HK40021168A

HK40021168A - Antisense oligonucleotides for modulating htra1 expression

Info

Publication number: HK40021168A
Application number: HK62020009573.0A
Authority: HK
Inventors: 鲁文‧阿尔瓦雷斯‧桑切斯; 罗伯托‧依阿哥尼; 彼得‧哈哲多恩; 苏珊娜‧凯姆乐; 索仁‧奥托森; 辛德瑞‧特劳斯塔森; 海迪‧赖伊‧胡德布施; 吕克‧彼泽森; 马尔科‧贝雷拉; 安德雷斯‧迪克曼
Original assignee: 豪夫迈·罗氏有限公司
Priority date: 2017-06-01
Filing date: 2018-05-30
Publication date: 2020-10-30

Description

Antisense oligonucleotides for modulating expression of HTRA1

Technical Field

The present invention relates to antisense oligonucleotides (oligomers) complementary to HTRA1, which result in modulation of HTRA1 expression. Modulation of HTRA1 expression is beneficial for a range of medical conditions such as macular degeneration, e.g., age-related macular degeneration.

Background

Human High Temperature Requirement A (HTRA) family of serine proteases ubiquitously expressed PDZ proteases, which are involved in maintaining protein homeostasis in extracellular compartments through the dual functions of proteases and chaperones. HTRA proteases are associated with organization of the extracellular matrix, cell proliferation and senescence. Modulation of HTRA activity has been associated with severe disease including Duchenne muscular dystrophy (Bakay et al 2002, Neurousacu. Disord.12: 125-, 2002, Oncogene 21: 6684-6688). In ovarian cancer, HTRA1 expression is also down-regulated. In ovarian cancer cell lines, overexpression of HTRA1 induced cell death, while expression of antisense HTRA1 promoted anchorage-independent growth (Chien et al, 2004, Oncogene 23: 1636-.

In addition to its effects on the IGF pathway, HTRA1 also inhibits signaling of TGF-beta family growth factors (Oka et al, 2004, Development 131: 1041-1053). HTRA1 is capable of cleaving Amyloid Precursor Protein (APP), and HTRA1 inhibitors cause accumulation of a β peptide in cultured cells. Thus, HTRA1 is also associated with Alzheimer's disease (Grau et al, 2005, Proc. nat. Acad. Sci. USA.102: 6021-6026).

Furthermore, HTRA1 has been observed to be upregulated and appears to be associated with Duchenne muscular dystrophy (Bakay et al 2002, Neurousacu. Disord.12: 125-61141) and osteoarthritis (Grau et al 2006, JBC 281: 6124-6129) and AMD (Fritsche, et al Nat Gen 201345 (4): 433-9).

A Single Nucleotide Polymorphism (SNP) in the HTRA1 promoter region (rs11200638) was associated with a 10-fold increase in the risk of developing age-related macular degeneration (AMD). Furthermore, the HTRA1 SNP is in linkage disequilibrium with the ARMS2 SNP (rs10490924) associated with an increased risk of developing age-related macular degeneration (AMD). The risk allele is associated with a 2-3 fold increase in HTRA1mRNA and protein expression, and HTRA is present in drusen in AMD patients (Dewan et al, 2006, Science 314: 989-. Overexpression of HtrA1 induced an AMD-like phenotype in mice. hHTRA transgenic mice (Veierkottn, Ploss 2011) revealed degradation of the Bruch's membranous elastic layer, identified choroidal vascular abnormalities (Jones, PNAS, 2011), and increased Polypoidal Choroidal Vasculopathy (PCV) lesions (Kumar, IOVS 2014). Furthermore, Bruch membrane damage has been reported in hHTRA1 Tg mice, which determines 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 the age of 65. With the onset of AMD, light-sensitive photoreceptor cells in the back of the eye, metabolically support their underlying pigment epithelium and the clear central vision they provide, are gradually lost. Age is a major risk factor for the onset of AMD: the likelihood of AMD is tripled after age 55. Smoking, light iris color, gender (greater risk for women), obesity, and repeated exposure to UV radiation also increase the risk of AMD. AMD progression can be divided into three stages: 1) early AMD, 2) intermediate AMD and 3) advanced AMD. Advanced AMD has two forms: dry AMD (also known as geographic atrophy, GA) and wet AMD (also known as exudative AMD). Dry AMD is characterized by the loss of photoreceptors and retinal pigment epithelial cells, which results in vision loss. Wet AMD is associated with pathological choroidal (also known as subretinal) neovascularization. Leakage of abnormal blood vessels formed during this process can damage the macula and impair vision, eventually leading to blindness. In certain instances, patients may exhibit pathologies associated with both types of advanced AMD. The treatment strategy for wet AMD requires frequent injections into the eye and focuses primarily on delaying the progression of the disease. There is currently no treatment for dry AMD. Thus, there is an unmet medical need to provide effective medicaments for the treatment of macular degenerative conditions such as wet and dry AMD. WO 2008/013893 claims a composition for treating a subject with age-related macular degeneration comprising a nucleic acid molecule comprising an antisense sequence that hybridizes to the HTRA1 gene or mRNA: antisense molecules are not disclosed.

WO2009/006460 provides sirnas targeting HTRA1 and their use in the treatment of AMD.

Object of the Invention

The invention provides antisense oligonucleotides that modulate HTRA1 in vivo or in vitro. The present invention identifies cryptic target sequence motifs present in human HTRA1mRNA (including pre-mRNA) that can be targeted by antisense oligonucleotides to produce potent HTRA1 inhibition. The invention also provides effective antisense oligonucleotide sequences and compounds capable of inhibiting HTRA1, and their use in treating diseases or disorders indicative of HTRA 1.

Disclosure of Invention

The present invention relates to oligonucleotides that target mammalian HTRA1 nucleic acids, i.e., are capable of inhibiting expression of HTRA1 and treating or preventing diseases associated with the function of HTRA 1. The oligonucleotide targeting HTRA1 is an antisense oligonucleotide, i.e., complementary to its HTRA1 nucleic acid target.

The oligonucleotides of the invention may be in the form of a pharmaceutically acceptable salt, for example a sodium or potassium salt.

Accordingly, the invention provides antisense oligonucleotides comprising a contiguous nucleotide sequence of 10-30 nucleotides in length having at least 90% complementarity, such as being fully complementary, to a mammalian HTRA1 nucleic acid, such as SEQ ID NO1, SEQ ID NO2, SEQ ID NO 3 or SEQ ID NO 4.

In another aspect, the invention provides a pharmaceutical composition comprising an oligonucleotide of the invention and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.

The present invention provides LNA antisense oligonucleotides, such as LNA gapmer oligonucleotides, comprising a contiguous nucleotide sequence of 10-30 nucleotides in length having at least 90% complementarity (such as being fully complementary) to an HTRA1 nucleic acid, such as a sequence selected from the group consisting of SEQ ID NO1, SEQ ID NO2, SEQ ID NO 3 or SEQ ID NO 4.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of 10-30 (such as 12 to 22) nucleotides, wherein the contiguous nucleotide region is at least 90% complementary, such as 100% complementary, to SEQ ID NO 113.

The present invention provides an antisense oligonucleotide 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide region of 10-30 (such as 12-22) nucleotides that is complementary to at least 90% (such as 100%) of: SEQ ID NO 113:

the reverse complement of SEQ ID NO 113 is SEQ ID NO 119:

the present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of 10-30 (such as 12 to 22) nucleotides, wherein the contiguous nucleotide region is at least 90% complementary, such as 100% complementary, to SEQ ID NO 114.

The present invention provides an antisense oligonucleotide 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide region of 10-30 (such as 12-22) nucleotides that is complementary to at least 90% (such as 100%) of: SEQ ID NO 114: 5'

The reverse complement of SEQ ID NO 114 is SEQ ID NO 120:

the present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of 10-30 (such as 12 to 22) nucleotides, wherein the contiguous nucleotide region is at least 90% complementary, such as 100% complementary, to SEQ ID NO 115.

The present invention provides an antisense oligonucleotide 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide region of 10-30 (such as 12-22) nucleotides that is complementary to at least 90% (such as 100%) of: SEQ ID NO 115: 5'

The reverse complement of SEQ ID NO 115 is SEQ ID NO 121:

the present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of 10-30 (such as 12 to 22) nucleotides, wherein the contiguous nucleotide region is at least 90% complementary, such as 100% complementary, to SEQ ID NO 116.

The present invention provides an antisense oligonucleotide 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide region of 10-30 (such as 12-22) nucleotides that is complementary to at least 90% (such as 100%) of: SEQ ID NO 116: 5'

The reverse complement of SEQ ID NO 116 is SEQ ID NO 122:

the present invention provides an antisense complementing nucleotide comprising a contiguous nucleotide region of 10-30 (such as 12 full 22) nucleotides, wherein said contiguous nucleotide region is at least 90% complementary, such as 100% complementary, to SEQ ID NO 117.

The present invention provides an antisense oligonucleotide 10-30 nucleotides in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide region of 10-30 (such as 12-22) nucleotides that is complementary to at least 90% (such as 100%) of: SEQ ID NO 117: 5'

The reverse complement of SEQ ID NO 117 is SEQ ID NO 123:

in some embodiments, the antisense oligonucleotide of the invention is not sequence 5 'gcaatgtgtaagaagt 3' (SEQ ID NO 112). In some embodiments, the antisense oligonucleotides of the invention do not comprise or consist of the sequence 5 'gcaatgtgtaagaagt 3'. In some embodiments, the antisense oligonucleotides of the invention do not comprise or consist of 10 or more contiguous nucleotides present in sequence 5 'gcaatgtgtaagaagt 3'. In some embodiments, the oligonucleotide of the invention is not 5 'GCAatgtgtaagaAGT 3', wherein the capital letters represent LNA nucleosides (using β -D-oxy LNA nucleosides), all LNA cytosines are 5-methylcytosine, the lowercase letters represent DNA nucleosides, and the DNA cytosine with the superscript m represents 5-methyl C-DNA nucleosides. All internucleoside linkages (linkages) are phosphorothioate internucleoside linkages.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 contiguous nucleotides present in any one of SEQ ID NOs 5 to 111. The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 12 contiguous nucleotides present in any one of SEQ ID NOs 5 to 111. The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 14 contiguous nucleotides present in any one of SEQ ID NOs 5 to 111. The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 15 or at least 16 contiguous nucleotides present in any one of SEQ ID NOs 5 to 111. The present invention provides an antisense oligonucleotide, wherein the contiguous nucleotide sequence of said oligonucleotide comprises or consists of a nucleobase sequence selected from the group consisting of any one of SEQ ID NOs 5 to 111.

The present invention provides an antisense oligonucleotide comprising the sequence presented in SEQ ID NO 118: a contiguous nucleotide region of at least 10 or at least 12, or at least 14 or at least 15 or at least 16 contiguous nucleotides of 5 'CTTCTTCTATCTACGCATTG 3'. The reverse complement of SEQ ID NO 118 is SEQ ID NO 231: CAATGCGTAGATAGAAGAAG are provided.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, at least 13, or at least 14 or at least 15 or at least 16 contiguous nucleotides complementary to SEQ ID NO 231.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or 16 contiguous nucleotides present in SEQ ID NO 67.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or 16 contiguous nucleotides present in SEQ ID NO 86.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or at least 16 or at least 17 or 18 contiguous nucleotides present in SEQ ID NO 73.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or 16 contiguous nucleotides complementary to SEQ ID NO 186.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or 16 contiguous nucleotides complementary to SEQ ID NO 205.

The present invention provides an antisense oligonucleotide comprising a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or at least 16 or at least 17 or 18 contiguous nucleotides complementary to SEQ ID NO 192.

The present invention provides an oligonucleotide comprising or consisting of an oligonucleotide selected from the group consisting of:

T_sT_s ^mC_st_sa_st_sc_st_sa_s ^mc_sg_sc_sa_sT_sT_sG(SEQ ID NO 67，1)，

^mC_sT_sT_s ^mC_st_st_sc_st_sa_st_sc_st_sa_s ^mc_sg_sc_sA_st (SEQ ID NO73, 1), and

T_sA_s ^mC_sT_st_st_sa_sa_st_sa_sg_sc_sT_s ^mC_sA_sA(SEQ ID NO 86，1)；

wherein capital letters represent beta-D-oxyLNA nucleosides, lowercase letters are DNA nucleosides, subscript s represents phosphorothioate internucleoside linkages,^mc represents a 5 methylcytosine beta-D-oxoLNA nucleoside, and^mc represents 5 methylcytosine DNA nucleoside.

The present invention provides oligonucleotides of the formula:

T_sT_s ^mC_st_sa_st_sc_st_sa_s ^mc_sg_sc_sa_sT_sT_sG(SEQ ID NO 67，1)，

The present invention provides oligonucleotides of the formula:

^mC_sT_sT_s ^mC_st_st_sc_st_sa_st_sc_st_sa_s ^mc_sg_sc_sA_sT(SEQ ID NO 73，1)

The present invention provides oligonucleotides of the formula:

T_sA_s ^mC_sT_st_st_sa_sa_st_sa_sg_sc_sT_s ^mC_sA_sA(SEQ ID NO 86，1)

The invention provides oligonucleotides provided in the examples.

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

The invention provides pharmaceutically acceptable salts of the oligonucleotides or conjugates of the invention.

In another aspect, the invention provides an in vivo or in vitro method of modulating HTRA1 expression in a cell expressing HTRA1 by administering to the cell an effective amount of an oligonucleotide, conjugate, or composition of the invention.

