WO2018178608A1

WO2018178608A1 - Methods of treatment of diabetic ulcers with microrna

Info

Publication number: WO2018178608A1
Application number: PCT/GB2017/050923
Authority: WO
Inventors: Sergiu-Bogdan CATRINA; Xiaowei ZHENG
Original assignee: Catrina Sergiu Bogdan; Zheng Xiaowei
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2018-10-04
Also published as: CN110494146A; CN110494146B

Abstract

There are herein provided methods for the treatment of diabetic ulcers comprising administering micro RNA-210, or related compounds, to a patient in need thereof.

Description

METHODS OF TREATMENT OF DIABETIC ULCERS WITH MICRORNA

Field of the Invention The present invention relates to novel methods of treatment of ulcers in diabetic patients comprising administration of microRNAs or vectors encoding the same. In particular, the invention relates to the use of microRNA-210 and precursors thereof, including in methods of treating and/or promoting the healing of diabetic ulcers. Background of the Invention

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Diabetes mellitus (diabetes) refers to a group of diseases in which the malfunction of glucose homeostasis results in hyperglycaemia experienced over a prolonged period. The major subtypes of diabetes are type 1 (or insulin-dependent diabetes) and type 2 (insulin- independent diabetes).

Patients with diabetes are at high risk of developing skin ulcers. The areas at greatest risk are the legs; particularly the feet. The peripheral neuropathy that often develops as a result of chronic hyperglycaemia causes a loss of coordination of muscle groups in the feet and legs, which increases mechanical stresses during ambulation, increasing the likelihood of the formation ulcers on the lower legs or feet. The sensation of pain is also reduced in extremities affected by peripheral neuropathy, further increasing the likelihood of ulcer formation due to the patient's lack of ability to identify the need to take preventative measures. In other cases, vascular disease developing in later stage diabetes may also contribute to lesion formation.

As well as increasing the likelihood of ulcer formation, biological factors associated with diabetes impair the normal healing of open wounds. Diabetic ulcers and particularly diabetic foot ulcers are therefore one of the gravest complications of diabetes. Indeed, diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Diabetes is the leading cause of non-traumatic lower extremity amputations in the United States, with approximately 5% of diabetics developing foot ulcers each year and 1 % requiring amputation. At present, there are no effective treatments for diabetic foot ulcers approved for use. Therefore, there exists a significant and unmet need for such treatments.

Description of the Invention

The inventors have now surprisingly found that the administration of mature microRNA- 210, or precursors thereto can improve the healing of diabetic ulcers in a patient in need thereof. Methods of medical treatment

In a first aspect of the invention, there is provided a method of treatment of an ulcer in a patient having diabetes, comprising administering a therapeutically-effective amount of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need of such treatment.

In an alternative first aspect of the invention, there is provided a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 for use in the treatment of an ulcer in a patient having diabetes.

In a further alternative first aspect of the invention, there is provided the use of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 for the manufacture of a medicament for the treatment an ulcer in a patient having diabetes.

Unless indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Preferences and options for a given aspect, embodiment, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For the avoidance of doubt, mature microRNA-210 and precursors to mature microRNA- 210, as defined herein, may be referred to herein as the "nucleic acids of the invention". The skilled person will be able to identify suitable mature micro-RNA and precursors thereto, as defined herein, by reference to sources available to those skilled in the art. For example, the skilled person may refer to databases such as the the NCBI database and the miRBase database (http://www.mirbase.org), the contents of which are incorporated herein in their entirety.

For example, the skilled person will understand that mature microRNA-210 (for example for use in various species such as mouse and human (e.g. human)) comprises or consists of (e.g. consists of) the nucleotide sequence CUGUGCGUGUGACAGCGGCUGA (SEQ ID NO: 1)

For the avoidance of doubt, microRNA-210 may refer to mature microRNA-210, pre- microRNA-210 and/or primary-microRNA-210 (pri-microRNA-210), which terms will be understood by those skilled in the art.

