CN112175945A

CN112175945A - NGF small interfering nucleic acid molecules and uses thereof

Info

Publication number: CN112175945A
Application number: CN201910587689.5A
Authority: CN
Inventors: 孔素东; 冷红
Original assignee: Suzhou Beixin Biotechnology Co ltd; Nuclear Industry General Hospital
Current assignee: Suzhou Beixin Biotechnology Co ltd; Nuclear Industry General Hospital
Priority date: 2019-07-02
Filing date: 2019-07-02
Publication date: 2021-01-05

Abstract

The present invention relates to double-stranded ribonucleic acid (dsRNA) for NGF genes. The dsRNA comprises a sequence having a length of 19-25 nucleotides and is substantially complementary to at least a portion of a gene selected from NGF. The invention also relates to pharmaceutical compositions comprising said dsRNA and a pharmaceutically acceptable carrier; methods of using the dsRNA and using the pharmaceutical composition for treating psoriasis; and methods of using the dsrnas and using the pharmaceutical compositions for inhibiting expression of an NGF gene in a cell.

Description

NGF small interfering nucleic acid molecules and uses thereof

Technical Field

The present invention relates to double-stranded ribonucleic acid (dsRNA), and to small interfering nucleic acid molecules directed against the NGFNGF gene and pharmaceutical compositions thereof for the treatment of psoriasis by RNA interference (RNAi) effect.

Background

Andrew z.fire and Craig c.mello, in 1998, have together discovered mechanisms of action for RNA interference in vivo and have collectively won the nobel prize for physiology in 2006. Thus opening a door for the development of a new generation of drugs (RNA interference drugs) for resisting serious diseases such as viruses, cancers and the like. The RNA interference medicine has the advantages of novel action mode, definite action mechanism, strong targeting property, small side effect and the like. RNA interference (RNAi) is the phenomenon whereby double-stranded RNA molecules turn off or silence the expression of homologous genes at the mRNA level. RNA interference techniques, also referred to figuratively as gene knock-down or gene silencing (PTGS), are a typical method of post-transcriptional gene regulation, also known as post-transcriptional gene silencing (PTGS). The first reports on RNA interference appeared in 1990, where RNA interference in transgenic plants was reported simultaneously by two different groups, and later in almost all eukaryotes such as nematodes, drosophila, zebrafish and mice. In 1999, Hamilton and Baulcombe detected RNA fragments of 21-25 nucleotides in length in plants undergoing RNA interference, which were shown to be essential for RNA interference, and were called small interfering nucleic acids (siRNA). The double-stranded siRNA forms an RNA-induced silencing complex (RISC) with associated enzymes and proteins of cell origin. During RNA interference, the sense strand of the double-stranded siRNA is excluded from the complex, the antisense strand directs RISC to bind to the homologous site of the target mRNA, and the target mRNA is then degraded by ribonuclease III in the complex, thereby turning off the expression of the target gene.

Psoriasis (Psoriasis), also known as "Psoriasis", presents erythema on the surface of the patient's skin, a chronic skin disease characterized by lesions and white Psoriasis covered by the skin, with the scalp, palms and soles of the feet being common sites of attack. The pathological features of the medicine are abnormal proliferation of keratinocyte and acanthocyte, inflammatory cell infiltration and vascular proliferation, and the disease is easy to recur and difficult to cure radically, and is one of the diseases which are mainly concerned at home and abroad. Psoriasis has a worldwide incidence of between 0.09 and 6.1%, and in most developed countries, the prevalence is 1.5 to 5%. Affects the normal life of about 1.25 million patients; the disease rate in China is increased from 0.12% in 1988 to 0.47% at present, about 650 ten thousand patients exist, the disease rate is in an increasing trend every year, and most patients suffer from anxiety and depression, which seriously affect the quality of life.

As the worldwide incidence of psoriasis increases year by year, in 2015, in order to improve the cognition and prevention and control of the psoriasis for the public, the world health organization sets the day of 10 months and 29 days (resolution EB133.R2) every year as the world psoriasis day, improves the public understanding of the psoriasis and loves the psoriasis patients. According to investigation, the medication expenditure of patients suffering from the psoriasis globosa in 2014 is up to $ 74.9 billion, and the medication expenditure is estimated to reach $ 90.4 billion in 2019, so that the research on psoriasis treatment medicines is of great significance.