In another aspect, the invention provides a method for treating or preventing a disease, disorder or dysfunction associated with the in vivo activity of HTRA1, the method comprising administering to a subject suffering from or susceptible to the disease, disorder or dysfunction a therapeutically or prophylactically effective amount of an oligonucleotide of the invention or a conjugate thereof.

In another aspect, the oligonucleotides or compositions of the invention are useful for treating or preventing macular degeneration, and other disorders associated with HTRA 1.

The invention provides an oligonucleotide or conjugate of the invention for use in the treatment of a disease or condition selected from the list comprising: duchenne muscular dystrophy, arthritis such as osteoarthritis, familial ischemic cerebrovascular-vascular disease, alzheimer's disease and parkinson's disease.

The invention provides an oligonucleotide or conjugate of the invention for use in the treatment of macular degeneration, such as wet or dry age-related macular degeneration (such as wAMD, dAMD, geographic atrophy, early AMD, intermediate AMD) or diabetic retinopathy.

The invention provides the use of an oligonucleotide, conjugate or composition of the invention in the manufacture of a medicament for the treatment of macular degeneration, such as wet or dry macular degeneration (wAMD, dAMD, geographic atrophy, mid-term dAMD) or diabetic retinopathy.

The present invention provides the use of an oligonucleotide, conjugate or compound of the invention in the manufacture of a medicament for the treatment of a disease or condition selected from the group consisting of: duchenne muscular dystrophy, arthritis such as osteoarthritis, familial ischemic cerebrovascular disease, alzheimer's disease and parkinson's disease.

The present invention provides a method of treating a subject having a disease or disorder selected from the group consisting of: duchenne muscular dystrophy, arthritis such as osteoarthritis, familial ischemic cerebrovascular disease, alzheimer's disease and parkinson's disease, said method comprising the step of administering to said subject an effective amount of an oligonucleotide, conjugate or composition of the invention.

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

The invention provides a method of treating a subject suffering from an ocular disease, such as macular degeneration, such as wet or dry age-related macular degeneration (e.g., wAMD, dAMD, geographic atrophy, intermediate AMD) or diabetic retinopathy, the method comprising administering at least two doses of an oligonucleotide of the invention, or a pharmaceutically acceptable salt thereof, in an intraocular injection at a dose of about 10 μ g to 200 μ g, wherein the dose interval between successive administrations is at least 4 weeks (i.e., "dose interval ≧ 4 weeks) or at least one month (i.e.," dose interval ≧ 1 month).

Brief Description of Drawings

Figure 1 screening of n-231 HTRA1 LNA oligonucleotide library at 5 μ M in U251 cell line. Residual HTRA1mRNA expression levels were determined by qPCR and are shown as% of control (PBS treated cells). The n-10 oligonucleotides located between positions 53113-53384 are relatively active.

Figure 2 screening of n-210 HTRA1 LNA oligonucleotide library at 5 μ M in U251 cell line. Residual HTRA1mRNA expression levels were determined by qPCR and are shown as% of control (PBS treated cells). The n-33 oligonucleotides located between positions 53113-53384 are relatively active.

Figure 3 n-305 HTRA1 LNA oligonucleotide libraries were screened at 5 and 25 μ M in U251 and ARPE19 cell lines, respectively. Residual HTRA1mRNA expression levels were determined by qPCR and are shown as% of control (PBS treated cells). The n 95 oligonucleotides located between positions 53113-53384 are relatively active compared to the remaining positions.

Fig. 4 dose response of HTRA1mRNA levels after treatment of human primary RPE cells with LNA oligonucleotides, treatment for 10 days. Scrambled (scrambled) sequences are control oligonucleotides having a sequence that is scrambled regardless of the Htra1 target sequence.

FIG. 5 NHP PK/PD study, IVT dosing, 25 μ g/eye. A) HTRA1mRNA levels in the retina as measured by qPCR. B) Oligonucleotide content in the retina measured by oligonucleotide ELISA. C) HTRA1mRNA levels as indicated by ISH. D-E) quantification of HTRA1 protein levels in retina and vitreous by IP-MS, respectively. Dots show data for individual animals. Error bars show the standard error of the technical replicates (n-3). F-G) reduction of the level of HTRA1 protein in retina and vitreous, which is illustrated by western blot (western blot), respectively.

FIG. 6A Compound of the present invention (Compound ID NO 67, 1). The compounds may be in the form of pharmaceutically acceptable salts, such as sodium or potassium salts.

FIG. 7 shows a compound A according to the present invention (Compound ID NO 86, 1). The compounds may be in the form of pharmaceutically acceptable salts, such as sodium or potassium salts.

FIG. 8 Compound (Compound ID NO73, 1) according to the invention. The compounds may be in the form of pharmaceutically acceptable salts, such as sodium or potassium salts.

Figure 9, compound 67, 1: examples of pharmaceutically acceptable salts of M + are suitable cations, typically positive metal ions, such as sodium or potassium ions. The stoichiometric ratio of cation to oligonucleotide anion will depend on the charge of the cation used. Suitably, a cation having one, two or three positive charges (M may be used)⁺，M⁺⁺Or M⁺⁺⁺). For illustrative purposes, with divalent cations (e.g., Ca)^2+.) In contrast, twice the amount of singly + charged cations (monovalent), such as Na + or K +, is required.

Figure 10 example of pharmaceutically acceptable salts of compound 86, 1: relating to cation M⁺Please refer to the legend of fig. 9.

Figure 11, examples of pharmaceutically acceptable salts of compound 73, 1: relating to cation M⁺Please refer to the legend of fig. 9.

Figure 12a intravitreal administration of compounds #15, 3 and #17 in cynomolgus monkeys (cynomolgus monkeys) and aqueous humor samples collected on days 3, 8, 15 and 22 post-injection. Proteins from undiluted samples were analyzed by capillary electrophoresis using a Peggy Sue device (protein samples). HTRA1 was detected using a custom polyclonal rabbit antiserum. Data from animals # J60154 (vehicle), J60158(c.id #15, 3), J60162(c.id #17) are presented.

Figure 12b signal intensity was quantified by comparison with purified recombinant (S328A mutant) HTRA1 protein (Origene, # TP 700208). The calibration curve is shown here.

Fig. 12c. top view: the calculated HTRA1 aqueous humor concentrations from individual animals were plotted against time post-injection. The following figures: the mean HTRA1 concentration for the vehicle group at each time point was determined and the corresponding relative concentrations for the treated animals were calculated. Hollow circle: individual values, filled circles: group mean values. A reduction in% HTRA1 at day 22 is shown.

Figure 13 HTRA1mRNA was plotted against HTRA1 protein levels in the aqueous humor (blue diamonds) or retina (red squares) of cynomolgus monkeys treated with various LNA molecules targeting HTRA1 transcripts. Values are expressed as percentages normalized to PBS control.

Figure 14 correlation of HTRA1 protein in cynomolgus monkey aqueous humor treated with various LNA molecules targeting HTRA1 transcripts with (a) HTRA1 protein in retina and (B) HTRA1mRNA in retina. Values are expressed as percentages normalized to PBS control.

Definition of

Oligonucleotides

As used herein, the term "oligonucleotide" is defined as a molecule commonly understood by a skilled artisan to comprise two or more covalently linked nucleosides. Such covalently bound 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 nucleobase portion of a covalently linked nucleotide or nucleoside or a modified sequence or order thereof. The oligonucleotides of the invention are artificial and chemically synthesized and are usually purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides or nucleotides.

Antisense oligonucleotides

The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly to a contiguous sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded, and thus are not sirnas. Preferably, the antisense oligonucleotides of the invention are single stranded.

Continuous nucleotide region

The term "contiguous nucleotide region" refers to the region of an oligonucleotide that is complementary to a target nucleic acid. The term may be used interchangeably herein with the term "contiguous nucleotide sequence" or "contiguous nucleobase sequence" and the term "oligonucleotide motif sequence". In some embodiments, all nucleotides of the oligonucleotide are present in a contiguous nucleotide region. In some embodiments, the oligonucleotide comprises a contiguous nucleotide region, and may optionally comprise other nucleotides, such as a nucleotide linker region that may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the internucleoside linkages present between nucleotides of the contiguous nucleotide region are all phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide region comprises one or more sugar modified nucleosides.

Nucleotide, its preparation and use

Nucleotides are building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, nucleotides include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, comprise a ribose moiety, a nucleobase moiety and one or more phosphate groups (which are not present in nucleosides). Nucleosides and nucleotides can also be interchangeably referred to as "units" or "monomers".

Modified nucleosides

The term "modified nucleoside" or "nucleoside modification" as used herein refers to a nucleoside that is modified by the introduction of one or more modifications of the sugar moiety or (nucleobase) moiety as compared to an equivalent DNA or RNA nucleoside. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer".

Modified internucleoside linkages

The term "modified internucleoside linkage" is defined as a linkage other than a Phosphodiester (PO) linkage, which covalently couples two nucleosides together, as is commonly understood by the skilled artisan. Nucleotides having modified internucleoside linkages are also referred to as "modified nucleotides". In some embodiments, the modified internucleoside linkages increase nuclease resistance of the oligonucleotide compared to phosphodiester linkages. For naturally occurring oligonucleotides, internucleoside linkages include phosphate groups that result in phosphodiester linkages between adjacent nucleosides. Modified internucleoside linkages are particularly useful for stabilizing oligonucleotides for use in vivo, and for preventing nuclease cleavage of a nucleotide region of DNA or RNA (such as within the gap region of a gapmer oligonucleotide, and within a modified nucleotide region) in an oligonucleotide of the invention.

In one embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from a native phosphodiester to a linkage that is, for example, more resistant to nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay, such as Snake Venom Phosphodiesterase (SVPD), both of which are well known in the art. An internucleoside linkage capable of enhancing nuclease resistance of an oligonucleotide is referred to as a nuclease-resistant internucleoside linkage. In some embodiments, all internucleoside linkages of the oligonucleotide or a contiguous nucleotide sequence thereof are modified. It will be appreciated that in some embodiments, the nucleoside linking the oligonucleotide of the invention to a non-nucleotide functional group (such as a conjugate) may be a phosphodiester. In some embodiments, all of the internucleoside linkages of the oligonucleotide or the contiguous nucleotide sequence thereof are nuclease resistant internucleoside linkages.

In some embodiments, the modified internucleoside linkage can be a phosphorothioate internucleoside linkage. In some embodiments, the modified internucleoside linkages are compatible with rnase H recruitment by the oligonucleotides of the invention, e.g., phosphorothioate.

In some embodiments, the internucleoside linkage comprises a sulfur (S), such as a phosphorothioate internucleoside linkage.

Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments, all of the internucleoside linkages of the oligonucleotide, or a contiguous nucleotide sequence thereof, are phosphorothioate.

Nucleobases

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 in nucleic acid hybridization. In the context of the present invention, the term nucleobase also covers modified nucleobases, which may differ from naturally occurring nucleobases, but which play a role during nucleic acid hybridization. As used herein, "nucleobase" refers to naturally occurring nucleobases, such as adenine, guanine, cytosine, thymine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research, volume 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry supply.371.4.1.

In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine (pseudoisocytosine), 5-methylcytosine, 5-thio-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolouracine, 2-thio-uracil, 2' thio-thymine, inosine, diaminopurine, 6-aminopurine, 2, 6-diaminopurine and 2-chloro-6-aminopurine.

Nucleobase moieties can be represented by the letter code of each corresponding nucleobase, e.g., a, T, G, C or U, wherein each letter can optionally include a modified nucleobase having an equivalent function. For example, in exemplary oligonucleotides, the nucleobase moiety is selected from the group consisting of A, T, G, C and 5-methylcytosine. Optionally, for LNA gapmer, 5-methylcytosine LNA nucleosides can be used. In some embodiments, the cytosine nucleobase in the 5 'cg 3' motif is a 5-methylcytosine.

Modified oligonucleotides

The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar modified nucleosides and/or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term that has been used in the literature to describe oligonucleotides having modified nucleosides.

Complementarity

The term complementarity describes the ability of a nucleoside/nucleotide to undergo Watson-Crick base pairing. Watson-Crick base pairs are guanine (G) -cytosine (C) and adenine (A) -thymine (T)/uracil (U). It is to be understood that the oligonucleotide may comprise a nucleoside having a modified nucleobase, e.g. cytosine is typically replaced with 5-methylcytosine, and thus the term complementarity encompasses watson-crick base pairing between unmodified and modified nucleobases (see e.g. Hirao et al (2012) Accounts of Chemical Research, volume 45, p.2055 and Bergstrom (2009) current protocols in Nucleic Acid Chemistry supply.371.4.1).

As used herein, the term "% complementary" refers to the percentage of nucleotides of a contiguous nucleotide region or sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is complementary at a given position to a contiguous nucleotide sequence at a given position in an individual nucleic acid molecule (e.g., a target nucleic acid) (i.e., forms watson-crick base pairs). The percentage is calculated by counting the number of aligned bases forming a pair between two sequences, dividing by the total number of nucleotides in the oligonucleotide, and then multiplying by 100. In this comparison, the alignment (not forming base pairs) of nucleobases/nucleotides is called mismatch.

It will be understood that when referring to complementarity between two sequences, the determination of complementarity is measured over the shorter of the two sequences, such as over a contiguous nucleotide region or length of the sequences.

The term "fully complementary" refers to 100% complementarity. In the absence of a term value or mismatch indication of% complementary means fully complementary.