As described herein, the nucleic acids of the invention include mature microRNA-210 and precursors thereto. Suitable precursors include biological precursors, such as nucleic acids that may be processed into mature microRNA-210 by means of one or more cellular processing steps and/or result in the formation of mature microRNA-210 following their addition to a cell. Such precursors include pri-microRNA-210, pre-microRNA-210, double-stranded mimics of mature microRNA-210, and vectors encoding microRNA-210. In certain embodiments, the nucleic acid is a double-stranded mimic of mature microRNA- 210. The skilled person will understand that pri-microRNA-210 and pre-microRNA-210 are upstream precursors to mature microRNA-210. Without wishing to be bound by theory, pri-microRNA-210 may be understood to refer to the primary transcript(s) produced by the action of RNA polymerase II on the gene encoding microRNA-210, and pre-microRNA- 210 may be understood to refer to the hairpin precursor(s) to mature microRNA-210 produced following the processing of pri-microRNA-210 by ribonuclease Ill-type enzyme Drosha. Both pri-microRNA-210 and pre-microRNA-210 comprise, within longer nucleotide sequences, the nucleotide sequence of mature microRNA-210 (CUGUGCGUGUGACAGCGGCUGA (SEQ ID NO: 1)). Double-stranded mimics of mature microRNA-210 include artificial double-stranded oligonucleotides designed to mimic the function of endogenous mature miRNA-210. Such mimics may be structurally similar to the double-stranded RNA produced following cleavage of the pre-microRNA-210 hairpin by Dicer ribonuclease and, therefore, may be loaded into RNA-induced silencing complex (RISC) and processed into single-stranded mature microRNA-210. An example of a suitable double-stranded mimic of microRNA- 210 is the miRIDIAN miR-210 mimic (miRIDIAN mmu-miR-210-3p; Cat #: C-310570-05, Dharmacon)

The skilled person will understand that vectors are recombinant nucleic acid constructs that are engineered to express polynucleotides in target cells. Vectors typically include the nucleic acid sequence encoding the polynucleotide to be expressed and one or more regulatory (or control) sequences that facilitate efficient transcription of that polynucleotide. Examples of vectors include plasmids, viral, and non-viral vectors, and the like.

As used herein, vectors encoding microRNA-210 may be understood to be nucleic acid molecules comprising a polynucleotide sequence encoding microRNA-210 (pri-microRNA- 210, pre-microRNA-210 or mature microRNA-210 (e.g. pri-microRNA-210)), and optionally one or more regulatory sequences. In some embodiments, the vectors are DNA molecules comprising a polynucleotide sequence encoding microRNA-210 (pri-microRNA-210, pre- microRNA-210 or mature microRNA-210 (e.g. pri-microRNA-210)), and optionally one or more regulatory sequences. Such vectors may be readily prepared using methods known to the person skilled in the art (such as, for example, those described in Green and Sambrook Molecular Cloning: A Laboratory Manual, Fourth Edition Coldspring Harbour Laboratory Press (2012) the contents of which is incorporated herein in its entirety).

In some embodiments, mature microRNA-210 comprises or consists of (e.g. consists of) the nucleotide sequence CUGUGCGUGUGACAGCGGCUGA (SEQ ID NO: 1). In alternative embodiments, analogues of the nucleic acids of the invention may be used, as long as the mature microRNA administered or produced following the administration of precursors thereto maintains the ability to regulate its target(s). Such analogues encompass miRNA sequences in which one or more bases are substituted or deleted. Preferably, in order to maintain the activity towards the target mRNA, the 'seed' sequence, which is a sequence completely complementary to a sequence within the mRNA targeted by microRNA-210, should remain unchanged. In some embodiments, an analogue has at least about 75% identity or complementarity to mature microRNA-210; for example, at least about 80%, at least about 85%, at least about 90%, at least about 99% identity or complementarity to mature microRNA-210. For the avoidance of doubt, each possibility represents a separate embodiment of the present invention. The skilled person will understand that references to the treatment of a particular condition (or, similarly, to treating that condition) take their normal meanings in the field of medicine. In particular, the terms may refer to achieving a reduction in the severity of one or more clinical symptom associated with the condition. For example, in the case of ulcers in patients having diabetes (i.e. diabetic ulcers) this may refer to promoting the healing of the ulcer such that the ulcer heals (i.e the skin or mucous membrane (e.g. skin) heals over) within an improved time frame relative to an untreated ulcer. This time frame may be in the range of three to twelve months (for example six to twelve months). In particular embodiments, such as for neuropathic diabetic ulcers, this time frame may be three months, and for ischemic diabetic ulcers, the time frame may be six months. Alternatively, a successful treatment may comprise achieving a measurable decrease in the size (e.g. width, depth, volume etc) of the ulcer within a certain time frame (for example within one month, such as within three months, e.g. within six months). In certain embodiments, the size of the ulcer may be reduced by at least 10%; for example, at least 20%, such as at least 50% (e.g. at least 75%).

