capture column (100 μm x 2 cm, C18, 5 μm, Thermo Scientific) in loading buffer (0.5% v/v formic acid, 2% v/v ACN) for 6 min. The peptides were then separated on a PepMap Easy-SPRAY analytical column (75 μm x 15 cm, 3 μm, Thermo Scientific) with an integrated electrospray emitter heated to 40 °C using the following gradients at a flow rate of 250 nL/min: 6 min, 98% buffer A (2% ACN, 0.1% formic acid), 2% buffer B (ACN + 0.1% formic acid); 36 min, 30% buffer B; 41 min, 60% buffer B; 43 min, 80% buffer B; 49 min, 80% buffer B; 50 min, 2% buffer B. The TSQ Quantiva was run in SRM mode with the following parameters: cycle time, 1.5 s; spray voltage, 1800 V; collision pressure, 2 mTorr; Q1 and Q3 resolution, 0.7 FWHM; ion transfer tube temperature, 300 °C. SRM conversions were obtained for the HTRA1 peptide “LHRPPVIVLQR” and its isotopically labeled synthetic form (L-[U-13C, U-15N]R) (which was used as an internal standard). Data analysis was performed using Skyline version 3.6.

Claims (17)

1. An antisense oligonucleotide of the formula T _s A _s T _{s t s t s} a _{s c s} c _{s t s g s} g s t _s T s G _s T _s T (SEQ ID NO 143), wherein uppercase letters represent LNA nucleoside units, lowercase letters represent DNA nucleoside units, and the subscript s represents the linkage between phosphate thioester nucleosides. 2. The antisense oligonucleotide according to claim 1, wherein the LNA nucleoside is β-D-oxyLNA nucleoside. 3. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide is of the following formula: 4. A pharmaceutically acceptable salt of the antisense oligonucleotide according to any one of claims 1-3. 5. The pharmaceutically acceptable salt according to claim 4, wherein the salt is a sodium salt. 6. The pharmaceutically acceptable salt of claim 4, wherein the salt is a potassium salt. 7. A pharmaceutical composition comprising the antisense oligonucleotide of any one of claims 1-3 and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises a pharmaceutically acceptable diluent. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable diluent is phosphate-buffered saline. 10. The pharmaceutical composition according to any one of claims 7-9, wherein the oligonucleotide is in the form of a pharmaceutically acceptable salt. 11. A pharmaceutical composition comprising a pharmaceutically acceptable salt and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant of any one of claims 4-6. 12. The pharmaceutical composition according to any one of claims 7-9, wherein the pharmaceutically acceptable salt is a sodium or potassium salt. 13. An antisense oligonucleotide according to any one of claims 1-3, or a pharmaceutically acceptable salt according to any one of claims 4-6, or a pharmaceutical composition according to any one of claims 7-12, for use in a medicament. 14. An antisense oligonucleotide according to any one of claims 1-3, or a pharmaceutically acceptable salt according to any one of claims 4-6, or a pharmaceutical composition according to any one of claims 7-12, for use in the treatment of macular degeneration. 15. Use of an antisense oligonucleotide of any one of claims 1-3, or a pharmaceutically acceptable salt of any one of claims 4-6, or a pharmaceutical composition of any one of claims 7-12 in the preparation of a medicament for the treatment or prevention of macular degeneration. 16. The antisense oligonucleotide of claim 14, a pharmaceutically acceptable salt or pharmaceutical composition, or the use of claim 15, wherein the macular degeneration is selected from wet AMD, dry AMD, geographic atrophy, and intermediate dAMD. 17. An in vitro method for regulating HTRA1 expression in target cells expressing HTRA1, the method comprising administering to the cells an effective amount of the antisense oligonucleotide of any one of claims 1-3, a pharmaceutically acceptable salt of any one of claims 4-6, or a pharmaceutical composition of any one of claims 7-12.