Identity of each other

As used herein, the term "identity" refers to the percentage of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is identical at a given position to the contiguous nucleotide sequence at a given position in an individual nucleic acid molecule (e.g., a target nucleic acid) (i.e., in terms of the ability to form watson-crick base pairs with complementary nucleotides). The percentage is calculated by counting the number of aligned bases that are identical between the two sequences (including the gap), dividing by the total number of nucleotides in the oligonucleotide, and then multiplying by 100. Percent identity is (match x 100)/length of alignment area (with gap).

When determining the identity of a contiguous nucleotide region of an oligonucleotide, the identity is calculated over the entire length of the contiguous nucleotide region. Thus, in embodiments where the entire contiguous nucleotide sequence of the oligonucleotide is a contiguous region of nucleotides, the identity is therefore calculated over the entire length of the nucleotide sequence of the oligonucleotide. In this regard, the contiguous region of nucleotides may be identical to a region of the reference nucleic acid sequence, or in some embodiments may be identical to the entire reference nucleic acid. Unless otherwise indicated, sequences that are 100% identical to a reference sequence are referred to as identical.

For example, the reference sequence may be selected from the group consisting of any one of SEQ ID NOs 5 to 111.

However, if the oligonucleotide comprises additional nucleotides flanking a contiguous nucleotide region (e.g., region D' or D "), these additional flanking nucleotides may be ignored in determining identity. In some embodiments, identity may be calculated over the entire oligonucleotide sequence.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide region of at least 10 contiguous nucleotides identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide region of at least 12 contiguous nucleotides identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide region of at least 13 contiguous nucleotides identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide region of at least 14 contiguous nucleotides identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide region of at least 15 contiguous nucleotides identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide region of at least 16 contiguous nucleotides identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

In some embodiments, the contiguous nucleotide region comprises or consists of at least 10 contiguous nucleotides, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides, of the sequence of the group consisting of SEQ ID NO 113-118 or SEQ ID NO 5-111. In some embodiments, the entire contiguous sequence of oligonucleotides comprises or consists of at least 10 contiguous nucleotides of SEQ ID NO, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides.

In some embodiments, the contiguous sequence of oligonucleotides comprises or consists of at least 10 contiguous nucleotides of SEQ ID NO 119, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides.

In some embodiments, the contiguous sequence of oligonucleotides comprises or consists of at least 10 contiguous nucleotides of SEQ ID NO 120, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides.

In some embodiments, the contiguous sequence of oligonucleotides comprises or consists of at least 10 contiguous nucleotides of SEQ ID NO 121, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides.

In some embodiments, the contiguous sequence of oligonucleotides comprises or consists of at least 10 contiguous nucleotides of SEQ ID NO 122, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides.

In some embodiments, the contiguous sequence of oligonucleotides comprises or consists of at least 10 contiguous nucleotides of SEQ ID NO 123, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous nucleotides, such as from 12-22, such as from 14-18 contiguous nucleotides.

The present invention provides an antisense oligonucleotide comprising the sequence presented in SEQ ID NO 118: a contiguous nucleotide region of at least 10 or at least 12, or at least 13, or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 contiguous nucleotides of 5 'cttcttctatctacgcattg 3'.

In some embodiments, the contiguous nucleotide region comprises 10, 11, 12, 13, 14, 15, or 16 contiguous nucleotides identical to SEQ ID NO 67.

In some embodiments, the contiguous nucleotide region comprises 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous nucleotides identical to SEQ ID NO 73.

In some embodiments, the contiguous nucleotide region comprises the same 10, 11, 12, 13, 14, 15, or 16 contiguous nucleotides as SEQ ID NO 86.

The present invention provides antisense oligonucleotides 11-30 nucleotides in length, such as 12 to 20 nucleotides in length, wherein the oligonucleotides comprise a contiguous nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs 5-111.

The present invention provides antisense oligonucleotides comprising or consisting of a contiguous nucleotide sequence that is identical to a reference sequence selected from the group consisting of SEQ ID NOs 5-111 across at least 10 contiguous nucleotides of the reference sequence.

The present invention provides antisense oligonucleotides comprising or consisting of a contiguous nucleotide sequence that is identical to a reference sequence selected from the group consisting of SEQ ID NOs 5-111 across at least 12 contiguous nucleotides of the reference sequence.

The present invention provides antisense oligonucleotides comprising or consisting of a contiguous nucleotide sequence that is identical to a reference sequence selected from the group consisting of SEQ ID NOs 5-111 across at least 14 contiguous nucleotides of the reference sequence.

The present invention provides antisense oligonucleotides comprising or consisting of a contiguous nucleotide sequence that is the same as a reference sequence selected from the group consisting of SEQ ID NOs 5-111 across the length of the reference sequence.

Hybridization of

As used herein, the term "hybridizing" or "hybridization" is understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form hydrogen bonds between base pairs on opposite strands, thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of hybridization. Usually by melting temperature (T)_m) Described, the melting temperature is defined as the temperature at which half of the oligonucleotide is duplexed with the target nucleic acid. Under physiological conditions, T_mNot strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13: 515-. The standard state gibbs free energy Δ G ° is a more precise representation of binding affinity and is related to the dissociation constant (K) of the reaction by Δ G ═ rtln (kd)_d) Related, where R is the gas constant and T is the absolute temperature. Thus, the very low Δ G ° of the reaction between the oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and the target nucleic acid. Δ G ° is the energy associated with the reaction at an aqueous solution concentration of 1M, pH 7, and temperature of 37 ℃. Hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction for which Δ G ° is less than zero. Δ G ° can be measured experimentally, for example, by subjectingIsothermal Titration Calorimetry (ITC) is performed as described, for example, in Hansen et al, 1965, chem. Co mM.36-38 and Holdgate et al, 2005, Drug Discov Today. The skilled person will know that commercial equipment can be used for the "Δ G ° measurement. Δ G can also be measured by using Santa Lucia, 1998, Proc Natl Acad Sci USA.95: the nearest neighbor model described in 1460-: 11211-11216 and McTigue et al, 2004, Biochemistry 43: 5388-. In order to have the possibility of modulating its intended nucleic acid target by hybridization, for oligonucleotides of 10-30 nucleotides in length, the oligonucleotides of the invention hybridize with the target nucleic acid with an estimated Δ G value of less than-10 kcal. In some embodiments, the degree or intensity of hybridization is measured by the gibbs free energy Δ G ° in the standard state. For oligonucleotides 8-30 nucleotides in length, the oligonucleotide can hybridize to the target nucleic acid with an estimated Δ G ° value in the range of less than-10 kcal (such as less than-15 kcal, such as less than-20 kcal, and such as less than-25 kcal). In some embodiments, the oligonucleotide hybridizes to a target nucleic acid at an estimated Δ G ° value of about-10 to-60 kcal, such as-12 to-40, such as from-15 to-30 kcal or-16 to-27 kcal, such as-18 to-25 kcal.

Target sequence

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

The oligonucleotides of the invention comprise a contiguous region of nucleotides that is complementary to a target nucleic acid, e.g., a target sequence.

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

In some embodiments, the target sequence is present within a sequence selected from the group consisting of SEQ ID NOs 113, 114, 115, 116, 117, and 118.

Target cell

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, e.g., a primate cell, such as a monkey cell or a human cell. In some embodiments, the target cell may be a retinal cell, such as a retinal pigment epithelium (PRE) cell. In some embodiments, the cell is selected from the group consisting of an RPE cell, a bipolar cell, an amacrine cell, an endothelial cell, a ganglion cell, and a microglia cell. For in vitro evaluation, the target cells may be primary cells or established cell lines, such as U251, arpe19.

Target nucleic acid

According to the invention, the target nucleic acid is a nucleic acid encoding mammalian HTRA1 and may be, for example, a gene, RNA, mRNA, and pre-, mature mRNA or cDNA sequence. This target may therefore be referred to as the HTRA1 target nucleic acid.

Suitably, the target nucleic acid encodes an HTRA1 protein, in particular a mammalian HTRA1, such as human HTRA1 (see, e.g., tables 1&2, which provide mRNA and pre-mRNA sequences for human and rat HTRA 1).

In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4, or a naturally occurring variant thereof (such as a sequence encoding a mammalian HTRA1 protein).

The target cell is a cell expressing the HTRA1 target nucleic acid. In preferred embodiments, the target nucleic acid is HTRA1mRNA, such as HTRA1 pre-mRNA or HTRA1 mature mRNA. For antisense oligonucleotide targeting, the poly a tail of HTRA1mRNA is generally not considered.

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

The target sequence may be a subsequence of the target nucleic acid. In some embodiments, the oligonucleotide or contiguous nucleotide region is fully complementary to an HTRA1 subsequence (such as a sequence selected from the group consisting of the sequences of SEQ ID NOs 113, 114, 115, 116, 117, or 231) or comprises only one or two mismatches.

The target sequence may be a subsequence of the target nucleic acid. In some embodiments, the oligonucleotide or contiguous nucleotide region is fully complementary to a HTRA1 subsequence (such as a sequence selected from the group consisting of SEQ ID NO 124-. In some embodiments, the oligonucleotide or contiguous nucleotide region is fully complementary to HTRA1 subsequence SEQ ID NO231 or comprises only one or two mismatches.

Complementarity to the target or a subsequence thereof is measured over the length of the oligonucleotide or a contiguous nucleotide region thereof.

For in vivo or in vitro applications, the oligonucleotides of the invention are generally capable of inhibiting the expression of the HTRA1 target nucleic acid in a cell expressing the HTRA1 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotides of the invention is typically complementary to the HTRA1 target nucleic acid, as measured over the entire length of the oligonucleotide, optionally except for one or two mismatches, and optionally excludes nucleotide-based linker regions that can attach the oligonucleotide to optional functional groups (such as conjugates), or other non-complementary terminal nucleotides (such as region D). In some embodiments, the target nucleic acid may be RNA or DNA, such as messenger RNA, such as mature mRNA or pre-mRNA. In some embodiments, the target nucleic acid is RNA or DNA encoding a mammalian HTRA1 protein (such as human HTRA1), for example a human HTRA1 human HTRA1mRNA sequence, such as disclosed in SEQ ID NO1 (NM-002775.4, GI: 190014575). Tables 1&2 provide more information about exemplary target nucleic acids.

TABLE 1 genomic and Assembly information for human and Cyno HTRA 1.

Fwd is the forward chain. The genomic coordinates provide the pre-mRNA sequence (genomic sequence). The NCBI reference provides mRNA sequences (cDNA sequences).

The National Center for biotechnology information reference sequence database is a comprehensive, complete, non-redundant, and well-annotated set of reference sequences, including genomes, transcripts, and proteins. It is located atwww.ncbi.nlm.nih.gov/refseq。

In the NCBI reference sequence, there is a stretch of 100 nucleotides from position 126 to position 227, the identity of which is unknown. In SEQ ID NO 3&4, this stretch has been replaced by the nucleotide present in the human and cynomolgus monkey (Macaca mulatta) HTRA1 pre-mRNA sequence of this region.

Table 2 sequence details of human and cynomolgus HTRA 1.

Species (II)	RNA type	Length (nt)	SEQ ID NO
				Human being	mRNA	2138	1
Human being	Precursor mRNA	53384	2
				Macaca fascicularis	mRNA	2123	3
Macaca fascicularis	Precursor mRNA	52575	4

Naturally occurring variants

The term "naturally occurring variant" refers to a variant of the HTRA1 gene or transcript that is derived from the same genetic locus as the target nucleic acid, but differs, for example, due to: the degeneracy of the genetic code (which results in a multiplicity of codons encoding the same amino acid), or due to alternative splicing of pre-mrnas, or the presence of polymorphisms, such as single nucleotide polymorphisms, and allelic variants. The oligonucleotides of the invention can thus target nucleic acids and naturally occurring variants thereof, based on the presence of a sufficient complementary sequence to the oligonucleotide. In some embodiments, the naturally occurring variant has at least 95%, such as at least 98% or at least 99% homology to a mammalian HTRA1 target nucleic acid (such as a target nucleic acid selected from the group consisting of SEQ ID NOs 1, 2, 3, or 4).

Modulation of expression

As used herein, the term "modulation of expression" shall be understood to be a generic term for the ability of an oligonucleotide to alter the amount of HTRA1 when compared to the amount of HTRA1 prior to administration of the oligonucleotide. Alternatively, modulation of expression may be determined by reference to a control experiment in which the oligonucleotide of the invention is not administered. One type of modulation is the ability of the oligonucleotide to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, alleviate, reduce, avoid, or terminate expression of HTRA1, e.g., by degrading mRNA or blocking transcription. The antisense oligonucleotides of the invention are capable of inhibiting, down-regulating, reducing, suppressing, abrogating, stopping, blocking, preventing, alleviating, reducing, avoiding, or terminating expression of HTRA 1.

High affinity modified nucleosides

A high affinity modified nucleoside is a modified nucleotide that, when incorporated into an oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, such as by melting temperature (T)^m) The measurement is performed. The high affinity modified nucleosides of the present invention preferably result in a melting temperature increase of between +0.5 and +12 ℃, more preferably between +1.5 and +10 ℃, most preferably between +3 and +8 ℃ per modified nucleoside. Many high affinity modified nucleosides are known in the art and include, for example, many 2' substituted nucleosides and locked nucleic acids (see, e.g., Freier nucleic acid (LNA)) (see, e.g., Freier @)&Altmann；Nucl.Acid Res.，1997，25，4429-4443 and Uhlmann；Curr.Opinion in DrugDevelopment，2000，3(2)，293-213)。

Sugar modification

Oligomers of the invention may comprise one or more nucleosides having a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

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

Such modifications include those in which the ribose ring structure is modified, such as by substitution with a hexose ring (HNA) or a bicyclic ring, which typically has a biradical bridge between the C2 and C4 carbons of the ribose ring (LNA), or an unlinked ribose ring that typically lacks a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, for example in the case of Peptide Nucleic Acid (PNA) or morpholino nucleic acid.