As used herein, references to patients will refer to a living subject being treated, including mammalian (e.g. human) patients. Thus, in particular embodiments of the first aspect of the invention, the treatment is in a mammal (e.g. a human).

In a second aspect of the invention, there is provided a method of promoting diabetic ulcer healing comprising administering a therapeutically-effective amount of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) to a patient in need of such treatment.

In an alternative second aspect of the invention, there is provided a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA- 210 (i.e. the nucleic acids of the invention, as defined herein) for use in promoting diabetic ulcer healing.

In a further alternative aspect of the invention, there is provided the use of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) for the manufacture of a medicament for promoting diabetic ulcer healing. In a third aspect of the invention, there is provided a method of promoting ulcer healing in a patient having diabetes comprising administering a therapeutically-effective amount of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) to a patient in need of such treatment.

In an alternative third aspect of the invention, there is provided a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) for use in promoting ulcer healing in a patient having diabetes.

In a further alternative third aspect of the invention, there is provided the use of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) for the manufacture of a medicament for promoting ulcer healing in a patient having diabetes.

For the avoidance of doubt, references to promoting diabetic ulcer healing and promoting ulcer healing in a patient having diabetes, as used herein, may both be understood to indicate improving the rate of healing of ulcers in patients having diabetes (i.e. diabetic ulcers) such that an increased number of patients heal within a certain time frame or the ulcer heals (i.e the skin or mucous membrane (e.g. skin) heals over) within an improved time frame relative to an untreated ulcer. In particular embodiments, such as for neuropathic diabetic ulcers, this time frame may be three months, and for ischemic diabetic ulcers, the time frame may be six months. Alternatively, promoting diabetic ulcer healing may comprise achieving a measurable decrease in the size (e.g. width, depth, volume etc) of the ulcer within a certain time frame (for example within one month, such as within three months, e.g. within six months). In certain embodiments, the size of the ulcer may be reduced by at least 10%, for example at least 20%, such as at least 50% (e.g. at least 75%) following treatment with the nucleic acids of the invention.

As used herein, the term diabetes may be understood to encompass type 1 diabetes, type 2 diabetes and less common subtypes such as gestational diabetes, maturity-onset diabetes in the young (MODY), latent autoimmune diabetes of adults (LADA), cystic fibrosis-related diabetes (CFRD) and the like.

In particular embodiments of the first to third aspects of the invention, references to diabetes (e.g. in a diabetic patient) will refer to type 1 or type 2 diabetes. The skilled person will understand that the term ulcer may refer to an open lesion on an internal or external body surface caused by a break in the skin or mucous membrane, which is slow to heal and/or fails to heal. Ulcers can form on a range of body surfaces, such as in the mouth, in the gastrointestinal tract or on the skin. In particular embodiments of the first to third aspects of the invention, the ulcer being treated with the nucleic acids of the invention occurs on the skin, such an ulcer may be referred to as a skin ulcer.

The skilled person will understand that skin ulcers may be positioned anywhere on the body of the patient being treated. In particular embodiments of the first to third aspects of the invention, the ulcer may be positioned on the leg or foot (e.g. the foot) of the patient being treated. Such ulcers may be referred to as 'leg ulcer(s)' and 'foot ulcer(s)', respectively. In particular embodiments of the first to third aspects of the invention, the ulcer is not an ischemic ulcer (i.e. the ulcer is a non-ischemic ulcer).