The pathogenesis of psoriasis is not clear at present, and the generation of psoriasis is generally considered to be caused by abnormal differentiation and hyperproliferation of epidermal keratinocytes, with neovascularization and marked inflammation of the skin, and is closely related to genetic, personal physical, endocrine and mental factors.

The psoriasis treating method mainly comprises drug treatment, physical treatment and psychological treatment. Psychological treatment is usually used as an adjuvant therapy for psoriasis. Physical therapy refers to applying photosensitizer to the skin of a patient and irradiating the skin with long-wave, broadband short-wave or broadband short-wave ultraviolet rays. The physical therapy is directed to psoriasis vulgaris and pustular psoriasis, and the physical therapy has obvious effect but is accompanied with the generation of skin aging, pigmentation and skin cancer.

In the psoriasis treatment guideline of China, the drug therapy is the first-push therapy. Therapeutic drugs are divided into botanical drugs, chemical drugs and antibody drugs. The disease is divided into three grades of mild, severe and severe according to the severity of the disease, and 80 percent of patients with psoriasis have mild to moderate disease. The plant medicines are generally applied to the treatment of mild psoriasis, the antibody medicines are applied to the treatment of severe psoriasis, and the chemical medicines are applied to the treatment of mild to moderate psoriasis, so that the chemical medicines are widely applied to the psoriasis.

The plant medicine comprises the following components: is mainly applied to mild to moderate psoriasis vulgaris and is mainly applied to external application in the form of ointment. Natural drugs and their derivatives usually include vitamin A derivatives, salicylic acid, camptothecin, triptolide, and the like. For example, tripterygium wilfordii and salicylic acid act in a mode of resisting inflammation and inhibiting the proliferation of keratinocytes to play a role in resisting psoriasis, but excessive use causes more side effects, such as headache, dizziness, arrhythmia and the like.

Chemical drugs: is mainly applied to mild to moderate psoriasis, and is applied in a form of cream smearing and matched with oral medicines. Chemical drugs include antibiotics and antiproliferative drugs such as: cyclosporin A (cyclosporine A), Methotrexate (Methotrexate), tacrolimus, dithranol and the like. The chemical medicine has the functions of regulating immunity, inhibiting proliferation and differentiation of epidermal cells and inhibiting activation of immune cells to resist psoriasis. The medicines have good curative effect and low cost; but the side effect is large, and the patient can not use the medicine for a long time, for example, the toxicity of methotrexate to the liver, the kidney and the bone marrow is large. The immunosuppressive drugs mainly comprise apremilast, adenosine A3 receptor agonist and the like, and the drugs obviously improve the symptoms of psoriasis, but have side effects of skin redness, swelling, vomiting and the like, and have certain embryotoxicity and teratogenicity.

Therefore, there is a need in the art to develop safe and effective therapeutic agents.

Disclosure of Invention

To solve the above problems, the present invention provides a solution for alleviating or treating psoriasis by a small interfering nucleic acid molecule directed against the NGFNGF gene.

In a first aspect of the invention, there is provided the use of a small interfering nucleic acid molecule which directs the cleavage of an NGF mRNA sequence present in a cell, in the preparation of an agent for inhibiting the expression of an NGF gene in said cell, whereby said inhibition is effected. In the present invention, the small interfering nucleic acid molecule comprises a sense nucleic acid fragment and an antisense nucleic acid fragment, wherein the sense nucleic acid fragment and the antisense nucleic acid fragment contain complementary regions capable of forming a double-stranded nucleic acid structure capable of inhibiting the expression of the NGF gene in a cell. The sense and antisense nucleic acid fragments of the small interfering nucleic acid may be present on two different nucleic acid strands or on the same nucleic acid strand. When the sense nucleic acid fragment and the antisense nucleic acid fragment are located on both strands, respectively, at least one strand of the small interfering nucleic acid has a3 'overhang of 0 to 6 nucleotides in length, preferably both strands have a 3' overhang of 2-3 nucleotides in length. When the sense nucleic acid fragment and the antisense nucleic acid fragment are present on the same nucleic acid strand, the small interfering nucleic acid is preferably a hairpin-type single-stranded nucleic acid molecule in which complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure. In the small interfering RNA molecule, the length of the sense nucleic acid fragment and the antisense nucleic acid fragment is 19 to 29 nucleotides, and can be 19, 20, 21, 23, 25, 27 and 29, preferably 19, 21, 25 and 27 nucleotides. The small interfering nucleic acid molecule can be directly introduced into the cell, or can be produced in the cell after introducing a nucleotide sequence encoding the small interfering nucleic acid molecule into the cell; the cell is preferably a mammalian cell, more preferably a human cell. The cells may be ex vivo, e.g., cell lines, or may be present in a mammalian body, e.g., a human body. The human is a patient with psoriasis and the small interfering nucleic acid molecule is administered in an amount sufficient to effect treatment of the condition.