Sugar modifications also include modifications by changing the substituents on the ribose ring to groups other than hydrogen or to the 2' -OH group naturally present in DNA and RNA nucleosides. Substituents may 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. Indeed, much attention has been focused on the development of 2 'substituted nucleosides, and many 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.

2' modified nucleosides

2 ' sugar modified nucleosides are nucleosides having a substituent other than H or-OH at the 2 ' position (2 ' substituted nucleosides) or a diradical comprising a 2 ' linkage, and nucleosides including 2 ' substituted nucleosides and LNA (2 ' -4 ' diradical bridged) nucleosides. For example, a 2' modified sugar may provide an oligonucleotide with enhanced binding affinity and/or increased nuclease resistance. Examples of nucleosides modified by 2 'substitution 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 further examples, see, e.g., Freier & Altmann; nucleic acids res, 1997, 25, 4429-; opinion in drug development, 2000, 3(2), 293-. The following is a schematic representation of some nucleosides modified with 2' substitutions.

Locked nucleic acid nucleosides (LNA)

LNA nucleosides are modified nucleosides that comprise a linking group (called a diradical or bridge) between C2 'and C4' of the ribose sugar ring of the nucleotide. These nucleosides are also referred to in the literature as bridged or Bicyclic Nucleic Acids (BNA).

In some embodiments, the modified nucleoside or LNA nucleoside of the oligomer of the invention has the general structure of formula I or II:

wherein W is selected from-O-, -S-, -N (R)^a)-，-C(R^aR^b) -, such as, in some embodiments, -O-;

b represents a nucleobase moiety;

z represents an internucleoside linkage to an adjacent nucleoside, or a 5' -terminal group;

z represents an internucleoside linkage to an adjacent nucleoside, or a 3' -terminal group;

x represents a group selected from the list consisting of: -C (R)^aR^b)-，-C(R^a)＝C(R^b)-，-C(R^a)＝N-，-O-，-Si(R^a)₂-，-S-，-SO₂-，-N(R^a) -and > C ═ Z.

In some embodiments, X is selected from the group consisting of: -O-, -S-, NH-, NR^aR^b，-CH₂-，CR^aR^b，-C(＝CH²) -and-C (═ CR)^aR^b)-。

In some embodiments, X is-O-.

Y represents a group selected from the group consisting of: -C (R)^aR^b)-，-C(R^a)＝C(R^b)-，-C(R^a)＝N-，-O-，-Si(R^a)₂-，-S-，-SO₂-，-N(R^a) -and > C ═ Z.

In some embodiments, Y is selected from the group consisting of: -CH₂-，-C(R^aR^b)-，-CH₂CH₂-，-C(R^aR^b)-C(R^aR^b)-，-CH₂CH₂CH₂-，-C(R^aR^b)C(R^aR^b)C(R^aR^b)-，-C(R^a)＝C(R^b) -and-C (R)^a)＝N-。

In some embodiments, Y is selected from the group consisting of: -CH₂-，-CHR^a-，-CHCH₃-，CR^aR^b-，

or-X-Y-together represent a divalent linking group (also referred to as a radical) together represent a divalent linking group consisting of 1, 2 or 3 groups/atom selected from the group consisting of: -C (R)^aR^b)-，-C(R^a)＝C(R^b)-，-C(R^a)＝N-，-O-，-Si(R^a)₂-，-S-，-SO₂-，-N(R^a) -and > C ═ Z.

In some embodiments, -X-Y-represents a diradical selected from the group consisting of: -X-CH₂-，-X-CR^aR^b-，-X-CHR^a-，-X-C(HCH₃)^-，-O-Y-，-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-，-NR^a-CH₂-，N-O-CH₂，-S-CR^aR^b-and-S-CHR^a-。

In some embodiments, -X-Y-represents-O-CH₂-or-O-CH (CH)₃)-。

Wherein Z is selected from the group consisting of-O-, -S-and-N (R)^a)-，

And R is^aAnd, when present, R^bEach independently selected from hydrogen, optionally substituted C_1-6-alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, optionally substituted C_1-6-alkoxy radical, C_2-6Alkoxyalkyl group, C_2-6Alkenoxy, carboxyl, C_1-6-alkoxycarbonyl, C_1-6Alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di (C)_1-6Alkyl) amino, carbamoyl, mono-and di (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6-alkanesRadical-aminocarbonyl, mono-and di (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6Alkyl-carbonylamino, carbamates, C_1-6Alkanoyloxy, sulfonyl (sulpho), C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C_1-6Alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and two geminal substituents R^aAnd R^bTogether may represent optionally substituted methylene (═ CH)₂) Where asymmetric groups can be found in either the R or S orientation for all chiral centers.

Wherein R is¹，R²，R³，R⁵And R^5*Independently selected from: hydrogen, optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, C_1-6-alkoxy radical, C_2-6Alkoxyalkyl group, C_2-6Alkenoxy, carboxyl, C_1-6Alkoxycarbonyl radical, C_1-6Alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di (C)_1-6Alkyl) amino, carbamoyl, mono-and di (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6Alkyl-aminocarbonyl, mono-and di (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6Alkyl-carbonylamino, ureido, C_1-6Alkanoyloxy, sulfonyl (sulpho), C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C_1-6Alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two geminal substituents together may represent oxo, thioxo (thioxo), imino, or optionally substituted methylene.

In some embodiments, R¹，R²，R³，R⁵And R^5*Independently selected from C_1-6Alkyl groups such as methyl, and hydrogen.

In some embodiments, R¹，R²，R³，R⁵And R^5*Are all hydrogen.

In some embodiments, R¹，R²，R³Are all hydrogen, and R⁵And R^5*Is also hydrogen, and R⁵And R^5*Is other than hydrogen, such as C_1-6Alkyl groups such as methyl.

In some embodiments, R^aIs hydrogen or methyl. In some embodiments, when present, R^bIs hydrogen or methyl.

In some embodiments, R^aAnd R^bOne or both of which are hydrogen.

In some embodiments, R^aAnd R^bOne is hydrogen and the other is not hydrogen.

In some embodiments, R^aAnd R^bOne is methyl and the other is hydrogen.

In some embodiments, R^aAnd R^bAre both methyl groups.

In some embodiments, the diradical-X-Y-is-O-CH₂-, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such LNA nucleosides are disclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160 (all of which are incorporated herein by reference) and include those commonly referred to as β -D-oxy LNA and α -L-oxy LNA nucleosides.

In some embodiments, the diradical-X-Y-is-S-CH₂-, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such thiolated LNA nucleosides are disclosed in WO99/014226 and WO2004/046160 (which are incorporated herein by reference).

In some embodiments, the diradical-X-Y-is-NH-CH₂-, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such aminoLNA nucleosides are disclosed in WO99/014226 and WO2004/046160 (which are incorporated herein by reference).

In some casesIn the embodiment, the diradical is O-CH₂-CH₂- - (O- -CH) or- -O- -CH₂-CH₂-CH₂-, W is O, and R₁，R₂，R₃，R⁵And R^5*All are hydrogen. Such LNA nucleosides are described in WO00/047599 and Morita et al, Bioorganic&Med, chem, lett, 1273-76 (which is incorporated herein by reference) and includes nucleic acids (ENA) commonly referred to as 2 '-O-4' C-ethylene bridged.

In some embodiments, the diradical-X-Y-is-O-CH₂-, W is O, and all R¹，R²，R³And R⁵And R^5*Are all hydrogen, and R⁵And R^5*Is not hydrogen, such as C_1-6Alkyl groups, such as methyl. Such 5' substituted LNA nucleosides are disclosed in WO2007/134181 (which is incorporated herein by reference).

In some embodiments, the diradical-X-Y-is-O-CR^aR^b-，Wherein R is^aAnd R^bOne or both of which are not hydrogen, such as methyl, W is O, and all R¹，R²，R³And R⁵And R^5*Are all hydrogen, and R⁵And R^5*Is not hydrogen, such as C_1-6Alkyl groups such as methyl. Such doubly modified LNA nucleosides are disclosed in WO2010/077578 (which is incorporated herein by reference).

In some embodiments, the diradical-X-Y-represents a divalent linker group-O-CH (CH)₂OCH₃) - (2' O-methoxyethyl bicyclic nucleic acid-Seth at al., 2010, j.org.chem.vol 75(5) pp.1569-81). In some embodiments, the diradical-X-Y-represents a divalent linker group-O-CH (CH)₂OCH₃) - (2' O-ethylethylbicyclo nucleic acid-Seth at al., 2010, j.org.chem.vol 75(5) pp.1569-81). In some embodiments, the diradical-X-Y-is-O-CHR^a-, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such 6' substituted LNA nucleosides are disclosed in WO10036698 and WO07090071 (which are incorporated herein by reference).

In some embodiments, the diradical-X-Y-is O-CH (CH)₂OCH₃) -, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such LNA nucleosides are also known in the art as cyclic moes (cmoe) and are disclosed in WO 07090071.

In some embodiments, the diradical-X-Y-represents a divalent linking group-O-CH (CH)₃) -. -in the R-or S-configuration. In some embodiments, the diradicals-X-Y-taken together represent a divalent linker group-O-CH₂-O-CH₂- (Seth at al., 2010, j. In some embodiments, the diradical-X-Y-is-O-CH (CH)₃) -, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such 6' methyl LNA nucleosides are also known in the art as cET nucleosides, and may be the (S) cET or (R) cET stereoisomers, as disclosed in WO07090071(β -D) and WO2010/036698(α -L) (both incorporated herein by reference).

In some embodiments, the diradical-X-Y-is-O-CR^aR^b-, in which at R^aOr R^bNone of (1) is hydrogen, W is O, and all R are¹，R²，R³，R⁵And R^5*Are all hydrogen. In some embodiments, R^aAnd R^bAre both methyl groups. Such 6' substituted LNA nucleosides are disclosed in WO 2009006478 (which is incorporated herein by reference).

In some embodiments, the diradical-X-Y-is-S-CHR^a-, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. Such 6' substituted thioalna nucleosides are disclosed in WO11156202 (which is incorporated herein by reference). In some 6' substituted thiaLNA embodiments, R^aIs methyl.

In some embodiments, diradical-X-Y-is-C (═ CH2) -C (R)^aR^b) -, such as-C (═ CH)₂)-CH₂-or-C (═ CH)₂)-CH(CH₃) W is O and all R¹，R²，R³，R⁵And R^5*Are all hydrogen. Such vinyl carbo LNA nucleosides are disclosed in WO08154401 and WO09067647 (both of which are incorporated herein by reference).

In some embodiments, the diradical-X-Y-is N (-OR)^a) -, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl. Such LNA nucleosides are also known as N-substituted LNAs and are disclosed in WO2008/150729 (which is incorporated herein by reference). In some embodiments, the diradicals-X-Y-taken together represent a divalent linker group-O-NR^a-CH₃- (Seth at al., 2010, j. In some embodiments, the diradical-X-Y-is N (R)^a) -, W is O, and R¹，R²，R³，R⁵And R^5*All are hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl.

In some embodiments, R⁵And R^5*One or both of which are hydrogen, and when substituted, R⁵And R^5*Is another of C_1-6Alkyl groups such as methyl. In such embodiments, R¹，R²，R³All may be hydrogen and the diradical-X-Y-may be selected from-O-CH 2-or-OC (HCR)^a) -, such as-OC (HCH3) -.

In some embodiments, the diradical is-CR^aR^b-O-CR^aR^b-, such as CH₂-O-CH₂-, W is O, and R¹，R²，R³，R⁵And R^5*Are all hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl. Such LNA nucleosides are also known as Conformational Restriction Nucleotides (CRNs) and are disclosed in WO2013036868, which is incorporated herein by reference.

In some embodiments, the diradical is-O-CR^aR^b-O-CR^aR^b-, such as CH₂-O-CH₂-, W is O, and R¹，R²，R³，R⁵And R^5*Are all hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al, Nucleic Acids Research 200937 (4), 1225-1238, which is incorporated herein by reference.

Unless otherwise indicated, it will be appreciated that LNA nucleosides can be either the beta-D or alpha-L stereoisomers.

Examples of LNA nucleosides are given in scheme 1.

Scheme 1

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

Nuclease-mediated degradation

Nuclease-mediated degradation refers to oligonucleotides that are capable of mediating degradation of complementary nucleotide sequences when duplexed with such sequences.

In some embodiments, the oligonucleotides can function by nuclease-mediated target nucleic acid degradation, wherein the oligonucleotides of the invention are capable of recruiting nucleases, particularly endonucleases, preferably endonucleases (rnases), such as RNase H. Examples of oligonucleotide designs that function by nuclease-mediated mechanisms are oligonucleotides that typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one or both sides by affinity-enhancing nucleosides, such as gapmers, head-bodies (headmers) and tail-bodies (tailmers).

Activity and recruitment of RNase H

The rnase H activity of an antisense oligonucleotide refers to its ability to recruit rnase H when duplexed with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Oligonucleotides are generally considered to recruit rnase H if: the oligonucleotides when provided with a complementary target nucleic acid sequence have an initial rate measured in pmol/1/min of at least 5%, such as at least 10%, or 20% or more of the initial rate determined using the oligonucleotide (which has the same base sequence as the modified oligonucleotide being tested but contains only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide) and using the methods provided in examples 91-95 of WO01/23613 (incorporated herein by reference).