Ulcers in patients suffering from diabetes may be referred to as diabetic ulcers. In certain embodiments, the ulcer is a diabetic ulcer. In more particular embodiments of the first to third aspects of the invention, the ulcer is a diabetic leg ulcer or a diabetic foot ulcer (e.g. a diabetic foot ulcer).

As used herein, the term (diabetic) leg ulcer may be understood to refer to an ulcer formed on the leg of an affected patient. Such an ulcer may form on the upper leg (e.g. in the groin and/or on the thigh), on/around the knee, and/or, more particularly on the lower leg (e.g. on the shin, the calf and/or on/around the ankle). The term (diabetic) foot ulcer may be understood to refer to an ulcer formed on the foot of an affected patient. Such an ulcer may form on/around the ankle, on the top of the foot, on/around the heel, on the toes and/or, more particularly, on the bottom (i.e. the sole) of the foot. For the avoidance of doubt, ulcers formed on/around the ankle of an affected patient may fall under the definitions of both (diabetic) leg and foot ulcers.

Diabetic ulcers (e.g. diabetic foot ulcers) may be divided into different subtypes depending on the underlying condition responsible for their cause. Ulcers occurring in patients suffering from diabetes-induced peripheral neuropathy (preferably in the substantial absence of vascular disease) in the affected limb may be referred to as neuropathic diabetic ulcers, and ulcers occurring in patients suffering from vascular disease (e.g. diabetes-induced vascular disease) in the affected limb may be referred to as ischemic diabetic ulcers. Diabetic ulcers may also have mixed aetiology, and such ulcers may be referred to as neuroischemic ulcers.

Without wishing to be bound by theory, it is believed that, as chronic hyperglycaemia causes the hypoxic response to be impaired in all diabetic ulcers, the administration of the nucleic acids of the invention may provide an effective treatment for diabetic ulcers (e.g. diabetic skin ulcers) regardless of their aetiology.

Accordingly, in certain embodiments of the first to third aspects of the invention, the (diabetic) ulcer (for example, the diabetic leg or foot ulcer (e.g. diabetic foot ulcer) is a neuropathic diabetic skin ulcer (for example, a neuropathic diabetic leg or foot ulcer (e.g. a neuropathic diabetic foot ulcer)). In particular embodiments, the neuropathic diabetic ulcer (for example, the neuropathic diabetic leg or foot ulcer (e.g. neuropathic diabetic foot ulcer)) is not an ischemic ulcer (i.e. the neuropathic diabetic ulcer is a non-ischemic ulcer).

In alternative embodiments of the first to third aspects of the invention, the diabetic skin ulcer (for example, the diabetic leg or foot ulcer (e.g. diabetic foot ulcer) is an ischemic diabetic skin ulcer (for example, an ischemic diabetic leg or foot ulcer (e.g. an ischemic diabetic foot ulcer)).

In further alternative embodiments, the diabetic skin ulcer (for example, the diabetic leg or foot ulcer (e.g. diabetic foot ulcer)) is a neuroischemic diabetic skin ulcer (for example a neuroischemic diabetic leg or foot ulcer (e.g. a neuroischemic diabetic foot ulcer)). For the avoidance of doubt, all preferences and embodiments described hereinabove with reference to the first to third aspects of the invention apply equally to the fourth to seventh aspects of the invention, as described herein below.

Further methods of medical treatment

Without wishing to be bound by theory, it is believed that microRNA-210 may enhance the healing of diabetic ulcers by enhancing the response to hypoxia, which is impaired by the chronically high blood glucose levels experienced by diabetic patients. One aspect of the hypoxic response that is impaired under hyperglycaemic conditions is the proliferation and migration of fibroblasts, the cells responsible for the production of fibrous tissue such as collagen and the extracellular matrix. According to a fourth aspect of the invention, there is provided a method of enhancing the hypoxic response in a diabetic ulcer, comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) to a patient in need thereof.