In a second aspect, the invention provides a small interfering nucleic acid molecule which directs cleavage of an NGF mRNA sequence present in a cell, thereby effecting inhibition of expression of the gene, preferably the small interfering nucleic acid molecule sequence is as follows:

NGF siRNA1

siRNA1 sense strand 5'-CCAUGUUGUUCUACACUCU dTdT-3' (SEQ ID No.2)

siRNA1 antisense strand 3 '-dTdTGGUACAAGAUGAGA-5' (SEQ ID No.3)

NGF siRNA2

siRNA2 sense strand 5 '-CCACAGACAUCAAGGGCAA dTdT-3' (SEQ ID No.4)

siRNA2 antisense strand 3'-dTdT GGUGUCUGUAGUUCCCGUU-5' (SEQ ID No.5)

NGF siRNA3

siRNA3 sense strand 5'-GGGCAAGGAGGUGAUGGUGdTdT-3' (SEQ ID No.6)

siRNA3 antisense strand 3' -dTdCCCGUUCCCUACCAC-5 "(SEQ ID No.7)

All nucleotides in the small interfering RNA molecule can be natural nucleotides which are not chemically modified, or at least one nucleotide can be chemically modified nucleotides, and the chemical modification mode is selected from at least one of the following modifications:

(1) a modification of the phosphodiester bond linking nucleotides in the nucleotide sequence of the small interfering nucleic acid molecule;

(2) a modification of the 2' -OH of the ribose sugar in the nucleotide sequence of the small interfering nucleic acid molecule;

(3) modifications to bases in the nucleotide sequence of the small interfering nucleic acid molecule.

After the NGF gene small interfering nucleic acid is injected into the edges of skin lesions, the psoriasis symptoms can be effectively relieved.

The invention also provides a composition comprising any one of the small interfering nucleic acid molecules described above and a pharmaceutically acceptable carrier, preferably a liposome or a high molecular polymer. Preferably, the composition further comprises a botanical drug and/or a chemical drug for treating psoriasis, wherein the chemical drug is selected from the group consisting of Yixepu and Allolopine, or a combination thereof.

The invention also provides the application of the small interfering RNA molecule or the composition in the preparation of the medicine for treating psoriasis. The small interfering nucleic acid molecules can be introduced directly into the diseased skin cells, preferably the cells are diseased human skin cells.

The invention has the advantages of

The small interfering RNA molecule aiming at the NGF gene expression can efficiently and specifically inhibit the expression of the NGF gene, has low toxic and side effects, and can be used for preparing medicines for inhibiting the NGF gene expression and treating psoriasis.

Drawings

Figure 1 is a graph of the therapeutic effect of NGF siRNA on psoriasis mice. The results show that the skin on the back of the control mice is not obviously changed, while the skin on the back of the model mice gradually appears red macula, plaque and scale after being externally applied with the IMQ cream for 3 days, and is most obviously changed in a typical psoriasis-like manner on the 7 th day. Compared with the model group, the blumea and Yisaipu administration group has reduced erythema and scale; after the NGF siRNA of the invention is respectively injected, the back skin of the mice in the administration group only has slight erythema and scale, and the change on the 7 th day is obviously lighter than that of the mice in the model group.

FIG. 2 is a graph of the effect of NGF siRNA on the pathological changes in skin lesions in psoriatic mice. In pathological changes, the skin lesions of the mice in the model group are manifested by obvious hyperkeratosis, acanthosis hypertrophy and dermal inflammatory cell infiltration, and are very similar to the pathological changes of the human psoriasis; the pathological changes of the mouse skin lesions in the group with the Alloloson and NGF-siRNA administration are obviously reduced, the effect of the group with the NGF-siRNA3 administration on the pathological changes of the mouse skin lesions is better than that of the group with the NGF-siRNA1 and the NGF-siRNA2 administration, but compared with the skin of a control group, the group with the NGF-siRNA administration still has slight hyperkeratosis, acanthosis pachynsis, inflammatory infiltration and the like.