Gapmer

As used herein, the term gapmer refers to an antisense oligonucleotide comprising a region of rnase H (gap) that recruits the oligonucleotide, which region is flanked 5 'and 3' by one or more affinity-enhancing modified nucleosides (flanking or wing). Various notch designs are described herein. The head and tail bodies are oligonucleotides capable of recruiting rnase H, with one flanking deletion, i.e. only one end of the oligonucleotide contains a nucleoside modified by affinity enhancement. For the head, the 3 'flank is deleted (i.e., the 5' flank comprises an affinity enhancing modified nucleoside) and for the tail, the 5 'flank is deleted (i.e., the 3' flank comprises an affinity enhancing modified nucleoside).

LNA gapmer

The term LNA gapmer is a gapmer oligonucleotide, wherein at least one of the affinity-enhanced modified nucleosides is an LNA nucleoside. In some embodiments, the LNA nucleoside in the LNA gapmer is a beta-D-oxy LNA nucleoside and/or a 6' methyl beta-D-oxy LNA nucleoside (such as (S) cET nucleoside.

Hybrid wing gapmer

The term mixed-wing gapmer refers to an LNA gapmer, wherein the flanking region comprises 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 '-substitution 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 gapmer has one flank comprising an LNA nucleoside (e.g., 5 ' or 3 '), and another flank comprising a nucleoside modified with a 2 ' substitution (3 ' or 5 ', respectively). In some embodiments, the LNA nucleoside in the mixed wing gapmer is a β -D-oxy LNA nucleoside and/or a 6' methyl β -D-oxy LNA nucleoside (such as (S) cET nucleoside.

Conjugates

The term conjugate, as used herein, refers to an oligonucleotide covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

In some embodiments, the non-nucleotide moiety is selected from the group consisting of: proteins, such as enzymes, antibodies or antibody fragments or peptides; lipophilic moieties such as lipids, phospholipids, sterols; polymers such as polyethylene glycol or polypropylene glycol; a receptor ligand; a small molecule; a reporter molecule; and non-nucleoside carbohydrates.

Joint

A linkage or linker is a linkage between two atoms that connects one chemical group or target fragment to another chemical group or target fragment through one or more covalent bonds. The conjugate moiety may be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or tether). The linker is used to covalently link the third region (e.g., the conjugate moiety) to the oligonucleotide (e.g., the end of region a or C).

In some embodiments of the invention, the conjugates or oligonucleotide conjugates of the invention may optionally comprise a linker region between the oligonucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and the oligonucleotide is bio-cleavable.

A cleavable linker comprising or consisting of a physiologically labile bond, which linker is cleavable under conditions normally encountered in the body of a mammal or conditions similar thereto. Conditions under which the physiologically labile linker undergoes chemical transformation (such as lysis) include chemical conditions such as pH, temperature, oxidizing or reducing conditions or agents, and salt concentrations found in mammalian cells or similar to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities typically present in mammalian cells, such as enzymatic activities from proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biologically cleavable linker is sensitive to S1 nuclease cleavage. In a preferred embodiment, the nuclease-sensitive linker comprises between 1 and 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably 2 to 6 nucleosides, most preferably between 2 and 4 linked nucleosides comprising at least two consecutive phosphodiester linkages, for example at least 3 or 4 or 5 consecutive phosphodiester linkages. Preferably, the nucleoside is DNA or RNA. Phosphodiesters containing a bio-cleavable linker are described in more detail in WO2014/076195 (incorporated herein by reference) and may be referred to herein as region D.

The conjugate may also be linked to the oligonucleotide by a non-cleavable linker, or in some embodiments, the conjugate may comprise a non-cleavable linker covalently attached to a bio-cleavable linker. The linker need not be cleavable, but is primarily used to covalently link the conjugate moiety to the oligonucleotide or to the cleavable linker. Oligomers that may comprise chain structures or repeating units (e.g., ethylene glycol, amino acid units or aminoalkyl groups). In some embodiments, the linker (region Y) is aminoalkyl, such as C₂-C₃₆Aminoalkyl radicals, including such as C₆To C₁₂An aminoalkyl group. In some embodiments, the linker (region Y) is C₆An aminoalkyl group. Conjugate linker groups can be routinely attached to oligonucleotides through the use of amino-modified oligonucleotides and activated ester groups on the conjugate groups.

Treatment of

The term 'treatment' as used herein refers to the treatment of an existing disease (e.g. a disease or disorder as described herein) or the prevention (prevention) of a disease, i.e. prophyylaxis. It will thus be appreciated that in some embodiments, the treatment referred to herein may be prophylactic.

Disclosure of Invention

Oligonucleotides of the invention

The present invention relates to oligonucleotides capable of inhibiting the expression of HTRA 1. Modulation can be achieved by hybridization to a target nucleic acid encoding HTRA1 or involved in HTRA1 modulation. The target nucleic acid may 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 invention are antisense oligonucleotides targeted to HTRA1 (e.g., mammalian HTRA 1).

In some embodiments, the antisense oligonucleotides of the invention are capable of modulating the expression of a target by inhibiting or down regulating the target. Preferably, such modulation results in at least 20% inhibition of expression compared to the normal expression level of the target, such as at least 30%, 40%, 50%, 60%, 70%, 80% or 90% inhibition compared to the normal expression level of the target. In some embodiments, the compounds of the invention may be capable of inhibiting the expression level of HTRA1mRNA in vitro by at least 60% or 70% using ARPE-19 cells. In some embodiments, the compounds of the invention may be capable of inhibiting the expression level of HTRA1mRNA in vitro by at least 60% or 70% using ARPE-19 cells. In some embodiments, using ARPE-19 cells, the compounds of the invention may be capable of inhibiting the expression level of HTRA1 protein by at least 50% in vitro. Suitably, the examples provide assays useful for measuring HTRA1 RNA or protein inhibition. Target modulation is triggered by hybridization between a contiguous nucleotide sequence of an oligonucleotide and a target nucleic acid. In some embodiments, the oligonucleotides of the invention comprise a mismatch between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid may be sufficient to show the desired modulation of HTRA1 expression. The reduced binding affinity caused by mismatches may advantageously be compensated by an increase in the number of nucleotides in the oligonucleotide and/or an increase in the number of modified nucleotides capable of increasing binding affinity to the target, such as 2' modified nucleotides present in the oligonucleotide sequence, including LNA.

One aspect of the invention relates to an antisense oligonucleotide comprising a continuous nucleotide region of 10 to 30 nucleotides in length that is at least 90% complementary to an HTRA1 target sequence, such as being fully complementary to an HTRA1 target sequence (e.g., a nucleic acid selected from the group consisting of SEQ ID NOs 1, 2, 3& 4).

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

In some embodiments, the oligonucleotide of the invention or a contiguous nucleotide sequence thereof is fully complementary (100% complementary) to a region of the target nucleic acid, or in some embodiments, may comprise one or two mismatches between the oligonucleotide and the target nucleic acid.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to a region of the sequence selected from the group consisting of SEQ ID NOs 119, 120, 121, 122, or 123.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to a sequence region selected from the group consisting of SEQ ID NO 124-230.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 186.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to a region of SEQ id no 192.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 12 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 205.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 13 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 186.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 13 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to a region of SEQ id no 192.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 13 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 205.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 14 nucleotides thereof is fully (or 100%) complementary to a sequence selected from seq id NOs 113, 114, 115, 116, 117 and 231.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 14 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 186.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 14 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to a region of SEQ id no 192.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 14 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 205.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 15 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 186.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 15 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to a region of SEQ id no 192.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 15 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 205.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 16 nucleotides thereof is fully (or 100%) complementary to a sequence selected from SEQ ID NOs 113, 114, 115, 116, 117 and 231. .

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 16 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 186.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 16, such as 16, 17 or 18 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary, to a region of SEQ ID NO 192.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 16 nucleotides thereof is at least 90% complementary, such as fully (or 100%) complementary to the region of SEQ id no 205.

In some embodiments, the oligonucleotide or contiguous nucleotide region thereof is fully (or 100%) complementary to a sequence selected from the group consisting of sequences selected from the group consisting of seq id nos: SEQ ID NO SEQ ID NO 113, 114, 115, 116, 117 and 231.

In some embodiments, the oligonucleotide or contiguous nucleotide region thereof is fully (or 100%) complementary to a sequence selected from the group consisting of sequences selected from the group consisting of SEQ ID NOs 124-230.

In some embodiments, the oligonucleotide or contiguous nucleotide region thereof is fully (or 100%) complementary to SEQ ID NO 186.

In some embodiments, the oligonucleotide or contiguous nucleotide region thereof is fully (or 100%) complementary to SEQ ID NO 192.

In some embodiments, the oligonucleotide or contiguous nucleotide region thereof is fully (or 100%) complementary to SEQ ID NO 205.

It will be appreciated that the oligonucleotide motif sequence may be modified, for example, to increase nuclease resistance and/or binding affinity for the target nucleic acid. Modifications are described in the definitions and "oligonucleotide design" section.

In some embodiments, the oligonucleotide of the invention or a contiguous region of nucleotides thereof is fully complementary (100% complementary) to a region of the target nucleic acid, or in some embodiments, may comprise one or two mismatches between the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotide, or a contiguous nucleotide sequence of at least 12 nucleotides thereof, is at least 90% complementary, such as fully (or 100%) complementary, to the target nucleic acid sequence.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 12 nucleotides thereof has 100% identity to a sequence selected from seq id NOs 5 to 111.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 14 nucleotides thereof has 100% identity to a sequence selected from seq id NOs 5 to 111.

In some embodiments, the oligonucleotide or a contiguous nucleotide sequence of at least 16 nucleotides thereof has 100% identity to a sequence selected from seq id NOs 5 to 111.

In some embodiments, the oligonucleotide or contiguous nucleotide region thereof comprises or consists of a sequence selected from SEQ ID NOs 5-111.

In some embodiments, the oligonucleotides of the invention are selected from the following group (note that the target subsequence is the reverse complement of the oligonucleotide motif):

or a conjugate thereof; wherein for the columns designated by the title compound, the capital letters are LNA nucleosides, the lowercase letters are DNA nucleosides, the cytosine nucleosides are optionally 5 methylcytosine, and the internucleoside linkages are at least 80%, such as at least 90% or 100% modified internucleoside linkages, such as phosphorothioate internucleoside linkages. In some embodiments, all of the internucleoside linkages of the compounds in the compound design columns of the above tables are phosphorothioate internucleoside linkages. The motif and target subsequences are nucleobase sequences.

The present invention provides the following oligonucleotides:

or a conjugate thereof; wherein in the compounds of the above table, the capital letters indicate beta-D-oxy LNA nucleosides, and all LNA cytosines are 5-methylcytosines (as indicated above)^mShown), lower case letters indicate DNA nucleosides, and the superscript m preceding the lower case letter c represents 5 methylcytosine DNA nucleosides. All internucleoside linkages (linkages) are phosphorothioate internucleoside linkages.

Oligonucleotide design

Oligonucleotide design refers to the pattern of nucleotide sugar modifications in the oligonucleotide sequence. The oligonucleotides of the invention comprise sugar modified nucleosides and may also comprise DNA or RNA nucleosides. In some embodiments, the oligonucleotide comprises a sugar modified nucleoside and a DNA nucleoside. Incorporation of modified nucleosides into the oligonucleotides of the invention can enhance the affinity of the oligonucleotides for the target nucleic acid. In that case, the modified nucleoside may be referred to as an affinity-enhancing modified nucleotide.

In one embodiment, the oligonucleotide comprises at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In one embodiment, the oligonucleotide comprises 1 to 10 modified nucleosides, such as 2 to 9 modified nucleosides, such as 3 to 8 modified nucleosides, such as 4 to 7 modified nucleosides, such as 6 or 7 modified nucleosides. In one embodiment, the oligonucleotide of the invention may comprise a modification independently selected from the three types of modified sugars, modified nucleobases and modified internucleoside linkages) or a combination thereof. Preferably, the oligonucleotide comprises one or more sugar modified nucleosides, such as 2' sugar modified nucleosides. Preferably, the oligonucleotide of the invention comprises 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, arabinonucleic acid (ANA), 2' -fluoro-ANA and LNA nucleosides. Even more preferably, the one or more modified nucleosides is LNA.

In some embodiments, at least 1 modified nucleoside is a Locked Nucleic Acid (LNA), such as at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 modified nucleosides are LNAs. In yet another embodiment, all modified nucleosides are LNAs.

In another embodiment, the oligonucleotide comprises at least one modified internucleoside linkage. In a preferred embodiment, the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boronate internucleoside linkages. In some embodiments, all of the internucleotide linkages in the contiguous sequence of the oligonucleotide are phosphorothioate linkages.

In some embodiments, the oligonucleotide of the invention comprises at least one modified nucleoside that is a 2 '-MOE-RNA, such as a 2, 3, 4, 5, 6, 7, 8, 9 or 102' -MOE-RNA nucleoside unit. In some embodiments, at least one of the modified nucleosides is a 2 '-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2' -fluoro DNA nucleoside units.