The skilled person will understand that enhancing the hypoxic response may refer to improving one or more processes mediated by hypoxia-inducible factor 1 (HIF-1) under hypoxic conditions. For example, an increase in angiogenesis, cell proliferation, cell survival, cell migration, erythropoiesis and cell differentiation (e.g. fibroblast proliferation, migration and differentiation), recruitment of endothelial precursors cells, antibacterial capacity.

According to a fifth aspect of the invention, there is provided a method of restoring fibroblast function (e.g. proliferation and migration and/or synthesis of extracellular matrix and collagen) in a diabetic ulcer, comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) to a patient in need thereof.

According to a sixth aspect of the invention, there is provided a method of promoting the formation of extracellular matrix and/or collagen in dermal tissue in a diabetic ulcer, comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) to a patient in need thereof. The skilled person will appreciate that a key process in the final stages of ulcer healing (e.g. diabetic ulcer healing) is the re-epithelialization of the wound.

According to a seventh aspect of the invention, there is provided a method of promoting the re-epithelialization of a diabetic ulcer comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA- 210 (i.e. the nucleic acids of the invention, as defined herein) to a patient in need thereof.

Pharmaceutical compositions As described herein, the nucleic acids of the invention are useful as pharmaceuticals. Such nucleic acids may be administered alone or may be administered by way of known pharmaceutical compositions/formulations. The skilled person will understand that in the uses and methods of treatment, promoting healing, enhancing the hypoxic response, restoring fibroblast function, promoting the formation of extracellular matrix and/or collagen and promoting re-epithelialization defined herein (e.g. for the first to seventh aspects of the invention) the nucleic acid of the invention as defined herein may be administered in the form of a pharmaceutical composition.

Accordingly, in an eighth aspect of the invention, there is provided a pharmaceutical composition comprising a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein), and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier, for use in the treatments and methods defined herein (e.g. for the first to seventh aspects of the invention). In an alternative eighth aspect of the invention, there is provided a method as defined herein (e.g. for the first to seventh aspects of the invention) comprising administering a (therapeutically-effective amount of) a pharmaceutical composition comprising a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 (i.e. the nucleic acids of the invention, as defined herein) and optionally one or more pharmaceutically-acceptable excipient to a patient in need thereof.

Pharmaceutically-acceptable excipients include vehicles, adjuvants, carriers, diluents, pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like.

The skilled person will understand that the nucleic acids of the invention may act locally (i.e. at the ulcer site). Therefore, the skilled person will understand that the nucleic acids and compositions as described for the first to eighth aspects of the invention may be administered topically in a pharmaceutically-acceptable dosage form. Suitable routes of administration may include direct administration to the skin (dermal) or mucous membrane surface, subcutaneous, intradermal or transdermal administration (e.g. via microneedle injection or transdermal patch).

Suitable dosage forms include, for example, liposomal systems (e.g. suspensions) and emulsions, and may be in the form of liquids, lotions, creams, ointments and/or aerosols. Liquid preparations may be aqueous or oil based, and may also be prepared in the form of a dry powder to be reconstituted with a suitable solvent prior to use. According to some embodiments, various delivery systems may be used to transfer/introduce the nucleic acids of the invention into cells, such as encapsulation in liposomes, targeted liposomes, dendritic polyglycerolamine nanocarriers, nanoparticles, microparticles, microcapsules, electroporation, nucleofection, ultrasound based, laser based, recombinant cells that are capable of expressing microRNA-210, receptor- mediated endocytosis, construction of microRNA-210 as part of a viral vector or other vector, viral vectors that are capable of being reproduced without killing the cell during the process of reproduction, viral vectors that are not capable of reproduction, injection of cells that produce viral vectors that comprise the nucleic acids of the invention, injection of polynucleotides, electroporation, calcium phosphate mediated transfection, and the like, or any other methods known to the skilled person.