Detailed Description

Psoriasis (psoriasis), also known as psoriasis, is a common and easily relapsed chronic inflammatory skin disease, clinically with erythematous squamous maculopapule as the major lesion, and a few patients can affect the joints. Histopathologically, psoriasis presents three major features: abnormal Keratinocyte (KC) proliferation, dermal superficial vascular proliferation, and lymphocyte infiltration.

With the development of the neuro-endocrine-immune network theory, the role of Nerve Growth Factor (NGF) in the pathogenesis of psoriasis has attracted increasing attention. NGF is one of the important members of the neurotrophin family, a protein characterized by a 118 amino acid dimer, consisting mainly of three subunits (α, β, γ), in which the biological activity is mainly in the β subunit. Since discovery to date, for half a century, early research focused on the field of the nervous system, and in recent years it was discovered that numerous non-neural cells also express NGF and its receptors, such as keratinocytes. Domestic and foreign studies have shown that three pathological features of psoriasis can be affected by NGF: KC generates NGF in an autocrine and paracrine mode, and the NGF can promote KC proliferation in a feedback mode; NGF activates T lymphocytes and causes secretion of inflammatory mediators, such as TNF- α, IL-2; NGF induces vascular proliferation by promoting endothelial cell proliferation.

The invention designs the siRNA medicament aiming at the NGF gene, can pertinently reduce or even silence the expression of the NGF gene, and reduce the content of NGF protein in vivo, thereby having the effect of treating psoriasis.

The embodiment of the invention discloses a method for designing and obtaining small interfering RNA molecules aiming at NGF genes and a method for preparing a medicament for treating psoriasis by using the small interfering RNA molecules. Those skilled in the art can now appreciate from this disclosure that appropriate modifications of the process parameters and adjuvant components can be made, and it is expressly intended that all such similar substitutes and modifications which are obvious to those skilled in the art are deemed to be within the scope of the invention. The invention of small interfering nucleic acid and pharmaceutical composition has been described through the preferred embodiment, but the related personnel obviously can not depart from the content and scope of the invention to the product and method of change or appropriate changes and combinations, to realize and apply the technology of the invention. The following examples are merely illustrative, and the present invention is not limited to these examples.

The invention designs and screens an active small interfering RNA (siRNA) sequence based on the NGF sequence shown in SEQ ID NO. 1. 5'-ATGTCCATGTTGTTCTACACTCTGATCACAGCTTTTCTGATCGGCATACAGGCGGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGCCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGA-3' (SEQ ID NO.1)

Example 1 NGF siRNA alleviates symptoms in psoriasis mouse models

BALB/c mice of 8 weeks of age were purchased and randomized into the following groups of 6 mice each, after weight numbering:

(1) control group (or normal group or Ctrl group) — applying vaseline on back

(2) IMQ group (imiquimod, imiquimod cream for mouse psoriasis model building)

(3) IMQ group + NGF siRNA injection group (3 pairs of siRNA)

(4) IMQ group + Arolopine ointment (amount determined according to reference)

(5) IMQ group + Yisaipu (dosage determined according to reference)

Each group of mice was shaved with an electric shaver by selecting a 2cm by 2cm area on the same site of the back of the mice (considering the reagent requirements and the size of the volume of the mice themselves, the 2cm by 2cm area was selected for IMQ application in the final experiment).

Administration dose of siRNA: treatment was initiated at day 8 after observation and detection success at day 7 of modeling, with drug injection around the skin lesion for 7 consecutive days, according to reference 25 nmol/day.

The administration dose of the allion ointment is as follows: each mouse was administered with 50 mg/day of allion ointment, and after observation and detection on day 7 of modeling were successful, the therapeutic yisaipu was administered from day 8: according to the calculation of the reference, the medicine is administered from day 8 after the observation, detection and modeling are successful on day 7 of modeling according to 0.1 mg/patient/day, and the medicine is injected around the skin lesion for 14 days continuously.