In some embodiments, the oligonucleotide of the invention comprises at least one LNA unit, such as 1, 2, 3, 4, 5, 6, 7 or 8 LNA units, such as 2 to 6 LNA units, such as 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 contiguous nucleotide region thereof has at least 1 LNA unit at the 5 'end of the nucleotide sequence and at least 2 LNA units at the 3' end of the nucleotide sequence. In some embodiments, all cytosine nucleobases present in an oligonucleotide of the invention are 5-methylcytosine.

In some embodiments, the oligonucleotide of the invention comprises at least one LNA unit and at least one nucleoside modified with a 2' substitution.

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

In one embodiment of the invention, the oligonucleotides of the invention are capable of recruiting rnase H.

In some embodiments, the oligonucleotide of the invention or a contiguous nucleotide region thereof is a gapmer oligonucleotide.

Gapmer design

In some embodiments, the oligonucleotides of the invention, or contiguous nucleotide regions thereof, have a Gapmer design or structure, also referred to herein as "gapmers". In the gapmer structure, the oligonucleotide comprises at least three distinct structural regions in the ' 5 → 3 "direction, a 5 ' -flank, a gap, and a 3 ' -flank, F-G-F ' in this design, the flanking regions F and F ' (also referred to as the wing regions) comprise at least one sugar modified nucleoside adjacent to region G, and in some embodiments may comprise a contiguous stretch of 2-7 sugar modified nucleosides, or a contiguous stretch of sugar modified and DNA nucleosides (comprising a mixed wing of sugar modified and DNA nucleosides). Thus, the nucleosides of the 5 ' flanking region and the 3 ' flanking region adjacent to the notch region are sugar modified nucleosides, such as 2 ' modified nucleosides. When the oligonucleotide is attached to the HTRA1 target nucleic acid duplex, the gap region G comprises a continuous nucleotide fragment capable of recruiting rnase H. In some embodiments, region G comprises a contiguous stretch of 5-16 DNA nucleosides. The gapmer region F-G-F' is complementary to the HTRA1 target nucleic acid and thus can be a contiguous nucleotide region of the oligonucleotide.

Regions F and F ' located at the 5 ' and 3 ' ends of region G may comprise one or more affinity-enhancing modified nucleosides. In some embodiments, the 3' flank comprises at least one LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments, the 5' flank comprises at least one LNA nucleoside. In some embodiments, both the 5 'and 3' flanking regions comprise LNA nucleosides. In some embodiments, all nucleosides in the flanking region are LNA nucleosides. In other embodiments, the flanking region may comprise LNA nucleosides and other nucleosides (mixed flanks), such as DNA nucleosides and/or non-LNA modified nucleosides, such as 2' substituted nucleosides. In this case, a gap is defined as at least 5 rnase H recruiting nucleosides (e.g. 5-16 DNA nucleosides) flanked at the 5 'and 3' end by a contiguous sequence of affinity enhancing modified nucleosides (e.g. LNA) as β -D-oxy-LNA.

Region F

Region F (5 ' flank or 5 ' flank) attached to the 5 ' end of region G comprises or consists of at least one sugar modified nucleoside, for example at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides. In some embodiments, region F comprises or consists of 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. Nucleosides, such as 1, 2, 3, or 4 modified nucleosides.

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 is selected from the group consisting of a 2 ' -O-alkyl-RNA unit, a 2 ' -O-methyl-RNA, a 2 ' -amino-DNA unit, a 2 ' -fluoro-DNA unit, a 2 ' -alkoxy-RNA, a MOE unit, an LNA unit, an Arabinose Nucleic Acid (ANA) unit, and a 2 ' -fluoro-ANA unit.

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 oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA in the β -D or α -L configuration or a combination thereof. In a preferred embodiment, region F has at least 1 β -D-oxyl LNA unit at the 5' end of the contiguous sequence.

Region G

Region G (the gap region) may comprise, contain or consist of 5-16 contiguous DNA nucleosides capable of recruiting RNase H. In another embodiment, region G comprises or consists of 5 to 12, or 6 to 10 or 7 to 9, such as 8, contiguous nucleotide units capable of recruiting RNaseH.

In another embodiment, at least one nucleoside unit in region G is a DNA nucleoside unit, for example 4 to 20 or 6 to 18 DNA units, for example 5 to 16. In some embodiments, all of the nucleosides of 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 of region G are DNA, such as at least 60%, at least 70% or at least 80% or at least 90% DNA.

Region F'

Region F (3 ' flank or 3 ' flank) attached to the 3 ' end of region G comprises or consists of at least one sugar modified nucleoside, for example at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides. In some embodiments, region F' comprises or consists of 1 to 7 modified nucleosides, e.g., 2 to 6 modified nucleosides, e.g., 2 to 5 modified nucleosides, e.g., 2 to 4 modified nucleosides, e.g., 1 to 3 modified nucleosides, e.g., 1, 2, 3, or 4 modified nucleosides.

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' is selected from the group consisting of a 2 '-O-alkyl-RNA unit, a 2' -O-methyl-RNA, a 2 '-amino-DNA unit, a 2' -fluoro-DNA unit, a 2 '-alkoxy-RNA, a MOE unit, an LNA unit, an Arabinose Nucleic Acid (ANA) unit, and a 2' -fluoro-ANA unit.

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 oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA in the β -D or α -L configuration or a combination thereof. In a preferred embodiment, region F 'has at least 1 β -D-oxyl LNA unit at the 5' end of the contiguous sequence.

Regions D, D' and D "

The oligonucleotides of the invention comprise a contiguous region of nucleotides complementary to the target nucleic acid. In some embodiments, the oligonucleotide may further comprise additional nucleotides located 5 'and/or 3' to the contiguous nucleotide region, which is referred to herein as region D. The regions D 'and D' may be linked to the 5 'end of the region F' or the 3 'end of the region F', respectively. In some embodiments, the D region (region D' or D ") may form part of a contiguous nucleotide sequence that is complementary to the target nucleic acid, or in other embodiments, the D region(s) may not be complementary to the target nucleic acid.

In some embodiments, the oligonucleotide of the invention comprises or consists of a contiguous nucleotide region and optionally 1-5 additional 5 'nucleotides (region D').

In some embodiments, the oligonucleotide of the invention comprises or consists of a contiguous nucleotide region and optionally 1-5 additional 3' nucleotides (region D ").

The regions D' or D "may independently comprise 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 invention may, in some embodiments, comprise a contiguous nucleotide sequence capable of modulating a target flanked at the 5 'and/or 3' end by additional nucleotides. Such additional nucleotides can serve as nuclease-sensitive, biologically cleavable linkers and thus can be used to attach functional groups, such as conjugate moieties, to the oligonucleotides of the invention. In some embodiments, the additional 5 'and/or 3' terminal nucleotide is linked to a phosphodiester, and may be DNA or RNA. In another embodiment, the additional 5 'and/or 3' terminal nucleotide is a modified nucleotide, which may for example be included to enhance nuclease stability or for ease of synthesis. In some embodiments, the oligonucleotide of the invention comprises region D' and/or D "in addition to the contiguous nucleotide region.

In some embodiments, the gapmer oligonucleotides of the invention can be represented by the formula:

F-G-F'; in particular F_1-7-G_4-12-F’_1-7

D ' -F-G-F ', in particular D '_1-3-F_1-7-G_4-12-F’_1-7

F-G-F '-D', in particular F_1-7-G_4-12-F’_1-7-D”_1-3

D '-F-G-F' -D ', especially D'_1-3-F_1-7-G_4-12-F’_1-7-D”_1-3。

Preparation method

In another aspect, the invention provides a method of preparing an oligonucleotide of the invention, the method comprising reacting nucleotide units, thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al, 1987, Methods in Enzymology, Vol.154, p.287-313). In another embodiment, the method further comprises reacting the contiguous nucleotide sequence with a conjugate moiety (ligand). In another aspect, there is provided a method of preparing a composition of the invention, the method comprising mixing an oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Pharmaceutically acceptable salts

For use as a therapeutic agent, the oligonucleotides of the invention may be provided as a suitable pharmaceutically acceptable salt, such as a sodium or potassium salt. In some embodiments, the oligonucleotide of the invention is a sodium salt.

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising any of the above oligonucleotides and/or oligonucleotide conjugates together with a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. Pharmaceutically acceptable diluents include Phosphate Buffered Saline (PBS), and pharmaceutically acceptable salts include, but are 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 50-300 μ M solution. In some embodiments, the oligonucleotides of the invention are administered at a dose of 10-1000 μ g.

WO2007/031091 provides suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Suitable doses, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO 2007/031091.

The oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on a number of criteria including, but not limited to, the route of administration, the extent of the disease or the dosage administered.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular for oligonucleotide conjugates, and in particular for oligonucleotide conjugates, the conjugate moiety is cleaved from the oligonucleotide upon delivery of the prodrug to the site of action, e.g., a target cell.

Applications of

The oligonucleotides of the invention are useful as research reagents, e.g., for diagnosis, treatment and prophylaxis.

In research, such oligonucleotides can be used to specifically modulate the synthesis of HTRA1 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or assessing its usefulness as a target for therapeutic intervention. Typically, target regulation is achieved by degradation or inhibition of the mRNA producing the protein, thereby preventing protein formation, or by degradation or inhibition of the regulator of the gene or mRNA producing the protein.

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

For therapeutic agents, animals or humans suspected of having a disease or disorder, may be treated by modulating the expression of HTRA 1.

The invention provides a method for treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention.

The invention also relates to an oligonucleotide, composition or conjugate as defined herein for use as a medicament.

The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition according to the invention is usually administered in an effective amount.

The invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the invention as described in the manufacture of a medicament for the treatment of a condition mentioned herein, or a method of treatment for a condition mentioned herein.

As mentioned herein, the disease or disorder is associated with the expression of HTRA 1. In some embodiments, the disease or disorder may be associated with a mutation in the HTRA1 gene or a gene whose protein product is associated with or interacts with HTRA 1. Thus, in some embodiments, the target nucleic acid is a mutant form of the HTRA1 sequence, while in other embodiments, the target nucleic acid is a modulator of the HTRA1 sequence.

The methods of the invention are preferably used to treat or prevent diseases caused by abnormal levels and/or activity of HTRA 1.

The invention further relates to the use of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of HTRA 1.

In one embodiment, the invention relates to an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition for use in the treatment of a disease or disorder selected from: ocular 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 invention can be used to treat geographic atrophy or mid-stage dAMD. HTRA1 has also been shown in alzheimer's disease and parkinson's disease, and thus in some embodiments, the oligonucleotide conjugates or pharmaceutical compositions of the invention can be used to treat alzheimer's disease or parkinson's disease. HTRA1 has also been shown to be in duchenne muscular dystrophy, arthritis, such as osteoarthritis, familial ischemic cerebrovascular disease, and thus, in some embodiments, the oligonucleotide conjugates or pharmaceutical compositions of the invention may be used to treat duchenne muscular dystrophy, arthritis, such as osteoarthritis, or familial ischemic cerebrovascular disease.

Administration of

The oligonucleotides or pharmaceutical compositions of the invention may be administered topically (such as to the skin, inhaled, ocular or otic) or enterally (such as orally or through the gastrointestinal tract) or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intracerebroventricularly or intrathecally).

In some embodiments, the oligonucleotide, conjugate or pharmaceutical composition of the invention is administered by a parenteral route, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular (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.

For the treatment of ocular diseases such as macular degeneration, AMD (wet or dry), intraocular injection may be used.

In some embodiments, a compound of the invention, or a pharmaceutically acceptable salt thereof, is administered by intraocular injection at a dose of about 10 μ g to about 200 μ g per eye, such as about 50 μ g to about 150 μ g per eye, such as about 100 μ g per eye. In some embodiments, the dosage interval, i.e., the period of time between successive administrations, is at least monthly, such as at least once every two months or at least once every three months.

Combination therapy

In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is used in combination therapy with another therapeutic agent. The therapeutic agent may be, for example, a standard of care for the above-mentioned disease or condition

Examples

Materials and methods

Oligonucleotide synthesis

Oligonucleotide synthesis is well known in the art. The following are applicable schemes. The oligonucleotides of the invention can be prepared by methods that vary somewhat in the apparatus, support and concentration used.

Oligonucleotides were synthesized on a1 μmol scale on Oligomaker 48 on a uridine universal support using the phosphoramidite method. At the end of the synthesis, the oligonucleotide was cleaved from the solid support using ammonia at 60 ℃ for 5-16 hours. The oligonucleotides were purified by reverse phase HPLC (RP-HPLC) or by solid phase extraction and characterized by UPLC and further confirmed for molecular weight by ESI-MS.

Extension of the oligonucleotide:

coupling of β -cyanoethyl phosphoramidite (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 by using a 0.1M solution of 5' -O-DMT protected imide in acetonitrile and a solution of DCI (4, 5-dicyanoimidazole) in acetonitrile (0.25M) as activators. For the last cycle, phosphoramidites with the desired modification (e.g., C6 linker for attaching a conjugate group or a conjugate group like this) can be used. The thiolation for the introduction of the phosphorothioate linkage was performed by using a hydrogenated flavonol (0.01M in acetonitrile/pyridine 9: 1). The phosphodiester linkage may be introduced using 0.02M iodine in THF/pyridine/water 7: 2: 1. The remaining reagents are reagents commonly used in oligonucleotide synthesis.

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

Purification by RP-HPLC:

the crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter C1810 μ 150X10mm column. 0.1M ammonium acetate pH 8 and acetonitrile were used as buffers at a flow rate of 5 mL/min. The collected fractions were lyophilized to give the purified compound as a generally white solid.