The skilled person will understand that the nucleic acids of the invention and compositions comprising the same, may be administered (for example, as formulations as described hereinabove) at varying doses, with suitable doses being readily determined by one of skill in the art. Dosage and frequency of administration may be selected in relation to the pharmacological properties of the nucleic acids to be delivered (i.e. naked RNA, vectors, delivery particles used, and the like). In some embodiments, the nucleic acids of the invention (alone or in combination with other agents) may be administered in a dose having an amount of between about 0.01 mg and about 10 mg per administration/treatment per day. For example, the amount may be between about 0.01 mg and about 8 mg per administration/treatment, such as between about 0.01 mg and about 2 mg per administration/treatment, for instance, between about 0.05mg and about 4 mg per administration/treatment, particularly, between about 0.05 mg and about 2 mg per administration/treatment, more particularly, between about 0.08 mg and about 2 mg per administration/treatment (e.g. be between about 0.08 mg and about 1 mg per administration/treatment). For example, the amount may be between about 0.5 mg and about 9 mg per administration/treatment. In some exemplary embodiments, the nucleic acids of the invention may be formulated in a saline solution (such as PBS). In some embodiments, the doses disclosed herein may be administered at any administration regime, such as, 1 to 5 times a day, 1 to 10 times a week or 1 to 15 times a month, wherein such administration may be at identical or different time intervals and/or at the same or different time of day. As used herein, the term about when being used to refer to a numerical value may be understood in indicate a variation of ±10%, in particular ±5%, such as ±2% (e.g. ±1 %) of the stated value. The nucleic acids of the invention may be introduced in any amount that allows delivery of at least one copy (i.e. of mature microRNA-210) per cell being treated. Higher doses for example at least 5 copies or at least 10 copies, such as at least 100 copies or at least 500 copies (e.g. at least 1000 copies) may give an improved effect. For the avoidance of doubt, the skilled person (e.g. the physician) will be able to determine the actual dosage which will be most suitable for an individual patient, which is likely to vary with the route of administration, the type and severity of the condition that is to be treated, as well as the species, age, weight, sex, renal function, hepatic function and response of the particular patient to be treated. The above-mentioned dosages are exemplary; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The nucleic acids and methods described herein may have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, and/or have other useful pharmacological properties over equivalent methods known in the art, whether for use in the above-stated indications or otherwise. In particular, such methods may have the advantage that they significantly improve the healing outcomes for and thus the prognosis for patients suffering from diabetic ulcers.

Brief Description of the Figures

Figure 1 illustrates the effect of the administration of a miR-210 mimic on wound healing in diabetic mice. A clear improvement in the rate of wound healing was observed for the miR-210 mimic compared to the negative control.

Figure 2 illustrates the effect of a miR-210 mimic on human fibroblast migration relative to a control. The effect was investigated under normoxic conditions at normal glucose levels (N5), under hypoxic conditions at normal glucose levels (H5) and under hypoxic conditions at high glucose levels (H30). A clear increase relative to the control was observed under hypoxic conditions at high glucose levels. Figure 3 illustrates the effect of a miR-210 mimic on human fibroblast proliferation relative to a control. The effect was investigated under normoxic conditions at normal glucose levels (N5), under hypoxic conditions at normal glucose levels (H5) and under hypoxic conditions at high glucose levels (H30). A clear increase relative to the control was observed under hypoxic conditions at high glucose levels.

Examples

The invention is illustrated, but in no way limited, by the following examples.

Example 1. The effect of the local injection of a microRNA-210 mimic on wound healing in diabetic mice

Intradermal injection of miR-210 mimic and negative control

Twelve-week-old male diabetic (db/db) mice on the C57BI/6J background (Charles Rivers, Jackson Labs, USA) were used. All experimental groups were age-, weight- and blood glucose-matched. The mice were anesthetized with 3% isoflurane (Abbott). The hair on the backs of the mice was removed using a shaver followed by a depilatory cream. After cleansing the skin with alcohol, wounds were made on the dorsum using a 6-mm biopsy punch on each side of the midline. Mice received buprenorphine (0.03 mg/kg) twice a day to relieve any potential pain and distress for the first two days and were placed in single cages after wounding. After wounding, 0.125nmol miRIDIAN miR-210 mimic (miRIDIAN mmu-miR-210-3p (based on the sequence MIMAT0000658 (CUGUGCGUGUGACAGCGGCUGA (SEQ ID NO: 1); Cat #: C-310570-05; Dharmacon) or negative control (miRIDIAN cel-miR-67 (based on the sequence MIMAT0000039 (UCACAACCUCCUAGAAAGAGUAGA (SEQ ID NO: 2); Cat #: CN-001000-01 ; Dharmacon) were injected along the edge of the wound intradermally. The injection was repeated 6 days after wounding. The wound biopsies and intact skin away from the injection site were taken after 12 days.