As can be seen from fig. 1: in the figure, the control group (Ctrl, control group) is the result of applying vaseline on the mouse denuded area, and the IMQ group (model group) is the result of continuously applying IMQ ointment on the mouse denuded area. The skin on the back of the control group mice is not obviously changed, while the skin on the back of the model group mice gradually appears red macula, plaque and scale after being externally applied with the IMQ cream for 3 days, and is most obviously changed in a typical psoriasis-like manner on the 7 th day. Compared with the model group, the blumea and Yisaipu administration group have reduced erythema and scale, mice in the NGF siRNA administration group are injected with the NGF siRNA respectively, the back skin of the mice in the NGF siRNA administration group has only slight erythema and scale, and the change of the mice on the 7 th day is obviously lighter than that in the model group.

Example 2 Effect of NGF siRNA on pathological changes in skin lesions in psoriatic mice

HE Experimental procedure

1. Selecting skin lesion tissues with the area of 1cm x 1cm after each group of experimental animals are taken, and fixing the skin lesion tissues with paraformaldehyde;

2. embedding the section in paraffin, and preparing for immunohistochemistry and HE staining;

3. paraffin tissue sections of 3 mice were arbitrarily extracted from each group and subjected to dewaxing treatment

Xylene I20 min → xylene II 20min → absolute ethanol 10min → 95% ethanol 10min → 85% ethanol 10min → 70% ethanol 10min → 50% ethanol 10min → ddH2O 10min

4. Staining with hematoxylin for 10min

5. Washing with double distilled water, and differentiating with hydrochloric acid-ethanol

6. Aqua ammonia blue

7. Eosin staining for 10min

8. Double distilled water flushing

9. Dehydrating the sealing sheet, taking a picture by a microscope

As shown in fig. 2, the skin lesions of the mice in the model group (IMQ group) showed significant hyperkeratosis, hypertrophy of the spinous layer and infiltration of inflammatory cells in the dermis in terms of pathological changes, which are very similar to those of human psoriasis, indicating successful modeling. The pathological changes of the mouse skin lesions in the group with the Alloloson and NGF-siRNA administration are obviously reduced, the effect of the group with the NGF-siRNA3 administration on the pathological changes of the mouse skin lesions is better than that of the group with the NGF-siRNA1 and the NGF-siRNA2 administration, but compared with the skin of a control group, the group with the NGF-siRNA administration still has slight hyperkeratosis, acanthosis pachynsis, inflammatory infiltration and the like.

Combining the above results, applicants have found a plurality of sirnas capable of significantly inhibiting the reduction of psoriasis symptoms by screening for sirnas targeting NGF genes. These results clearly suggest that the use of siRNA targeting NGF gene would be a promising strategy for the treatment of psoriasis.

Claims

1. A small interfering nucleic acid molecule directing cleavage of NGF mRNA sequence present in a cell, thereby effecting inhibition of NGF expression, the sense strand of the small interfering nucleic acid molecule being represented by any one of the nucleotide sequences selected from SEQ ID No.2 or SEQ ID No.4 or SEQ ID No.6, and the antisense strand being represented by any one of the nucleotide sequences selected from SEQ ID No.3 or SEQ ID No.5 or SEQ ID No. 7.

2. The small interfering nucleic acid molecule of claim 1, wherein at least one nucleotide is a chemically modified nucleotide.

3. The small interfering nucleic acid molecule of claim 2 wherein the chemical modification is at least one of:

4. A composition comprising the small interfering nucleic acid molecule of any one of claims 1-3 and a pharmaceutically acceptable carrier.

5. The composition of claim 4, wherein the pharmaceutically acceptable carrier is a liposome or a high molecular weight polymer.

6. A composition according to any one of claims 4 to 5, characterised in that the composition may also contain a botanical and/or chemical drug for the treatment of psoriasis.

7. The composition of claim 6, wherein the chemical for treating psoriasis is selected from the group consisting of cypress or Allolone or a combination thereof.

8. Use of a small interfering nucleic acid molecule according to any one of claims 1 to 3 or a composition according to any one of claims 4 to 7 for the manufacture of a medicament for the treatment of psoriasis.

9. The use according to claim 8, characterized in that the small interfering nucleic acid molecule is introduced directly into the diseased skin cells.

10. Use according to claim 9, characterized in that the cells are diseased human skin cells.