Abbreviations:

DCI: 4, 5-dicyanoimidazole

DCM: methylene dichloride

DMF: dimethyl formamide

DMT: 4, 4' -Dimethoxytrityl radical

THF: tetrahydrofuran (THF)

Bz: benzoyl radical

Ibu: isobutyryl radical

RP-HPLC: reversed phase high performance liquid chromatography

T_mAnd (3) determination:

oligonucleotide and RNA target (phosphate-linked, PO) duplexes were diluted to 3mM in 500ml RNase-free water and mixed with 500ml 2x T_mBuffer (200mM NaCl, 0.2mM EDTA, 20mM sodium phosphate, pH 7.0). The solution was heated to 95 ℃ for 3 minutes and then annealed at room temperature for 30 minutes. The duplex melting temperature (Tm) was measured on a Lambda 40UV/VIS spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature was raised from 20 ℃ to 95 ℃ and then lowered to 25 ℃ and the absorption at 260nm was recorded. First derivative and local maxima of melting and annealing for evaluation of duplex T_m。

The oligonucleotides used:

for the compound: capital letters represent LNA nucleosides (beta-D-oxy LNA nucleosides were used), all LNA cytosines are 5-methylcytosine, lowercase letters represent DNA nucleosides preceded by a superscript^mRepresents 5-methyl C-DNA nucleosides. All internucleoside linkages (linkages) are phosphorothioate internucleoside linkages. In EP16177508.5 and EP17170129.5, compound a is disclosed as compound 143, 1 and compound B is disclosed as 145, 1, and is used as a positive control compound.

Example 1 LNA oligonucleotides were tested at a single concentration for in vitro efficacy in the U251 cell line.

Promising "hot spot" areas for HTRA1 are determined. After 6 days of treatment, a library of n-231 HTRA1 LNA oligonucleotides was screened in a 5 μ M U251 cell line. From this library we identified a series of active oligonucleotides targeting the human HTRA1 precursor mRNA (between positions 53113-53384) as shown in FIG. 1 (SEQ ID NO 116 or 117).

Human glioblastoma U251 cell line was purchased from ECACC and recommended by the supplier at 37 ℃ with 5% CO₂Is maintained in a humidified incubator. For the assay, 15000U 251 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. Concentration of oligonucleotide: 5 μ M. 3-4 days after addition of the oligonucleotide, the medium was removed and fresh medium (without oligonucleotide) was added. Cells were harvested 6 days after the addition of the oligonucleotide. Using PureLink Pro 96RNA was extracted using RNA purification kit (Ambion, according to manufacturer's instructions). The cDNA was then synthesized using M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion, according to the manufacturer's instructions), 100mM dNTP panel (PCR grade, Invitrogen) and DNase/RNase free water (Gibco). For gene expression analysis, qPCR was performed using a TagMan Fast Advanced Master Mix (2X) (Ambion) in a doublex setting. The following TaqMan primer assays were used for qPCR: HTRA1, Hs01016151_ m1(FAM-MGB) and housekeeping genes TBP, Hs4326322E (VIC-MGB) from Life technologies. n-2 independent biological replicates. The residual HTRA1mRNA expression level in the table is shown as% of control (PBS treated cells).

Example 2: LNA oligonucleotides were tested at a single concentration for in vitro efficacy in the U251 cell line.

The "hot spot" region 53113-53384 described in example 1 was further validated in a new n-210 HTRA1 LNA oligonucleotide library, which had been screened at a concentration of 5 μ M in the U251 cell line. n-33 LNA oligonucleotides targeted human HTRA1 pre-mRNA (between positions 53113-53384), which were relatively active compared to the remaining oligonucleotides, as shown in figure 2.

The assay was performed as described in example 1. n-2 independent biological replicates. The remaining HTRA1mRNA expression levels are shown in the table as% of control (PBS treated cells).

Example 3 LNA oligonucleotides were tested at a single concentration for in vitro efficacy in the U251 and ARPE19 cell lines.

The "hot spot" region 53113-53384 described in examples 1 and 2 was further validated in a new n-305 HTRA1 LNA oligonucleotide library that had been screened at concentrations of 5 μ M and 25 μ M in the U251 and ARPE19 cell lines, respectively. n 95 LNA oligonucleotides targeted human HTRA1 pre-mRNA (between positions 53113-53384), which were relatively active compared to the remaining oligonucleotides, as shown in figure 3.

The human retinal pigment epithelium ARPE19 cell line was purchased from ATCC and 5% CO at 37 ℃₂In a humidified incubator, maintained in DMEM-F12(Sigma, D8437), 10% FBS, 1% pen/strep. The U251 cell line is described in example 1. For the assay, 2000U251 or ARPE19 cells/well were seeded in 96-well plates in the medium recommended by the supplier. Cells were incubated for 2 hours and then oligonucleotides dissolved in PBS were added. The concentration of the oligonucleotide was 5 and 25. mu.M in U251 and ARPE19 cells, respectively. Cells were harvested 4 days after the addition of the oligonucleotide. RNA extraction was performed as described in example 1, using qScriptXLT one-step RT-qPCR Toughmix Low ROX, 95134-100(Quanta Biosciences) for cDNA synthesis and qPCR. Next, TaqMan primer analysis was used for U251 and ARPE19 cells, set at douplex: HTRA1, Hs01016151_ m1(FAM-MGB) and housekeeping genes GAPDH, Hs4310884E (VIC-MGB). All primer sets were purchased from Life Technologies. n-1 biological replicates. The relative HTRA1mRNA expression levels are shown in the table as% of control (PBS treated cells).

Example 4. selected compounds were tested in a dose response curve for in vitro potency and efficacy in the U251 and ARPE19 cell lines.

The U251 and ARPE19 cell lines are described in examples 1 and 3, respectively. The U251 assay was performed as described in example 1. The ARPE19 was determined as follows: 5000 ARPE19 cells/well were seeded into 96-well plates in the medium recommended by the supplier (except 5% FBS instead of 10%). Cells were incubated for 2 hours and then oligonucleotides dissolved in PBS were added. Concentration of oligonucleotide: from 50 μ M, half log dilution, 8 dots. Cells were harvested 4 days after the addition of the oligonucleotide. RNA extraction, cDNA synthesis and qPCR were performed as described in example 1. n-2 independent biological replicates. EC50 values at 50 μ M and residual HTRA1mRNA levels are shown in the table as% of control (PBS).

Example 5, selected compounds were tested for in vitro potency and efficacy in the U251 and ARPE19 cell lines in a dose response curve.

The assay was performed as described in example 3. Concentration of oligonucleotide: from 50 μ M, half log dilution, 8 points. For U251 and ARPE19, n-2 and n-1 independent biological replicates, respectively. EC50 values at 50 μ M and residual HTRA1mRNA levels are shown in the table as% of control (PBS).

Example 6. selected compounds were tested in a dose response curve for in vitro potency and efficacy in the U251 cell line.

The assay was performed as described in example 3. Concentration of oligonucleotide: from 50 μ M, half log dilution, 8 points. n-2 independent biological replicates. EC50 values at 50 μ M and residual HTRA1mRNA levels are shown in the table as% of control (PBS).

Example 7. selected compounds were tested in a dose response curve for in vitro potency and efficacy in the U251 cell line.

The ARPE19 cell line is described in example 3. For the assay, ARPE19 cells (24000 cells per well) were seeded at a density of 100 μ Ι _ in starvation medium (supplier recommended medium except 1% FBS instead of 10%) in 96-well plates. Cells were incubated for 2 hours and then oligonucleotides dissolved in PBS were added. Concentration of oligonucleotide: from 50 μ M, half log dilution, 8 points. On days 4 and 7 after addition of the oligonucleotide compound, 75 μ L of fresh starvation medium without oligonucleotides was added to the cells (without removal of old medium). RNA extraction, cDNA synthesis and qPCR were performed as described in example 3. n-2 independent biological replicates. EC50 values at 50 μ M and residual HTRA1mRNA levels are shown in the table as% of control (PBS).

Example 8.

In vitro efficacy in human primary RPE cells was tested.

Human primary retinal pigment epithelium (hpRPE) cells were purchased from Sciencell (Cat # 6540). For the assay, 5000 hprppe cells/well were seeded in 96-well plates coated with Laminin (Laminin521, BioLamina Cat # LN521-03) in culture medium (EpiCM, Sciencell Cat # 4101). They were expanded in this medium for one week and differentiated in the following medium for 2 weeks: MEM α medium (Sigma Cat # M-4526), glutamine-penicillin-streptomycin (Sigma Cat # G-1146), nonessential amino acids (NEAA, Sigma Cat # M-7145), taurine (Sigma Cat # T-0625), hydrocortisone (Sigma Cat # H-03966), triiodothyroxine (Sigma Cat # T-5516), and bovine serum albumin (BSA, Sigma Cat # a-9647) supplemented with N1 supplement (MEM Cat # N-6530). Cells were incubated at 37 ℃ with 5% CO₂In a humid incubator。

On the day of the experiment, cells were incubated with fresh differentiation medium for 1 hour before addition of oligonucleotides. They were dissolved in PBS and applied to cells on day 0 and day 4. On day 7, the medium was changed and on day 10, the cells were harvested with 50 μ l RLT buffer with β -mercaptoethanol (Qiagen Cat # 79216). RNA extraction, including DNase I treatment (catalog No. 79254; batch No. 151042674), was performed according to the user's manual of the Qiagen RNeasy mini kit (catalog No. 74104; batch No. 151048073). RNA quality control was performed using the Agilent Bioanalyzer Nano Kit (Agilent; Cat # 5067-. Total RNA was reverse transcribed into cDNA (cDNA synthesis) using a high capacity cDNA reverse transcription kit (based on random hexamer oligonucleotides) according to the manufacturer's instructions (Thermo Fisher scientific, Cat # 4368814; Lot 00314158). Measurement of the cDNA samples was performed 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 genes, GAPDH, Hs99999905_ m1 and PPIA, Hs99999904_ m 1. n-3 biological replicates. The remaining HTRA1mRNA expression levels are shown in fig. 4 and the table below as% of control (PBS).

Example 9. in vivo pharmacokinetic and pharmacodynamic studies in cynomolgus monkeys, treatment 21 days, Intravitreal (IVT) injection, single dose.

Knockdown was observed for 3 HTRA1 LNA oligonucleotides directed against "hot spots" in human HTRA1 pre-mRNA (between positions 53113-53384) at both retinal mRNA and retinal and intravitreal protein levels (see FIG. 5)

Animal(s) production

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

Four animals were included in each study group for a total of 20.

Compounds and dosing procedures

Buprenorphine analgesia was administered two days before and two days after the test compound injection. Animals were anesthetized with an intramuscular injection of ketamine and xylazine. Test article and negative control (PBS) were administered intravitreally (50 μ L per administration) in both eyes of anesthetized animals following topical tetracaine (tetracaine) anesthetic administration on study day 1.

Death by peace and happiness

At the end of life stage (day 22), all monkeys were euthanized by intraperitoneal over-injection of pentobarbital.

Oligonucleotide content and quantification of Htra1 RNA expression by qPCR

Immediately after euthanasia, eye tissues were quickly and carefully sectioned on ice and stored at-80 ℃ until shipment. Retina samples were lysed in 700 μ L of MagNa Pure 96 LC RNA isolation tissue buffer and homogenized by adding 1 stainless steel ball per 2ml tube using a precellys evolution homogenizer for 2X 1, 5min, followed by incubation at room temperature for 30 min. The samples were centrifuged at 13000rpm for 5 min. Half was left for biological analysis and the other half was directly subjected to RNA extraction.

For biological analysis, samples were diluted 10 to 50 fold and oligonucleotide content was measured by hybridization ELISA. 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) were mixed with diluted homogenate or related standard, incubated at room temperature for 30 minutes and then added to streptavidin-coated ELISA plates (Nunc cat No. 436014).

The plate at room temperature 1h incubation, in 2x SSCT (300mM sodium chloride, 30mM sodium citrate and 0.05% v/vTween-20, pH 7.0) washing. Captured LNA duplexes were detected using anti-DIG antibodies conjugated with alkaline phosphatase (Roche Applied sciences cat. No.11093274910) and alkaline phosphatase substrate system (Blue Phos substrate, KPL product code 50-88-00). The amount of oligomeric complex was measured as absorbance at 615nm on a Biotek reader.

Cellular RNA bulk kit (05467535001, Roche) was used in MagNA Pure 96 system, programmed as follows: organization FF standard LV3.1 included DNAse processing according to the manufacturer's instructions. RNA quality control and concentration were measured using Eon readings (Biotek). RNA concentrations were normalized between samples and subsequently cDNA synthesis and qPCR were performed in a one-step reaction using qScript XLT one-step RT-qPCRToughMix Low ROX, 95134-100(Quanta Biosciences). The following TaqMan primer assay was used in single stranded body reactions: htra1, Mf01016150_, Mf01016152_ m1 and Rh02799527_ m1 and housekeeping genes from Life Technologies, ARFGAP2, Mf01058488_ g1 and Rh01058485_ m1, and ARL1, Mf02795431_ m 1. qPCR analysis was performed on a ViiA7 machine (Life Technologies). Eye/group: n-3 eyes. Each eye was considered an individual sample. The relative Htra1mRNA expression levels are shown in% of control (PBS) in the table.

Histology

The eyes were removed and fixed in 10% neutral buffered formalin for 24 hours, trimmed and embedded in paraffin.