Evaluation of wound healing Photographs of the wounds were obtained on the day of wounding and every alternate day until sacrifice. A circular reference of known area was also placed alongside the wounded mice. Wound area was calculated using ImageJ software (National Institute of Health) as a proportion of the known circular reference area and expressed as a percentage of initial wound area. As can be seen from Figure 1 , the miR-210 mimic improved the rate of wound healing compared to the negative control. Example 2. The effect of a microRNA-210 mimic on fibroblast migration under normal and high glucose and hypoxic and normoxic conditions

Transfection of miR-210 mimic in HDF cells: Human dermal fibroblast (HDF) cells were seeded with 80-90% confluency on the first day. Next day, 1 , 10 and 20nM miR-210 mimic (miRIDIAN mmu-miR-210-3p; Cat #: C-310570- 05; Dharmacon) or negative control (miRIDIAN cel-miR-67; Cat #: CN-001000-01; Dharmacon) were transfected using RNAiMAX transfection reagent (Thermo Fisher Scientific, Cat # 13778075). The cells were allowed to recover for 48 hours before performing the analyses.

Migration

HDF cells were seeded and transfected in 24 wells. After 48 hours, scratches were made and the cells were exposed to normal (5.5mM) or high glucose (30mM) in normoxia (21 %) or hypoxia (1 %). Cells were serum starved and mitomycin-C (10ug/ml_, Roche, Cat # 10107409001) was added to abolish the effects of proliferation. The migration was calculated from the area free of cells at 0 hours and 24 hours. As can be seen from Figure 2, the microRNA-210 mimic increased fibroblast migration significantly under hypoxic and high glucose conditions.

Example 3. The effect of a microRNA-210 mimic on fibroblast proliferation under normal and high glucose and hypoxic and normoxic conditions Transfection of miR-210 mimic in HDF cells

Human dermal fibroblast (HDF) cells were seeded with 80-90% confluency on the first day. Next day, 1 , 10 and 20nM miR-210 mimic (miRIDIAN mmu-miR-210-3p; Cat #: C-310570- 05; Dharmacon) or negative control (miRIDIAN cel-miR-67; Cat #: CN-001000-01; Dharmacon) were transfected using RNAiMAX transfection reagent (Thermo Fisher Scientific, Cat # 13778075). The cells were allowed to recover for 48 hours before performing the analyses. Cell proliferation assay

HDF cells were starved for 24 hours and exposed to normal (5.5mM) or high glucose in normoxia (21 %) or hypoxia (1 %) for 24 hours. The cell proliferation was measured using the BrdU Cell Proliferation ELISA kit (Abeam, ab-126556). As can be seen from Figure 3, the microRNA-210 mimic increased fibroblast proliferation significantly under hypoxic and high glucose conditions.

Claims

1. A method of treatment of an ulcer in a patient having diabetes, comprising administering a therapeutically-effective amount of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need of such treatment.

2. A nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 for use in the treatment of a skin ulcer in a patient having diabetes.

3. The use of a nucleic acid selected from the group consisting of mature microRNA- 210 and precursors to mature microRNA-210 for the manufacture of a medicament for the treatment a skin ulcer in a patient having diabetes.

4. The method, compound for use or use as claimed in any one of Claims 1 to 3, wherein the precursors to mature microRNA-210 are selected from the group consisting of pri-microRNA-210, pre-microRNA-210, a double-stranded mimic of mature microRNA- 210 or a vector encoding microRNA-210.

5. The method, compound for use or use as claimed in any one of Claims 1 to 4, wherein the nucleic acid is a double-stranded mimic of mature microRNA-210.