For ISH analysis, formalin-fixed, paraffin-embedded sections of rhesus macaque retinal tissue were processed to a thickness of 4 μm using a fully automated Ventana dictionary ULTRA staining module (program: mRNADDiscovery ULTRA Red 4.0-v0.00.0152), using an RNAscope 2.5VS Probe-mMu-HTRA1, REF486979, Advanced Cell Diagnostics, Inc. The chromogen used was fasted, hematoxylin II counterstain.

Quantification of HTRA1 protein using a plate-based immunoprecipitation mass spectrometry (IP-MS) method

Sample preparation, retina

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

Sample preparation, vitreous body

The vitreous (300 μ l) was diluted with 5x RIPA buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1mM EDTA) with protease inhibitor (complete absence of EDTA, Roche) and homogenized using Precellys 25 (5500, 15s, 2 cycles). The homogenate was centrifuged (13,000rpm, 3min) and the protein content of the supernatant determined (Pierce BCA protein assay).

Plate-based HTRA1 immunoprecipitation and trypsin digestion

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

Quantification of HTRA1 peptide by targeted mass spectrometry (Selective response monitoring, SRM)

Mass spectrometry was performed on UltimateRSLCnano LC coupled to a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Scientific). Samples (20 μ L) were injected directly from a 96-well plate for IP and loaded at 5 μ L/min in loading buffer (0.5% v/v formic acid, 2% v/v ACN) onto an Acclaim Pepmap 100 capture column (100 μm x2em, C18, 5 μm,thermo Scientific) for 6 min. The peptides were then purified on a PepMap Easy-SPRAY analytical column (75 μm x 15cm, 3 μm,ThermoScientific) was separated at a flow rate of 250nL/min using the following gradient: 6min, 98% buffer A (2% ACN, 0.1% formic acid), 2% buffer B (ACN + 0.1% formic acid); 36min, 30% buffer B; 41min, 60% buffer B; 43min, 80% buffer B; 49min, 80% buffer B; 50min, 2% buffer B. The TSQ Quantiva runs in SRM mode with the following parameters: cycle time, 1.5 s; spray voltage, 1800V; collision air pressure, 2 mTorr; q1 and Q3 resolutions, 0.7 FWHM; the ion transfer tube temperature was 300 ℃. HTRA1 peptide "LHRPPVIVLQR" and isotopically labeled (L- [ U-13C, U-15N)]R) SRM conversion of the synthetic form (which serves as an internal standard).

Data analysis was performed using Skyline version 3.6.

Western blot

The sectioned retinal samples were lysed in 0.5precell tubes (CK 14-0.5 ml, Bertin Technologies) in RIPA lysis buffer (20-188, Milipore) with protease inhibitors (protease-inhibitor mini, 11836170001, Roche without EDTA at all) and homogenized.

The vitreous samples were added to 0.5Precellyses tubes (CK14 — 0.5ml, berthin Technologies) and lysed and homogenized in 1/4xrip lysis buffer (20-188, Milipore) with protease inhibitors (protease-inhibitor mini, 11836170001, Roche without EDTA at all).

Samples (20. mu.g protein in retina, 40. mu.g protein in vitreous) were analyzed on a 4-15% gradient gel (#567-8084 Bio-Rad) under reducing conditions and transferred to nitrocellulose (#170-4159 Bio-Rad) using a Trans-Blot Turbo apparatus from Bio-Rad.

A first antibody: rabbit anti-human HTRA1(SF1) is a friendly gift to sasha house (university of cologne) and mouse anti-human Gapdh (#98795 Sigma-Aldrich). Secondary antibody: goat anti-rabbit 800CW and goat anti-mouse 680RD were from Li-Cor.

The blot was imaged and analyzed on an Odyssee CLX from Li-Cor.

Example 10-in vivo assessment of cynomolgus monkeys: HTRA1 protein assay in aqueous humor and comparison to HTRA1mRNA and protein inhibition in retina.

The experimental method comprises the following steps: please see the above examples. Aqueous humor samples were collected and samples were prepared according to the vitreous body sample of example 9. Aqueous humor samples (AH) of cynomolgus monkeys were prepared by sizing in Analytical methods: capillary Electrophoresis System (Peggy Sue)^TMProtein) was analyzed.

The samples were thawed on ice and used undiluted. For quantification, recombinant HTRA1-S328A mutant (Origene # TP 700208). Prepared as described in the provider.

The first rabbit anti-human HTRA antibody SF1 was provided by professor sasca Fauser and was raised at a rate of 1: 300 parts by weight. All other reagents were from proteimple.

Samples were technically processed in triplicate, using 12-230kDa separation modules, and curves were calibrated in duplicate. The area under the peak was calculated and analyzed using Xlfit (IDBS software).

Results

Note that the compound IDs shown in fig. 12-14 utilize different numbering systems as the remaining examples. The above table provides an index to the numbering used in fig. 12-14 as compared to the numbering used in the previous embodiments and elsewhere herein.

Figure 12A shows visualization of HTRA1 protein levels in the aqueous humor of monkeys administered compound B and #73, 1, samples collected on days 3, 8, 15, and 22 post-injection. Fig. 12B provides a calibration curve for calculating HTRA1 protein levels. Fig. 12C provides calculated HTRA1 levels of aqueous humor from individual animals plotted against time post-injection.

Figure 13 shows a direct correlation between HTRA1 protein levels in aqueous humor and HTRA1mRNA levels in retina. Thus, the aqueous humor HTRA1 protein level can be used as a biomarker for HTRA1 retinal mRNA levels or HTRA1 retinal mRNA inhibition.

Figure 14 illustrates that there is also a correlation between the level of HTRA1 protein in the retina and the level of HTRA1 protein in the aqueous humor, although in this experiment this correlation is less strong than the correlation between HTRA1mRNA inhibition in the retina and HTRA1 protein level in the aqueous humor, indicating that aqueous HTRA1 protein level is particularly suitable as a biomarker for HTRA1mRNA antagonists.

The claims (modification according to treaty clause 19)

1. An oligonucleotide of the formula:

T_sA_s ^mC_sT_st_st_sa_sa_st_sa_sg_sc_sT_s ^mC_sA_sA(SEQ ID NO 86)；

wherein the capital letters represent beta-D-oxyLNA nucleosides, the lowercase letters are DNA nucleosides, the subscript s represents a phosphorothioate internucleoside linkage, and^mc represents 5 methylcytosine beta-D-oxoLNA nucleoside.

2. The oligonucleotide of claim 1, wherein the oligonucleotide is of the formula:

3. a pharmaceutically acceptable salt of the oligonucleotide of claim 1 or 2.

4. The pharmaceutically acceptable salt of claim 3, wherein the salt is a potassium salt.

5. The pharmaceutically acceptable salt of claim 4, wherein the salt is a sodium salt.

6. A pharmaceutical composition comprising an oligonucleotide of the formula:

T_sA_s ^mC_sT_st_st_sa_sa_st_sa_sg_sc_sT_s ^mC_sA_sA(SEQ ID NO 86)；

wherein the capital letters represent beta-D-oxyLNA nucleosides, the lowercase letters are DNA nucleosides, the subscript s represents a phosphorothioate internucleoside linkage, and^mc represents 5 methylcytosine beta-D-oxoLNA nucleoside; and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises a pharmaceutically acceptable diluent.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable diluent is phosphate buffered saline.

9. The pharmaceutical composition of any one of claims 6-8, wherein the oligonucleotide is in the form of a pharmaceutically acceptable salt.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable salt is a sodium salt.

11. A conjugate comprising the oligonucleotide of claim 1 or 2, or the pharmaceutically acceptable salt of any one of claims 3-5, and at least one conjugate moiety covalently attached to the oligonucleotide.

12. Use of the oligonucleotide of claim 1 or 2, or the pharmaceutically acceptable salt of any one of claims 3-5, or the pharmaceutical composition of any one of claims 6-10, or the conjugate of claim 11, for use in medicine.

13. Use of the oligonucleotide of claim 1 or 2, or the pharmaceutically acceptable salt of any one of claims 3-5, or the pharmaceutical composition of any one of claims 6-10, or the conjugate of claim 11, for the treatment or prevention of macular degeneration.

14. The use of claim 13, wherein the use is for the treatment of wet AMD, dry AMD, geographic atrophy, intermediate dAMD or diabetic retinopathy.

15. The use of claim 14, wherein the use is for the treatment of geographic atrophy or mid-range dAMD.

Claims

1. An antisense oligonucleotide 10-30 nucleotides in length, wherein the antisense oligonucleotide targets an HTRA1 nucleic acid and comprises a contiguous nucleotide region of 10-22 nucleotides which is at least 90%, such as 100%, complementary to SEQ ID NO 113.

2. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide region is fully complementary to a sequence selected from the group consisting of SEQ ID Nos 231, 186, 192 and 205.

3. The antisense oligonucleotide of claim 1 or 2, wherein the contiguous nucleotide region comprises at least 12 contiguous nucleotides that are fully complementary to a sequence selected from the group consisting of SEQ id no 124-230.

4. The antisense oligonucleotide of any one of claims 1-3, wherein the contiguous nucleotide region comprises at least 12 contiguous nucleotides that are fully complementary to SEQ ID NO 113.

5. The antisense oligonucleotide of any one of claims 1-4, wherein the contiguous nucleotide region comprises at least 14 contiguous nucleotides that are fully complementary to SEQ ID NO 113.

6. The antisense oligonucleotide of any one of claims 1-5, wherein the contiguous nucleotide region of the oligonucleotide consists of or comprises the sequence: selected from any one of SEQ ID NOs 67, 73 and 86, or at least 12 contiguous nucleotides thereof.

7. The antisense oligonucleotide of any one of claims 1-6, wherein the contiguous nucleotide region of the oligonucleotide comprises one or more 2 'sugar modified nucleosides, such as 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, arabinonucleic acid (ANA), 2 ' -fluoro-ANA, and LNA nucleosides.

8. The antisense oligonucleotide of any one of claims 1-7, wherein a contiguous nucleotide region of the oligonucleotide comprises at least one modified internucleoside linkage, such as one or more phosphorothioate internucleoside linkages, or such as all internucleoside linkages within the contiguous nucleotide region are phosphorothioate internucleoside linkages.

9. The antisense oligonucleotide of any one of claims 1-8, wherein the oligonucleotide or a contiguous nucleotide sequence thereof is or comprises a gapmer, such as of formula 5 ' -F-G-F ' -3 ', wherein regions F and F ' independently comprise 1-7 sugar modified nucleosides and G is a region of 6-16 nucleotides capable of recruiting RNase H, wherein the nucleotides of regions F and F ' adjacent to region G are sugar modified nucleosides.

10. The antisense oligonucleotide of claim 9 wherein at least one or both of regions F and F' each comprise at least one LNA nucleoside.

11. The antisense oligonucleotide of any one of claims 1-10, wherein the contiguous nucleotide region is selected from the group of:

TTCtatctacgcaTTG(SEQ ID NO 67)，

CTTCttctatctacgcAT (SEQ ID NO 73), and

TACTttaatagcTCAA(SEQ ID NO86)；

wherein the capital letters are LNA nucleotides, the lowercase letters are DNA nucleosides, and the cytosine residue is optionally 5-methylcytosine.

12. The antisense oligonucleotide of claim 10 or 11, wherein the LNA nucleoside is a β -D-oxy LNA nucleoside.

13. The antisense oligonucleotide of any one of claims 1-12, wherein the internucleoside linkages between the nucleotides of the contiguous region of nucleotides are all phosphorothioate internucleotide linkages.

14. An oligonucleotide comprising or consisting of an oligonucleotide selected from the group consisting of:

T_sT_s ^mC_st_sa_st_sc_st_sa_s ^mc_sg_sc_sa_sT_sT_sG(SEQ ID NO 67，1)，

T_sA_s ^mC_sT_st_st_sa_sa_st_sa_sg_sc_sT_s ^mC_sA_sA(SEQ ID NO 86，1)；

15. A pharmaceutically acceptable salt of the oligonucleotide of any one of claims 1-14.

16. A conjugate comprising the oligonucleotide of any one of claims 1-15 and at least one conjugate moiety covalently attached to the oligonucleotide.

17. A pharmaceutical composition comprising the oligonucleotide of claims 1-15 or the conjugate of claim 16 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

18. An in vivo or in vitro method for modulating HTRA1 expression in a target cell expressing HTRA1, the method comprising administering to the cell an effective amount of the oligonucleotide of any one of claims 1-15 or the conjugate of claim 16 or the pharmaceutical composition of claim 17.

19. A method of treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of the oligonucleotide of any one of claims 1-15 or the conjugate of claim 16 or the pharmaceutical composition of claim 17.

20. An oligonucleotide of any one of claims 1-15 or a conjugate of claim 16 or a pharmaceutical composition of claim 17 for use in medicine.

21. The oligonucleotide of any one of claims 1-15 or the conjugate of claim 16 or the pharmaceutical composition of claim 17 for use in the treatment or prevention of a disease selected from the group consisting of: macular degeneration (such as wet AMD, dry AMD, geographic atrophy, mid-term dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Duchenne muscular dystrophy, arthritis (such as osteoarthritis) and familial ischemic cerebrovascular and cerebrovascular diseases.

22. Use of the oligonucleotide of any one of claims 1-15 or the conjugate of claim 16 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for treating or preventing a disease selected from the group consisting of: macular degeneration (such as wet AMD, dry AMD, geographic atrophy, mid-term dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Duchenne muscular dystrophy, arthritis, such as osteoarthritis, and familial ischemic cerebrovascular and cerebrovascular diseases.

23. The use or method of any one of claims 19-22, wherein the method or use is for the treatment of macular degeneration.