6. The method, compound for use or use as claimed in any one of Claims 1 to 5, wherein the diabetes is type 1 or type 2 diabetes.

7. The method, compound for use or use as claimed in any one of Claims 1 to 6, wherein the ulcer is a skin ulcer.

8. The method, compound for use or use as claimed in any one of Claims 1 to 7, wherein the ulcer is a diabetic leg or foot ulcer.

9. The method, compound for use or use as claimed in any one of Claims 1 to 8, wherein the skin ulcer is a diabetic foot ulcer.

10. A method of promoting diabetic ulcer healing comprising administering a therapeutically-effective amount of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need of such treatment.

1 1. A nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 for use in promoting diabetic ulcer healing.

12. The use of a nucleic acid selected from the group consisting of mature microRNA- 210 and precursors to mature microRNA-210 for the manufacture of a medicament for promoting diabetic ulcer healing.

13. The method, compound for use or use as claimed in any one of Claims 10 to 12, wherein the precursors to mature microRNA-210 are selected from the group consisting of pri-microRNA-210, pre-microRNA-210, a double-stranded mimic of mature microRNA- 210 or a vector encoding microRNA-210.

14. The method, compound for use or use as claimed in any one of Claims 10 to 13, wherein the nucleic acid is a double-stranded mimic of mature microRNA-210.

15. The method, compound for use or use as claimed in any one of Claims 10 to 14, wherein the diabetic ulcer is in a patient with type 1 or type 2 diabetes.

16. The method, compound for use or use as claimed in any one of Claims 10 to 15, wherein the diabetic ulcer is a skin ulcer.

17. The method, compound for use or use as claimed in any one of Claims 10 to 16, wherein the diabetic ulcer is a diabetic leg or foot ulcer.

18. The method, compound for use or use as claimed in any one of Claims 10 to 17, wherein the diabetic ulcer is a diabetic foot ulcer.

19. A method of promoting ulcer healing in a patient having diabetes comprising administering a therapeutically-effective amount of a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need of such treatment.

20. A nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 for use in promoting ulcer healing in a patient having diabetes.

21. The use of a nucleic acid selected from the group consisting of mature microRNA- 210 and precursors to mature microRNA-210 for the manufacture of a medicament for promoting ulcer healing in a patient having diabetes.

22. The method, compound for use or use as claimed in any one of Claims 19 to 21 , wherein the precursors to mature microRNA-210 are selected from the group consisting of pri-microRNA-210, pre-microRNA-210, a double-stranded mimic of mature microRNA- 210 or a vector encoding microRNA-210.

23. The method, compound for use or use as claimed in any one of Claims 19 to 22, wherein the nucleic acid is a double-stranded mimic of mature microRNA-210.

24. The method, compound for use or use as claimed in any one of Claims 19 to 23, wherein the diabetes is type 1 or type 2 diabetes.

25. The method, compound for use or use as claimed in any one of Claims 19 to 24, wherein the ulcer is a skin ulcer.

26. The method, compound for use or use as claimed in any one of Claims 19 to 25, wherein the skin ulcer is a diabetic leg or foot ulcer.

27. The method, compound for use or use as claimed in any one of Claims 19 to 26, wherein the skin ulcer is a diabetic foot ulcer.

28. A method of enhancing the hypoxic response in a diabetic ulcer, comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need thereof.

29. A method of restoring fibroblast function in a diabetic ulcer, comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need thereof.

30. A method of promoting the formation of extracellular matrix and/or collagen in dermal tissue in a diabetic ulcer, comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need thereof.

31. A method of promoting the re-epithelialization of a diabetic ulcer comprising administering a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 to a patient in need thereof.

32. A pharmaceutical composition comprising a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 as defined in any one of the preceding claims and optionally one or more pharmaceutically-acceptable excipient for use as defined in any one of the preceding claims.

33. The method as claimed in any one of the preceding claims, wherein the nucleic acid is administered in the form of a pharmaceutical composition comprising a nucleic acid selected from the group consisting of mature microRNA-210 and precursors to mature microRNA-210 as defined in any one of the preceding claims and optionally one or more pharmaceutically-acceptable excipient.